TWI837437B - Chimeric antigen receptor t cell therapy - Google Patents

Abstract

Provided herein are methods for preparing, producing, processing, culturing, isolating, or making cells suitable for immune or cell therapy, and for their use in cell therapy.

Description

Chimeric Antigen Receptor T Cell Therapy

This application relates to CAR-T cells, methods of making them, and methods of using them to treat cancer.

Human cancers, by their nature, consist of normal cells that undergo genetic or epigenetic transformation into abnormal cancer cells. Cancer cells express proteins and other antigens that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells employ a variety of mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells. Human T cell therapy relies on ex vivo enriched or modified human T cells to target and kill cancer cells in an individual, such as a patient. A variety of techniques have been developed to prepare T cell populations with enriched concentrations of naturally occurring T cells capable of targeting tumor antigens, remove circulating tumor cells and/or genetically engineer T cells to specifically target known cancer antigens, thereby generating chimeric antigen receptor (CAR)-T cell populations for cancer therapy. Some of these therapies have shown promising effects on tumor size and patient survival.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. Although the present invention has been described in conjunction with its embodiments, the description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the accompanying claims. Other aspects, advantages and modifications are within the scope of the following embodiments/claims.

Example 1. A method for treating mantle cell lymphoma (MCL) or B-cell ALL in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR).

Embodiment 2. The method of Embodiment 1, wherein the MCL and B-cell ALL are relapsed or refractory MCL and B-cell ALL, and optionally the MCL is typical, blastoid, and polymorphic MCL.

Embodiment 3. The method of any one of Embodiments 1 and 2, wherein the MCL and B-cell ALL are refractory to or relapsed after one or more of the following: chemotherapy, radiation therapy, immunotherapy (including T cell therapy and/or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof.

Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the individual has received 1-5 prior treatments, wherein at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibodies, chemotherapy containing anthracycline or bendamustine, and/or Bruton Tyrosine Kinase inhibitor (BTKi).

Embodiment 5. The method of Embodiment 4, wherein the BTKi is ibrutinib or acalabrutinib.

Embodiment 6. The method of any one of Embodiments 1 to 5, wherein R/R B-cell ALL is defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≤12 months after first remission, relapsed or refractory after ≥2 lines of prior systemic therapy, or relapsed after allogeneic stem cell transplantation (SCT), where appropriate, the individual is required to have ≥5% bone marrow blasts, an Eastern Cooperative Oncology Group performance status of 0 or 1, and/or adequate renal, liver, and heart function.

Embodiment 7. The method of any one of Embodiments 1 to 6, wherein if the B-cell ALL individual has received prior blinatumomab, the individual is required to have leukemic blasts with CD19 expression ≥90%.

Embodiment 8. The method of any one of Embodiments 1 to 7, wherein the individual receives bridging therapy after leukapheresis and before conditioning/lymphodepleting chemotherapy.

Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the MCL individual receives the following lymphodepleting chemotherapy regimen: 500 mg/m ² cyclophosphamide intravenously and 30 mg/m ² fludarabine intravenously, both given on the fifth day before, the fourth day before, and the third day before the T cell infusion.

Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the B-cell ALL individual receives the following lymphodepleting regimen: 25 mg/m ² of fludarabine per day administered intravenously (IV) on the fourth day, the third day, and the second day prior to T cell infusion; and 900 mg/m ² of cyclophosphamide per day administered IV on the day prior to infusion.

Embodiment 11. The method of any one of Embodiments 8 to 10, wherein the MCL transition therapy is selected from dexamethasone (e.g., 20-40 mg PO or IV once daily or equivalent for 1-4 days); methylprednisolone, ibrutinib (e.g., 560 mg PO once daily) and/or acalabrutinib (e.g., 100 mg PO twice daily); immunomodulators; R-CHOP, bendamustine; alkylating agents; and/or platinum-based agents, wherein the transition therapy is administered after leukapheresis and completed, e.g., 5 days or less, prior to conditioning chemotherapy.

Embodiment 12. The method of any one of Embodiments 8 to 10, wherein the B-cell ALL individual may receive any one or more of the following transitional chemotherapy regimens: Predefined transitional chemotherapy regimen Weakened VAD Vincristine non-liposomal (IV 1-2 mg once a week) or liposomal (IV 2.25 mg/m ² once a week) and dexamethasone IV or PO once daily 20-40 mg × 3-4 days a week. Doxorubicin 50 mg/m ² IV × 1 (first week only) as needed Mercaptopurine (6-MP) Oral 50-75 mg/m ² per day (take on an empty stomach at bedtime to improve absorption) Hydroxyurea ​ Titrated dose between 15-50 mg/kg per day (rounded to the nearest 500 mg capsule and given continuously as a single daily oral dose) DOMP Dexamethasone 6 mg/m ² divided BID PO (or IV) on days 1-5, vincristine 1.5 mg/m ² IV (maximum dose 2 mg) on day 1, methotrexate 20 mg/m ² PO once a week, 6-MP 50-75 mg/m ² PO once daily Weakened FLAG/FLAG-IDA IV 30 mg/m ² fludarabine on days 1-2, IV 2 g/m ² cytarabine on days 1-2, SC or IV 5 μg/kg G-CSF started on day 3 and may be continued until the day before starting conditioning chemotherapy. With or without IV 6 mg/m ² idarubicin on days 1-2 Mini -hyper CVAD ( method A and / or B) Routine A: 150 mg/m ² cyclophosphamide every 12 hours for 3 days, 20 mg/d dexamethasone IV or PO once daily on days 1-4 and 11-14, 2 mg vincristine IV × 1 Routine B: 250 mg/m ² methotrexate IV 24 hours on day 1, 0.5 g/m ² cytarabine IV every 12 hours × 4 doses on days 2 and 3

Embodiment 13. The method of any one of Embodiments 1 to 12, wherein the T cell product comprises CD4+ and CD8+ CAR T cells, which are prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment and subsequent partial or complete depletion of circulating cancer cells.

Embodiment 14. The method of Embodiment 13, wherein the PBMCs are enriched for T cells by positive selection for CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing the FMC63-28Z CAR, which is a chimeric antigen receptor (CAR) comprising an anti-CD19 single-chain variable fragment (scFv), CD28 and CD3-ζ domains.

Embodiment 15. The method of any one of Embodiments 13 and 14, wherein the T cell product comprises fewer cancer cells than a T cell product comprising T cells from a leukocyte separation product that has not been positively selected for CD4+ and CD8+ T cells.

Embodiment 16. The method of any one of Embodiments 13 to 15, wherein the T cell product has other superior product properties relative to a T cell product comprising T cells from a leukocyte separation product that has not been positively selected/enriched for CD4+ and CD8+ T cells.

Embodiment 17. The method of Embodiment 16, wherein the superior product attributes are selected from an increased percentage of CDRA45+CCR7+ (naive-like) T cells, a decreased percentage of differentiated T cells, an increased percentage of CD3+ cells, a decreased IFN-γ production, a decreased percentage of CD3- cells.

Embodiment 18. The method of any one of Embodiments 1 to 17, wherein one or more doses of 1.8×10 ⁶ , 1.9×10 ⁶ or 2×10 ⁶ CAR-positive live T cells per kilogram of body weight are administered to the MCL individual, wherein the maximum is 2×10 ⁸ CAR-positive live T cells (for patients over 100 kg), and 0.5×10 ⁶ , 1×10 ⁶ or 2×10 ⁶ CAR-positive live T cells per kilogram of body weight are administered to the B-cell ALL individual, wherein the maximum is 2×10 ⁸ CAR-positive live T cells (for patients over 100 kg).

Embodiment 19. The method of any one of Embodiments 1 to 17, wherein if the individual has achieved a complete response to the first infusion, then if remission evolves after the next >3 months, the individual may receive a second infusion of anti-CD19 CAR T cells, provided that CD19 expression has been retained and there are no suspected neutralizing antibodies against the CAR, wherein the response is assessed using the Lugano classification.

Embodiment 20. The method of any one of embodiments 1 to 19, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and neurotoxicity following administration of the T cells.

Embodiment 21. The method of Embodiment 20, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably four weeks following infusion.

Embodiment 22. The method of any one of Embodiments 20 and 21, wherein the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and hypotension, and the signs and symptoms associated with neurological events include encephalopathy, seizures, changes in mental status, speech disorders, tremors and confusion.

Embodiment 23. The method of any one of Embodiments 20 to 22, wherein the interleukin release syndrome in the MCL individual is managed according to the following regimen: CRS Level Tocilizumab ​ Corticosteroids Grade 1 : Symptoms requiring symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgia, malaise). If there is no improvement after 24 hours, give 8 mg/kg tocilizumab intravenously over 1 hour (not to exceed 800 mg). Not applicable Grade 2 Symptoms requiring and responsive to moderate intervention. Oxygen requirement less than 40% FiO2 or hypotension or Grade 2 organ toxicity responsive to fluids or low doses of one vasopressor. Administer 8 mg/kg tosilimab (not to exceed 800 mg) intravenously over 1 hour. Repeat tosilimab every 8 hours as needed if unresponsive to intravenous fluids or increased oxygenation. Limit to a maximum of 3 doses in a 24-hour period; if signs and symptoms of CRS do not improve clinically, give a maximum of 4 doses total. If improved, discontinue tosilimab. If no improvement within 24 hours of starting tocilizumab, manage according to Grade 3. If improvement, taper corticosteroids. Grade 3 Symptoms requiring and responding to aggressive intervention. Oxygen requirement greater than or equal to 40% FiO2 or hypotension requiring high-dose or multiple vasopressors or Grade 3 organ toxicity or Grade 4 transaminase elevations. According to Level 2 Administer 1 mg/kg methylprednisolone or equivalent dexamethasone twice daily (e.g., 10 mg IV every 6 hours) until Grade 1, then taper corticosteroids. If improvement, manage as for Grade 2. If no improvement, manage as for Grade 4. Grade 4 Life-threatening symptoms. Respiratory support or continuous venovenous hemodialysis (CVVHD) required, or grade 4 organ toxicity (excluding elevated transaminases). According to Level 2 Administer 1000 mg methylprednisolone IV daily for 3 days. If improvement, taper corticosteroids and manage as Grade 3. If no improvement, consider another immunosuppressant.

Embodiment 24. The method of any one of Embodiments 20 to 23, wherein neurotoxicity in MCL subjects is managed according to the following regimen: Graded Assessment Concurrent CRS No concurrent CRS Level 2 Administer tocilizumab according to Example 15 to manage Grade 2 CRS. If there is no improvement within 24 hours after starting tocilizumab, administer 10 mg dexamethasone intravenously every 6 hours until the event is Grade 1 or less, followed by a gradual taper of corticosteroids. If there is still no improvement, manage as for Grade 3. Administer 10 mg dexamethasone intravenously every 6 hours until the event is grade 1 or less, followed by a gradual taper of corticosteroids. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 3 Administer tosilimab according to Example 15 to manage Grade 2 CRS. In addition, administer 10 mg dexamethasone intravenously with the first dose of tosilimab, and repeat dexamethasone doses every 6 hours. Continue dexamethasone until the event is Grade 1 or lower, then taper corticosteroids. If improved, discontinue tosilimab and manage as Grade 2. If still no improvement, manage as Grade 4 (or less). Administer 10 mg dexamethasone intravenously every 6 hours. Continue dexamethasone until events are Grade 1 or less, then taper corticosteroids. If no improvement, manage as for Grade 4. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 4 Administer tocilizumab according to Example 15 to manage Grade 2 CRS. Administer 1000 mg methylprednisolone intravenously daily with the first dose of tocilizumab and continue with 1000 mg methylprednisolone intravenously daily for 2 additional days. If improved, manage as for Grade 3. If no improvement, consider another immunosuppressive agent. Administer 1000 mg methylprazosin IV daily for 3 days. If improved, manage as for grade 3. If not improved, consider another immunosuppressant. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures.

Embodiment 25. The method of any one of embodiments 1 to 24, wherein the MCL individual is a high-risk patient as determined by a Ki-67 tumor proliferation index ≥ 50% and/or the presence of a TP53 mutation.

Embodiment 26. The method of any one of Embodiments 20 to 22, wherein CRS in the B-cell ALL individual is managed according to the following regimen: CRS Level Tocilizumab Corticosteroids Grade 1 : Symptoms requiring symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgia, malaise). If there is no improvement after 24 hours, give 8 mg/kg tocilizumab intravenously over 1 hour (not to exceed 800 mg). Not applicable. Grade 2 Symptoms requiring and responsive to moderate intervention. Oxygen requirement less than 40% FiO2 or hypotension or Grade 2 organ toxicity responsive to fluids or low doses of one vasopressor. Administer 8 mg/kg tosilimab (not to exceed 800 mg) intravenously over 1 hour. Repeat tosilimab every 8 hours as needed if unresponsive to intravenous fluids or increased oxygenation. Limit to a maximum of 3 doses in a 24-hour period; if signs and symptoms of CRS do not improve clinically, give a maximum of 4 doses total. If improved, discontinue tosilimab. If no improvement within 24 hours of starting tocilizumab, manage according to Grade 3. If improvement, taper corticosteroids. Grade 3 Symptoms requiring and responding to aggressive intervention. Oxygen requirement greater than or equal to 40% FiO2 or hypotension requiring high-dose or multiple vasopressors or Grade 3 organ toxicity or Grade 4 transaminase elevations. According to Level 2 Administer 1 mg/kg methylprednisolone or equivalent dexamethasone (e.g., 10 mg IV every 6 hours) twice daily until Grade 1, then taper corticosteroids. If improvement, manage as for Grade 2. If no improvement, manage as for Grade 4. Grade 4 Life-threatening symptoms. Respiratory support or continuous venovenous hemodialysis (CVVHD) required, or grade 4 organ toxicity (excluding elevated transaminases). According to Level 2 Administer 1000 mg methylprednisolone IV daily for 3 days. If improvement, taper corticosteroids and manage as Grade 3. If no improvement, consider another immunosuppressant.

Embodiment 27. The method of any one of Embodiments 20 to 22 and 26, wherein neurotoxicity in the B-cell ALL subject is managed according to one of the following two regimens: NE level Original Management Guidelines Revised Management Guidelines Level 1 ● Supportive care ● Neurological examination and additional management as clinically indicated ● Supportive care ● Close monitoring of neurological status ● Consider prophylactic anti-epileptic drugs Level 2 Supportive Care and Assessment ● Neurological examination, brain MRI, and CSF evaluation; consider EEG as clinically indicated ● Consider prophylactic antiepileptic medications Supportive care and assessment ● Continuous cardiac telemetry and pulse oximetry as indicated ● Continuous neurological examinations, including funduscopy and Glasgow Coma Score, brain MRI, CSF evaluation, EEG; consider neurological society consultation ● Anti-epileptic drugs for epileptic patients Tosilimab ● Consider 8 mg/kg tosilimab IV over 1 hour (not to exceed 800 mg) for patients with comorbidities (e.g., grade ≥2 CRS) Tosilimab ● For patients with concurrent CRS , give 8 mg/kg tosilimab IV over 1 hour (not to exceed 800 mg); if unresponsive to IV fluids or increased supplemental oxygen, repeat every 4-6 hours as needed, up to 3 doses in 24 hours ● If patient improves, discontinue tosilimab Corticosteroids ● N/A Corticosteroids ● Dexamethasone 10 mg IV every 6 hours for patients without concurrent CRS ● Dexamethasone 10 mg IV every 6 hours for patients with concurrent CRS who do not improve within 24 hours of starting tocilizumab ● Taper corticosteroids if patient improves Level 3 Supportive care and assessment ● Based on Level 2 ● Monitoring with continuous cardiac telemetry and pulse oximetry Supportive care and assessment ● Management in monitored care or ICU Tosilimab ● Consider 8 mg/kg tosilimab IV over 1 hour (not to exceed 800 mg); repeat every 4-6 hours if symptoms are not stable or not improving Tosilimab ● Based on Grade 2 ● If patient improves, discontinue Tosilimab Corticosteroids ● Consider corticosteroids (eg, dexamethasone 10 mg IV q 6 h or methylprednisolone 1 mg/kg BID) for worsening symptoms despite tocilizumab Corticosteroids ● Dexamethasone 10 mg IV every 6 hours ● If patient improves, taper corticosteroids Level 4 Supportive care and assessment ● Based on Level 2 ● Monitoring with continuous cardiac telemetry and pulse oximetry Supportive care and assessment ● Based on level 3 ● May require ventilator ● Administer immunosuppressive agents if patient does not improve Tosilimab ● If not previously administered, administer tosilimab based on grade 3 Tocilizumab ● Based on level 2 Corticosteroids ● Administer corticosteroids (e.g., methylprednisolone 1 g/d x 3 days, then 250 mg BID x 2 days, then 125 mg BID x 2 days, then 60 mg BID x 2 days) Corticosteroids ● Administer high doses of corticosteroids (e.g., methylprednisolone 1 g/d x 3 days) ● If the patient improves, taper the corticosteroid

Embodiment 28. The method of any one of Embodiments 1 to 27, wherein the B-cell ALL individual may receive any one or more of the following transitional chemotherapy regimens: Predefined transitional chemotherapy regimen Weakened VAD Vincristine non-lipidic (IV 1-2 mg once a week) or liposomal (IV 2.25 mg/m ² once a week) and dexamethasone IV or PO once daily 20-40 mg × 3-4 days a week. Doxorubicin 50 mg/m ² IV × 1 (first week only) as needed 6- MP Oral 50-75 mg/m ² per day (take on an empty stomach at bedtime to improve absorption) Hydroxyurea Titrated dose between 15-50 mg/kg per day (rounded to the nearest 500 mg capsule and given continuously as a single daily oral dose) DOMP Dexamethasone 6 mg/m ² divided BID PO (or IV) on days 1-5, vincristine 1.5 mg/m ² IV (maximum dose 2 mg) on day 1, methotrexate 20 mg/m ² PO once a week, 6-MP 50-75 mg/m ² PO once a day Weakened FLAG/FLAG-IDA IV 30 mg/m ² fludarabine on days 1-2, IV 2 g/m ² cytarabine on days 1-2, SC or IV 5 μg/kg G-CSF started on day 3 and may be continued until the day before starting conditioning chemotherapy. With or without IV 6 mg/m ² idarucizumab on days 1-2 Mini-hyper CVAD ( method A and / or B) Routine A: 150 mg/m ² cyclophosphamide every 12 hours for 3 days, 20 mg/d dexamethasone IV or PO once daily on days 1-4 and 11-14, 2 mg vincristine IV × 1 Routine B: 250 mg/m ² methotrexate IV 24 hours on day 1, 0.5 g/m ² cytarabine IV every 12 hours × 4 doses on days 2 and 3

Embodiment 29. An autologous T cell expressing an anti-CD19 CAR, for use in the method of treating mantle cell lymphoma (MCL) or B cell ALL according to any one of Embodiments 1 to 28.

Embodiment 30. Use of autologous T cells expressing anti-CD19 CAR for the manufacture of a medicament for treating mantle cell lymphoma (MCL) or B cell ALL according to any one of Embodiments 1 to 28.

Embodiment 31. A method for predicting (i) an objective response of an individual to CAR T cell therapy (according to any of the methods of embodiments 1 to 28, as appropriate), the method comprising measuring the peak CAR T cell level and comparing it to a reference standard, wherein the objective response is positively correlated with the peak CAR T cell level, wherein the objective response includes a complete response and a partial response, and wherein all responses are assessed using the Lugano classification. (ii) minimal residual disease (e.g., at week 4) in response to CAR T cell therapy (according to any of the methods of embodiments 1 to 28, as appropriate), the method comprising measuring the peak CAR T cell level and comparing it to a reference standard, wherein negative minimal residual disease is associated with a higher peak CAR T cell level. (iii) Grade ≥3 CRS and/or Grade ≥3 neurological events (NE) in an individual receiving CAR T cell therapy (according to the method of any one of Embodiments 1 to 28, as appropriate), the method comprising measuring the peak CAR T cell expansion after treatment and comparing the level to a reference value, wherein the higher the CAR T cell expansion, the higher the probability of Grade ≥3 CRS and/or Grade ≥3 NE events. (iv) Level ≥3 CRS and/or Level ≥3 NE, the method comprising measuring the peak levels of GM-CSF and IL-6 after CAR T cell therapy (according to the method of any one of Examples 1 to 28, as appropriate) and comparing them to reference levels, wherein the higher the peak levels of these interleukins, the higher the probability of Level ≥3 CRS and/or Level ≥3 NE. (v) Level ≥3 CRS in an individual receiving CAR T cell therapy (according to the method of any one of Examples 1 to 28, as appropriate), the method comprising measuring the peak level of serum ferritin after CAR T cell therapy and comparing it to a reference level, wherein the higher the peak level of ferritin, the higher the probability of Level ≥3 CRS. (vi) Level ≥3 CRS, the method comprising measuring the peak levels of serum IL-2 and IFN-γ after CAR T cell therapy (as appropriate, any one of Examples 1 to 28) and comparing them with reference levels, wherein the higher the peak levels of IL-2 and IFN-γ, the higher the probability of level ≥3 NE. (vii) Grade ≥3 CRS, the method comprising measuring the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or vascular cell adhesion molecule (VCAM) after CAR T cell therapy (as appropriate, any one of Examples 1 to 28) and comparing them to reference levels, wherein the higher the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or vascular cell adhesion molecule (VCAM), the higher the probability of Grade ≥3 NE (viii) Grade ≥3 CRS after receiving CAR T cell therapy (as appropriate, according to the method of any one of Examples 1 to 28), the method comprising measuring IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B and/or perforin and compared these levels with the reference levels, wherein these peak serum levels of IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B and/or perforin were positively correlated with grade ≥3 CRS. (ix) Grade ≥3 CRS after CAR T cell therapy of B-cell ALL (according to the method of any one of Embodiments 1 to 28, as appropriate), the method comprising measuring the peak serum level of IL-15 after anti-CD19 CAR T treatment and comparing the level to a reference level, wherein the peak serum level of IL-15 is negatively correlated with Grade ≥3 CRS. (x) level ≥3 CRS and/or level ≥3 NE after CAR T cell therapy (according to the method of any one of Embodiments 1 to 28, as appropriate), the method comprising measuring the peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b and granzyme B after anti-CD19 CAR T therapy and comparing the levels with reference levels, wherein the peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b and granzyme B are positively correlated with level ≥3 CRS and level ≥3 NE. (xi) whether the patient will be MRD negative ( ^10-5 sensitivity) 4 weeks/one month after CAR T cell therapy (as appropriate any one of Examples 1 to 28), the method comprising measuring the peak serum levels of IFN-γ, IL-6 and/or IL-2 after treatment and comparing the levels to a reference standard, wherein the peak serum levels of IFN-γ, IL-6 and/or IL-2 are positively correlated with MRD negativity at one month.

Embodiment 32. The method of any one of embodiments 20 to 24, 26, 27 and 30 to 31, wherein CRS and NE are graded by the method described in Lee et al., Blood 2014; 124: 188-195.

Embodiment 33. The method of Embodiment 31, wherein the reference standard is established by any method commonly used in biomarker technology, such as quartile analysis of patient populations with known responses, toxicity grade, and MRD level.

Embodiment 34. The method of Embodiment 31, wherein the CAR T cell level is measured by the CAR gene copies per microgram of DNA in the blood.

Embodiment 35. The method of any one of Embodiments 1 to 34, further comprising reducing the level/activity of cytokines positively correlated with level ≥3 CRS and/or level ≥3 NE after CAR T cell infusion to reduce level ≥3 CRS and/or level ≥3 NE.

Embodiment 36. A method of improving the efficacy of CAR T cell therapy (e.g., typical, blastoid and polymorphic MCL, and B cell ALL) in an individual in need thereof, comprising manipulating the T cell phenotype of a T cell product administered to the individual, optionally wherein the manipulation comprises increasing the number of CD3+ T cells, decreasing the number of CD3- cells, increasing the number/percentage of CDRA45+CCR7+ (naive-like) T cells, and/or decreasing the number/percentage of differentiated cells in the T cell product during production, decreasing the amount of IFN-γ produced by the T cells, wherein the number of CDRA45+CCR7+ (naive-like) T cells in the T cell product is increased relative to the number of CDRA45+CCR7+ (naive-like) T cells in the T cell product without any deliberate manipulation. The improvement is observed in the potency of T cell products prepared at a higher number/percentage of T cells and/or a higher number/percentage of differentiated cells.

Unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide those skilled in the art with a general glossary of many of the terms used in the present invention.

Units, prefixes and symbols are expressed in the form accepted by the International System of Units (Système International de Unites (SI)). Numerical ranges include the values limiting the range. The disclosure provided herein is not a limitation of the various aspects of the present application, which can be understood by reference to the specification as a whole. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd edition, (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th edition, (2013), Academic Press; and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et al., eds., 2nd edition, (2006), Oxford University Press, provide those skilled in the art with a general glossary of many of the terms used in the present invention.

The article "a" or "an" refers to "one or more" of any descriptive or enumerative component.

The terms "about" or "consisting essentially of" refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within 1 or more than 1 standard deviation, as practiced in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any value between 2.7 mg and 3.3 mg (for 10%). In terms of biological systems or methods, the terms can mean up to an order of magnitude or up to 5 times the value. When a specific value or composition is provided in the application and the claims, unless otherwise indicated, the meaning of "about" or "consisting essentially of" includes an acceptable error range for the value or composition. Unless otherwise indicated, any concentration range, percentage range, ratio range or integer range includes any integer value and, where appropriate, its fraction (such as one tenth and one hundredth of an integer) within the stated range.

Unless expressly stated or obvious from the context, the term "or" as used herein is to be understood as inclusive and covers both "or" and "and". The term "and/or" refers to each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases such as "A and/or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and/or" as used in phrases such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The terms "such as" and "ie" are used by way of example only and are not intended to be limiting, and should not be construed as referring to only those items expressly listed in this specification.

The terms “or more”, “at least”, “more than” and similar terms such as “at least one” include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 ,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,8 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 29, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more than the stated values. Any greater number or fraction therebetween is also included. Conversely, the term "not more than" includes values that are less than the stated values. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller number or fractions therebetween.

The terms “plurality,” “at least two,” “two or more,” “at least a second,” and the like include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 ,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also including any larger number or fraction therebetween.

Throughout this specification, the word "comprising" or variations such as "comprises/comprising" should be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. It should be understood that whenever the language "comprising" is used herein to describe an aspect, similar aspects described by the terms "consisting of" and/or "consisting essentially of" are also provided. The term "consisting of" excludes any elements, steps or ingredients not specified in the scope of the patent application. In re Gray, 53 F.2d 520, 11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448, 450 (Bd. App. 1948) (“consisting of” is defined as “a closed solution, consisting of no matter other than the substance described, except impurities customarily associated therewith”). The term “consisting essentially of” limits the scope of the solution to the specified substances or steps and those substances or steps that do not materially affect the basic and novel characteristics of the claimed invention.

Unless specifically stated or apparent from the context, as used herein, the term "about" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" may mean within one or more than one standard deviation, according to practice in the art. "About" or "approximately" may mean a range of up to 10% (i.e., ±10%). Thus, "about" may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. In addition, particularly in the context of biological systems or methods, the terms may mean up to an order of magnitude or up to 5-fold of a value. When specific values or compositions are provided in the present invention, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for the specific value or composition.

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer) within the stated range.

The terms "activation", "activated" or similar terms refer to a state in which cells, including but not limited to immune cells (e.g., T cells), are sufficiently stimulated to induce detectable cell proliferation. Activation can be associated with induced interleukin production and detectable effector functions. The term "activated T cells" refers in particular to T cells that are undergoing cell division. The characteristics of T cell activation can be increased T cell expression of one or more biomarkers including (but not limited to) CD57, PD1, CD107a, CD25, CD137, CD69 and/or CD71. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication No. WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. Generally, such methods involve contacting cells (such as T cells) with activating, stimulating, or co-stimulatory agents (such as anti-CD3 and/or anti-CD28 antibodies) that may be attached, coated, or bound to beads or other surfaces in a solution (such as a feed, culture medium, and/or growth medium) with certain interleukins (such as IL-2, IL-7, and/or IL-15). Activators (such as anti-CD3 and/or anti-CD28 antibodies) attached to the same beads act as "surrogate" antigen presenting cells (APCs). One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In one embodiment, T cells are activated and stimulated to proliferate with certain antibodies and/or cytokines using the methods described in U.S. Patent Nos. 6,040,177, 5,827,642, and PCT Publication No. WO 2012/129514, the contents of which are incorporated herein by reference in their entirety.

The term "administering," "administering," or similar terms refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of immune cells prepared by the methods disclosed herein include intravenous (i.v. or IV), intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. Non-parenteral routes of administration refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In one embodiment, immune cells (e.g., T cells) prepared by the methods of the present invention are administered by injection or infusion. Non-parenteral routes include topical, epidermal, or transmucosal routes of administration, such as intranasal, vaginal, rectal, sublingual, or topical. Administration may also be performed once, twice, and/or multiple times over one or more prolonged periods of time. In the case of administration of one or more therapeutic agents (e.g., cells), administration can be simultaneous or sequential. Sequential administration includes administration of one agent only after administration of another or other agents has been completed.

The term "antibody" (Ab) includes but is not limited to immunoglobulins that specifically bind to an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains, CH1, CH2, CH3 and/or CH4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. The VH and VL regions may be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulins may be derived from any commonly known isotype, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of Ab (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, for example, naturally occurring and non-naturally occurring Abs, single and multiple Abs, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs, and single-chain Abs. Non-human Abs may be humanized by recombinant methods to reduce their immunogenicity in humans. Where not expressly stated, and unless the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or antigen-binding portions, monovalent and divalent fragments or portions, and single-chain Abs of any of the above-mentioned immunoglobulins.

"Antigen binding molecule", "antibody fragment" or similar terms refer to any portion of an antibody that is smaller than the entire antibody. Antigen binding molecules may include antigen complementation determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed by antigen binding molecules. In one embodiment, the CD19 CAR construct comprises an anti-CD 19 single-chain FV. The "single-chain Fv" or "scFv" antibody binding fragment comprises the variable heavy chain ( _VH ) and variable light chain ( _VL ) domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains, which enables the scFv to form the desired structure for antigen binding. All antibody-related terms used herein have the customary meanings in this technology and are well understood by those of ordinary skill in the art.

"Antigen" refers to any molecule that evokes an immune response or is capable of being bound by an antibody or antigen-binding molecule. The immune response may involve the production of antibodies, or the activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, may serve as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or may be expressed recombinantly. Antigens may be specific to a certain tissue, such as cancer cells, or they may be ubiquitously expressed. In addition, fragments of larger molecules may serve as antigens. In some embodiments, the antigen is a tumor antigen.

The term "neutralize" refers to the binding of an antigen binding molecule, scFv, antibody, or fragment thereof to a ligand and preventing or reducing the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or otherwise alters the ability of the ligand to bind by indirect means (such as structural or energetic changes in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it binds from performing a biological function.

The term "autologous" means any material that is derived from the same individual as the individual into which it is subsequently reintroduced. For example, the engineered autologous cell therapy described herein involves a population of lymphocytes from an individual (such as a donor or patient) that are then engineered to express a CAR construct and then administered back into the same individual.

The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species, such as an allogeneic T cell transplant.

"Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors at various stages. In one embodiment, the cancer or tumor is at stage 0, so, for example, the cancer or tumor is at a very early stage of development and has not yet metastasized. In another embodiment, the cancer or tumor is at stage I, so, for example, the cancer or tumor is relatively small in size, has not spread into neighboring tissues, and has not yet metastasized. In other embodiments, the cancer or tumor is in stage II or stage III, so, for example, the cancer or tumor is larger than stage 0 or stage I, and it has grown into adjacent tissues, but it has not yet metastasized, except perhaps to the lymph nodes. In additional embodiments, the cancer or tumor is in stage IV, so, for example, the cancer or tumor has metastasized. Stage IV may also be referred to as advanced or metastatic cancer.

As used herein, "anti-tumor effect" refers to biological effects that may exist and are not limited to: reduction in tumor size, inhibition of tumor growth, reduction in tumor cell number, reduction in tumor cell proliferation, reduction in the number/extent of cancer metastasis, increase in overall or progression-free survival, increase in life expectancy, and/or improvement in various physiological symptoms associated with tumors. Anti-tumor effect may also refer to the prevention of tumor occurrence, such as vaccines.

The term "progression-free survival" (PFS) refers to the time from the date of self-treatment to the date of disease progression (according to general criteria, such as modified IWG response criteria for malignant lymphoma) or death from any cause. The term "disease progression" may be assessed by measurement of malignant lesions on radiographs or other methods and should not be reported as an adverse event. Death due to disease progression in the absence of signs and symptoms may be reported for the primary tumor type (e.g., DLBCL). The term "duration of response" (DOR) refers to the time period from the individual's first objective response to the date of confirmation of disease progression (according to general criteria, such as modified IWG response criteria for malignant lymphoma) or death. The term "overall survival" (OS) refers to the time from the date of self-treatment to the date of death.

"Cytokine" refers to non-antibody proteins that can be released by immune cells including macrophages, B cells, T cells, and mast cells to propagate an immune response. In one embodiment, one or more cytokines are released in response to a therapy. In other embodiments, those cytokines secreted in response to a therapy may indicate or suggest an effective therapy. In one embodiment, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to modulate the response in the second cell. As used herein, "cytokine" means a protein released by a cell population that acts on another cell, such as an intercellular mediator. Interleukins can be expressed endogenously by cells or administered to an individual. Interleukins can be released by immune cells including macrophages, B cells, T cells, and mast cells to propagate immune responses. Interleukins can induce various responses in receptor cells. Interleukins can include homeostatic interleukins, trending factors, proinflammatory interleukins, effectors, and acute phase proteins. For example, homeostatic interleukins, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and proinflammatory interleukins can promote inflammatory responses. Examples of homeostatic interleukins include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) γ. Examples of proinflammatory interleukins include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, fibroblast growth factor (FGF) 2, granulocyte macrophage colony stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM -1), soluble vascular adhesion molecule 1 (sVCAM -1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

"Tendokines" are a type of interleukin that mediates cell tropism or directional movement. Examples of tropokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived tropism factor (MDC or CCL22), monocyte tropism protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducing protein 10 (IP-10), and thymus and activation-regulated tropism factor (TARC or CCL17).

"Therapeutically effective amount", "therapeutically effective dose" or similar terms refer to the amount of cells (such as immune cells or engineered T cells) produced by the methods of the present invention (producing T cell products) and which, when used alone or in combination with another therapeutic agent, prevent or treat the onset of disease in an individual or promote disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, and/or prevention of damage or disability caused by the disease. The ability to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in individuals during clinical trials, in animal model systems that predict efficacy in humans, or by analyzing the activity of the agent in an in vitro assay. In some embodiments, donor T cells used in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells used in T cell therapy are obtained from an individual other than the patient. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ cells, or at least about 10 ¹⁰ cells. In another embodiment, the therapeutically effective amount of T cells is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, about 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 ⁶ cells/kg, about 9×10 ⁶ cells/kg, about 1×10 ⁷ cells/kg, about 2×10 ⁷ cells/kg, about 3×10 ⁷ cells/kg, about 4×10 ⁷ cells/kg, about 5×10 ⁷ cells/kg, about 6×10 ⁷ cells/kg, about 7×10 ⁷ cells/kg, about 8×10 ⁷ cells/kg or about 9×10 ⁷ cells/kg. In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 1×10 ⁶ CAR-positive live T cells/kg body weight and about 2×10 ⁶ CAR-positive live T cells/kg body weight, up to a maximum dose of about 1×10 ⁸ CAR-positive live T cells. In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 0.4×10 ⁸ CAR-positive live T cells and about 2×10 ⁸ CAR-positive live T cells. In some embodiments, the therapeutically effective amount of CAR-positive live T cells is about 0.4×10 ⁸ , about 0.5×10 ⁸ , about 0.6×10 ⁸ , about 0.7×10 ⁸ , about 0.8×10 ⁸ , about 0.9×10 ⁸ , about 1.0×10 ⁸ , about 1.1×10 ⁸ , about 1.2×10 ⁸ , about 1.3×10 ⁸ , about 1.4×10 ⁸ , about 1.5×10 ⁸ , about 1.6×10 ⁸ , about 1.7×10 ⁸ , about 1.8×10 ⁸ , about 1.9×10 ⁸ , or about 2.0×10 ⁸ CAR-positive live T cells.

As used herein, the term "lymphocyte" may include natural killer (NK) cells, T cells, NK-T cells, or B cells. NK cells are a type of cytotoxic/cell toxic lymphocyte that represents a major component of the innate immune system. NK cells resist tumors and virally infected cells through a process of apoptosis or planned cell death. They are called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). T cell receptors (TCR) distinguish them from other lymphocyte types. The thymus (a specialized organ of the immune system) is primarily responsible for the maturation of T cells.

There are several types of "immune cells", including, but not limited to, macrophages (e.g., tumor-associated macrophages), neutrophils, basophils, eosinophils, granulocytes, natural killer cells (NK cells), B cells, T cells, NK-T cells, mast cells, tumor infiltrating lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and dendritic cells. The term also includes the precursor cells of these immune cells. Hematopoietic stem cells and/or progenitor cells can be obtained from bone marrow, cord blood, adult peripheral blood after interleukin mobilization, and the like by methods known in the art. Some progenitor cells are cells that can differentiate into lymphoid lineages, such as hematopoietic stem cells or progenitor cells of the lymphoid lineage. Additional examples of immune cells that can be used for immunotherapy are described in U.S. Publication No. 20180273601, which is incorporated herein by reference in its entirety.

There are also several types of T cells, namely: helper T cells (e.g., CD4+ cells, effector T _EFF cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem cell memory T _SCM cells, such as naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (i) central memory T CM cells express L-selectin and are CCR7+ and CD45RO+, and they secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T _EM cells _, however, do not express L-selectin or CCR7, but express CD45RO and produce effector cytokines, such as IFNγ and IL-4), regulatory T cells (T cells, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. T cells found in tumors are called "tumor infiltrating lymphocytes" (TIL). On the other hand, B cells play a major role in humoral immunity (with the involvement of antibodies). They produce antibodies and antigens and perform the role of antigen presenting cells (APCs) and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow from which they are named.

"Naive" T cells refer to mature T cells that remain immunologically undifferentiated. After positive and negative selection in the thymus, T cells appear as CD4 ⁺ or CD8 ⁺ naive T cells. In their naive state, T cells express L-selectin (CD62L ⁺ ), IL-7 receptor-α (IL-7R-α), and CD132, but they do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" may also refer to T cells that exhibit a phenotype that characterizes naive or immature T cells, such as T _SCM cells or T _CM cells. For example, immature T cells may express one or more of L-selectin (CD62L ⁺ ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive or immature T cells may be contrasted with terminally differentiated effector T cells, such as _TEM cells and T _EFF cells.

As referred to herein, "T cell function" refers to the normal characteristics of healthy T cells. T cell function may include T cell proliferation, T cell activity and/or cytolytic activity. In one embodiment, the method of preparing T cells under certain oxygen and/or pressure conditions of the present application will increase one or more T cell functions, thereby making the T cells more suitable and/or more effective for achieving therapeutic purposes. In some embodiments, T cells prepared according to the methods of the present invention have increased T cell function compared to T cells under conditions lacking certain oxygen and/or pressure. In other embodiments, T cells prepared according to the methods of the present invention have increased T cell proliferation compared to T cells cultured under conditions lacking certain oxygen and/or pressure. In additional embodiments, T cells prepared according to the methods of the present invention have increased T cell activity compared to T cells cultured under conditions lacking a certain amount of oxygen and/or pressure. In another embodiment, T cells prepared according to the methods of the present invention have increased cytolytic activity compared to T cells cultured under conditions lacking a certain amount of oxygen and/or pressure.

The terms cell "proliferation", "proliferating" or similar terms refer to the ability of a cell to grow in large quantities through cell division. Proliferation can be measured by staining the cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation can occur in vitro, such as during T cell culture, or in vivo, such as after administration of an immunocytotherapy (e.g., T cell therapy). Cell proliferation can be measured or determined by methods described herein or known in the art. For example, cell proliferation can be measured or determined by viable cell density (VCD) or total viable cells (TVC). The VCD or TVC can be theoretical (removing an aliquot or sample from the culture at a certain time point to determine the number of cells, then multiplying the number of cells by the volume of the culture at the beginning of the study) or actual (removing an aliquot or sample from the culture at a certain time point to determine the number of cells, then multiplying the number of cells by the actual volume of the culture at that certain time point). The term "T cell activity" refers to any activity common to healthy T cells. In one embodiment, T cell activity comprises interleukin production (such as INFγ, IL-2 and/or TNFα). In other embodiments, the T cell activity comprises the production of one or more interleukins selected from interferon gamma (IFNγ or IFN-γ), tissue necrosis factor alpha (TNFα or IFNα), and both. The term "cytolytic activity", "cytotoxicity" or similar terms refer to the ability of T cells to destroy target cells. In one embodiment, the target cell is a cancer cell, such as a tumor cell. In other embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cell expresses a target antigen.

The terms "genetically engineered", "gene editing" or "engineered" refer to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is incorporated into the genome of the cell.

The terms "transduction" and "transduced" refer to a method of introducing foreign DNA into a cell via a viral vector (see Jones et al., "Genetics: principles and analysis", Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a bacillary viral vector, an Epstein Barr viral vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

The "chimeric antigen receptors" (CAR or CAR-T) and T cell receptors (TCR) of this application are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. In the case of CAR, a single receptor can be programmed to recognize a specific antigen and, when bound to the antigen, activate immune cells to attack and destroy cells bearing or expressing the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells. In one embodiment, the cell prepared according to the present application is a cell having a chimeric antigen receptor (CAR) or a T cell receptor comprising an antigen binding molecule, a synergistic stimulatory domain, and an activation domain. The synergistic stimulatory domain may comprise an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular domain comprises a hinge or a truncated hinge domain.

"Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, interleukins, and complements) that result in the selective targeting, binding to, damage, destruction, and/or elimination from the vertebrate body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.

The terms "immunotherapy," "immune therapy," or similar terms refer to the treatment of an individual suffering from a disease or at risk of infection or recurrence of a disease by methods that include inducing, enhancing, suppressing, or otherwise improving an immune response. Examples of immunotherapy include, but are not limited to, T cell and NK cell therapy. T cell therapy may include donor T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy, and allogeneic T cell transplantation. Those skilled in the art will recognize that the methods of preparing immune cells disclosed herein will enhance the effectiveness of any cancer or transplantation T cell therapy. Examples of T cell therapy are described in U.S. Patent Publication Nos. 2014/0154228 and 2002/0006409, U.S. Patent Nos. 7,741,465, 6,319,494, and 5,728,388, and PCT Publication No. WO 2008/081035, which are incorporated by reference in their entireties.

The term "engineered autologous cell therapy" can be abbreviated as "eACT™", also known as donor-acceptor cell transfer, is a method in which a patient's own T cells are collected and subsequently genetically altered to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or cells of malignant diseases. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for certain tumor antigens, which is linked to an intracellular signaling portion that includes a co-stimulatory domain and an activation domain. The synergistic stimulatory domain can be derived from, for example, CD28, CTLA4, CD16, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, planned death-1 (PD-1), planned death ligand-1 (PD-L1), inducing T cell synergistic stimulator (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin proteins, interleukin receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or a signaling region of any combination thereof. The activation domain can be derived from, for example, CD3, such as CD3 ζ, ε, δ, γ or the like. In one embodiment, the CAR is designed to have two, three, four or more synergistic stimulatory domains. The CAR scFv can be designed to target, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignancies, including but not limited to NHL, CLL and non-T cell ALL. Example CAR+ T cell therapies and constructs are described in U.S. Patent Publications Nos. 2013/0287748, 2014/0227237, 2014/0099309 and 2014/0050708, which are incorporated herein by reference in their entirety.

As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR/CD3 engagement, causes a T cell response, proliferation, and/or up- or down-regulation of key molecules such as but not limited to.

As used herein, "co-stimulatory ligands" include molecules on antigen presenting cells that specifically bind to cognate co-stimulatory molecules on T cells. Binding of co-stimulatory ligands provides signals that mediate T cell responses, including but not limited to proliferation, activation, differentiation, and the like. Co-stimulatory ligands induce signals in addition to primary signals provided by stimulatory molecules, such as by binding of the T cell receptor (TCR)/CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule. Co-stimulatory ligands may include, but are not limited to, 3/TR6, 4-1BB ligands, agonists or antibodies that bind to ferroxine ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligands, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducing co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligands that specifically bind to B7-H3, lymphotoxin beta receptor, MHC class I chain-associated protein A (MICA), MHC class I chain-associated protein B (MICB), OX40 ligand, PD-L2 or planned death (PD) L1. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen 1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

"Co-stimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as (but not limited to) proliferation. Co-stimulatory molecules include, but are not limited to, "Co-stimulatory molecules" are cognate binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as (but not limited to) proliferation. Co-stimulatory molecules include (but are not limited to): 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (α; β; δ; ε; γ; ζ), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, thiabendazim ligand receptor, TRANCE/RANKL, VLA1 or VLA-6, or fragments, truncations or combinations thereof.

In some aspects, the cells of the present application can be obtained from T cells obtained from an individual. In one aspect, T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell strains available in this technology. T cells can also be obtained from blood units collected from an individual using a variety of techniques known to those skilled in the art, such as FICOLL™ separation and/or hemacytosis. In some aspects, cells collected by hemolysis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some aspects, the cells are washed with any solution (e.g., a solution with a neutralized pH or PBS) or medium. As will be appreciated, a washing step can be used, such as by using a semi-automatic flow-through centrifuge, such as a Cobe ^™ 2991 cell processor, a Baxter CytoMate ^™ , or the like. In some aspects, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In some aspects, undesirable components of the hemolysis sample are removed. Additional methods for isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, which is incorporated herein by reference in its entirety.

In some embodiments, T cells are separated from PBMCs by lysing erythrocytes and depleting monocytes, for example, by using centrifugation through a PERCOLL ^™ gradient. In some embodiments, specific T cell subsets (such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells) are further separated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry can be used, using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are used to isolate the relevant cell populations used in the present invention.

In one embodiment, CD3+ T cells are isolated from PBMC using Dynabeads coated with anti-CD3 antibodies. CD8+ and CD4+ T cells are further isolated by positive selection using CD8 microbeads (e.g., Miltenyi Biotec) or CD4 microbeads (e.g., Miltenyi Biotec), respectively.

In some embodiments, PBMCs are used directly for genetic modification using immune cells (such as CARs) using methods as described herein. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subsets before or after genetic modification and/or expansion.

One or more immune cells described herein can be obtained from any source including, for example, a human donor. The donor can be an individual in need of anti-cancer treatment, such as treatment with one of the immune cells produced by the methods described herein (i.e., an autologous donor), or can be an individual to whom a lymphocyte sample is given, which will be used to treat a different individual or cancer patient when the cell population produced by the methods described herein is produced (i.e., an allogeneic donor). The immune cells can be differentiated in vitro from a hematopoietic stem cell population, or the immune cells can be obtained from a donor. The immune cell population can be obtained from a donor by any suitable method used in this technology. For example, lymphocyte populations can be obtained by any suitable in vitro method, venous puncture, or other blood collection method that obtains a blood sample with or without lymphocytes. Lymphocyte populations are obtained by hemopheresis. One or more immune cells can be collected from any tissue containing one or more immune cells, including (but not limited to) a tumor. A tumor or a portion thereof is collected from an individual, and one or more immune cells are isolated from the tumor tissue. Any T cell can be used in the methods disclosed herein, including any immune cell suitable for T cell therapy. For example, one or more cells that can be used in the present application can be selected from the group consisting of tumor infiltrating lymphocytes (TIL), cytotoxic T cells, CAR T cells, engineered TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines that can be used in this technology. T cells may also be obtained from blood units collected from an individual using a variety of techniques known to those skilled in the art, such as FICOLL™ separation and/or hemapheresis. T cells may also be obtained from an artificial thymic organoid (ATO) cell culture system that replicates the human thymus environment to support efficient ex vivo differentiation of T cells from primary and reprogrammed multipotential stem cells. Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, PCT Publication No. WO2015/120096, and WO2017/070395, all of which are incorporated herein by reference for the purpose of describing such methods. In one embodiment, the T cells are tumor infiltrating leukocytes. In one embodiment, one or more T cells express CD8, such as CD8 ⁺ T cells. In other embodiments, one or more T cells express CD4, such as CD4 ⁺ T cells. Additional methods for isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, PCT Publication No. WO2015/120096, and WO2017/070395, all of which are incorporated herein by reference in their entirety and in their entirety for the purpose of describing such methods.

Immune cells and their progenitor cells can be isolated by available methods (see, e.g., Rowland-Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York (1999)). Sources of immune cells or their progenitor cells include, but are not limited to, peripheral blood, cord blood, bone marrow, or other sources of hematopoietic cells. Negative selection methods can be used to remove cells that are not desirable immune cells. In addition, positive selection methods can isolate or enrich for desirable immune cells or their progenitor cells, or a combination of positive and negative selection methods can be used. For both positive and negative selections, monoclonal antibodies (MAbs) are used to identify markers associated with certain cell lineages and/or differentiation stages. If certain types of cells are to be isolated, such as certain types of T cells, a variety of cell surface markers or combinations of markers may be used to separate the cells, including but not limited to CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells), and the like, as is well known in the art (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa N.J. (2000); De Libero, T Cell Protocols, Methods in Molecular Biology Vol. 514, Humana Press, Totowa N.J. (2009)).

PBMCs can be used directly for genetic modification using immune cells such as CARs. After the PBMCs are isolated, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subsets before or after genetic modification and/or expansion. In one embodiment, CD8+ cells can be further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In other embodiments, the expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In one embodiment, effector T cells are negative for CCR7, CD28, CD62L, and CD127 and positive for granzyme B and perforin. In additional embodiments, CD4+ T cells can be further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

The methods described herein further comprise enriching or preparing a population of immune cells obtained from a donor between harvesting from the donor and exposing one or more cells obtained from the donor individual. Enrichment of immune cell populations, such as one or more T cells, can be achieved by any suitable separation method, including but not limited to the use of separation media (e.g., FICOLL-PAQUE ^™ , ROSETTESEP™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Catalog No. 0850494X), or the like), cell size, shape or density separation by filtration or elutriation, immunomagnetic separation (e.g., magnetic activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

In one embodiment, T cells are obtained from a donor individual. In other embodiments, the donor individual is a human patient suffering from cancer or tumor. In additional embodiments, the donor individual is a human patient not suffering from cancer or tumor. The present application also provides a composition or formulation comprising a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative and/or adjuvant. In a certain embodiment, the composition or formulation comprises a formulator. The terms composition and formulation are used interchangeably herein. The terms composition, therapeutic composition, therapeutically effective composition, pharmaceutical composition, pharmaceutically effective composition and pharmaceutically acceptable composition are used interchangeably herein. The composition may be selected for parenteral delivery, inhalation, or delivery via the digestive tract (e.g., oral). The composition may be prepared by methods known to those skilled in the art. Buffers are used to maintain the composition at physiological pH or slightly lower pH, generally in the pH range of about 5 to about 8. When parenteral administration is contemplated, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution containing the composition described herein with or without an additional therapeutic agent in a pharmaceutically acceptable vehicle. For example, the vehicle for parenteral injection is sterile distilled water, in which the composition described herein with or without at least one additional therapeutic agent is formulated as a sterile isotonic solution, appropriately preserved. Preparation involves formulating the desired molecule with a polymeric compound (such as polylactic acid or polyglycolic acid), beads or liposomes that provide controlled or sustained release of the product, which is then delivered via depot injection. In addition, implantable drug delivery devices can be used to introduce the desired therapeutic agent.

In some embodiments, donor T cells used in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells used in T cell therapy are obtained from an individual other than the patient. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ cells, or at least about 10 ¹⁰ cells. In another embodiment, the therapeutically effective amount of T cells is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In some embodiments, the therapeutically effective amount of CAR T cells is about 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, about 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 ⁶ cells/kg, about 9×10 ⁶ cells/kg, about 1×10 ⁷ cells/kg, about 2×10 ⁷ cells/kg, about 3×10 ⁷ cells/kg, about 4×10 ⁷ cells/kg, about 5×10 ⁷ cells/kg, about 6×10 ⁷ cells/kg, about 7×10 ⁷ cells/kg, about 8×10 ⁷ cells/kg or about 9×10 ⁷ cells/kg. In some embodiments, the therapeutically effective amount of CAR-positive live T cells is between about 1×10 ⁶ CAR-positive live T cells/kg body weight and about 2×10 ⁶ CAR-positive live T cells/kg body weight, up to a maximum dose of about 1×10 ⁸ CAR-positive live T cells.

As used herein, "patient" includes any human suffering from a disease or condition including cancer (e.g., lymphoma or leukemia). The terms "individual" and "patient" are used interchangeably herein. The term "donor individual" herein refers to an individual from whom cells are obtained for further ex vivo engineering. The donor individual may be a cancer patient to be treated with a cell population produced by the methods described herein (i.e., an autologous donor), or may be an individual to whom a lymphocyte sample is given, which lymphocyte sample will be used to treat a different individual or cancer patient when the cell population produced by the methods described herein is produced (i.e., an allogeneic donor). Those individuals who receive cells prepared by the methods of the present invention may be referred to as "recipient individuals."

"Stimulation", "stimulating" or similar terms refer to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell, such as a T cell receptor (TCR)/CD3 complex. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., an artificial antigen presenting cell (aAPC), a dendritic cell, a B cell, and the like), can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response of the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. As used herein, "activated" or "active" refers to stimulated T cells. Active T cells may be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

The term "exogenous activating substance" refers to any activating substance that originates from an external source. For example, exogenous anti-CD3 antibodies, anti-CD28 antibodies, IL-2, exogenous IL-7, or exogenous IL-15 can be commercially purchased or recombinantly produced. "Exogenous IL-2", "exogenous IL-7", or "exogenous IL-15", when added to or in contact with one or more T cells, indicates that such IL-2, IL-7, and/or IL-15 is not produced by those T cells. T cells may contain trace amounts of "exogenous" IL-2, IL-7 or IL-15 prior to mixing with these substances, which are produced by these T cells or isolated from the individual possessing these T cells (i.e., endogenous "exogenous" IL-2, IL-7 or IL-15). One or more T cells described herein can be contacted with exogenous anti-CD3 antibodies, anti-CD28 antibodies, "exogenous" IL-2, IL-7 and/or IL-15 by any method known in the art, including adding isolated "exogenous" IL-2, IL-7 and/or IL-15 to the culture, including anti-CD3 antibodies, anti-CD28 antibodies, "exogenous" IL-2, IL-7 and/or IL-15 in the culture medium, or by expressing "exogenous" IL-2, IL-7 and/or IL-15 by one or more cells in the culture other than one or more T cells, such as by a feeder.

As used herein, the term "ex vivo cell" refers to any cell cultured in vitro. In one embodiment, the ex vivo cell comprises a T cell.

The term "persistence" refers to the ability of, for example, one or more transplanted immune cells or their progeny (e.g., differentiated or mature T cells) administered to an individual to remain at detectable levels in the individual for a period of time. As used herein, increasing the persistence of one or more transplanted immune cells or their progeny (e.g., differentiated or mature T cells) refers to increasing the amount of time that the transplanted immune cells can be detected in an individual after administration. For example, the in vivo persistence of one or more transplanted immune cells can be increased by at least about at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. In addition, the in vivo persistence of one or more transplanted immune cells can be increased by at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to one or more transplanted immune cells not prepared by the methods of the invention disclosed herein.

The terms "reduce" and "reduced" are used interchangeably herein and indicate any change that is less than the original. "Reduce" and "reduced" are relative terms and require a comparison between before and after the measurement. "Reduce" and "reduced" include complete depletion. As used herein, the term "regulating" T cell maturation refers to the use of any intervention described herein to control the maturation and/or differentiation of one or more cells such as T cells. For example, regulating refers to inactivating, delaying or inhibiting T cell maturation. In another example, regulating refers to accelerating or promoting T cell maturation. The term "delaying or inhibiting T cell maturation" refers to maintaining one or more T cells in an immature or undifferentiated state. For example, "delaying or inhibiting T cell maturation" may refer to maintaining T cells in a naive or _TCM state, as opposed to evolving to a _TEM or T _EFF state. In addition, "delaying or inhibiting T cell maturation" may refer to increasing or enriching the total percentage of immature or undifferentiated T cells (e.g., naive T cells and/or _TCM cells) within a mixed population of T cells. The state of a T cell (e.g., mature or immature) can be determined, for example, by screening for the expression of multiple genes and the presence of multiple proteins expressed on the surface of T cells. For example, the presence of one or more markers selected from the group consisting of: L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof may indicate less mature, undifferentiated T cells.

"Treatment" or "treating" of an individual/patient refers to any type of intervention or method performed on an individual/patient, or the administration of one or more T cells prepared by this application to an individual/patient, with the goal of reversing, alleviating, improving, inhibiting, reducing or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions, or biochemical markers associated with the disease. In one embodiment, "treatment" includes partial remission. In another embodiment, "treatment" includes complete remission.

Various aspects of this application are described in further detail in the following subsections.

Patients with B-cell malignancies who are loaded with high levels of circulating CD19-expressing tumor cells represent a population with extremely high unmet needs. For example, mantle cell lymphoma (MCL) is challenging to treat and remains incurable in the relapsed or refractory setting. No standard of care exists for second-line and higher chemotherapy. Treatment options include cytotoxic chemotherapy, proteasome inhibitors, immunomodulatory drugs, tyrosine kinase inhibitors, and stem cell transplantation (autologous [ASCT] and allogeneic stem cell transplantation [allogeneic-SCT]). The choice of regimen is influenced by prior therapy, comorbidities, and tumor chemosensitivity. Despite high initial response rates observed with Bruton's tyrosine kinase inhibitors (BTK inhibitors), the majority of patients will eventually develop progressive disease. New treatment strategies are needed to improve the dismal prognosis of r/r MCL patients whose disease has not been effectively controlled by chemoimmunotherapy, stem cell transplantation, and BTK inhibitors.

Anti-CD19 CAR T cell therapy or products used in CD19 CAR-T can be made from the patient's own T cells via leukapheresis that is tailored for B cell malignancies with circulating tumor cell burden to minimize CD19 expressing tumor cells in the final product. T cells from leukocytes harvested from the leukapheresis product can be enriched by selection against CD4+/CD8+ T cells, activated with anti-CD3 and anti-CD28 antibodies, and/or transduced with a viral vector containing the anti-CD19 CAR gene. Further details of the method can be found in PCT/US2015/014520 published as WO2015/120096 and PCT/US2016/057983 published as WO2017/070395. In one embodiment, the cells are not treated with AKT inhibitors, IL-7, and IL-15. These engineered T cells can proliferate to produce a sufficient number of cells to achieve a therapeutic effect. Such a process removes malignant and normal B cells expressing CD19, which reduce the activation, expansion, and clearance of anti-CD19 CAR T cells.

Activation, transduction and/or expansion of immune cells can be performed at any suitable time that allows for the production of (i) a sufficient number of cells in a population of engineered immune cells for at least one dose to be administered to a patient; (ii) a population of engineered immune cells having a favorable ratio of naive cells compared to a typical longer course, or (iii) both (i) and (ii). The appropriate time may depend on a number of parameters, including the population of one or more cells, the cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and/or other variables. The activation time can be 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more than 21 days. The activation time according to the method of the present application is reduced compared to the expansion method known in the art. For example, the activation time can be shortened by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or can be shortened by more than 75%. In addition, the expansion time can be 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more than 21 days. The expansion time according to the method of the present application is reduced compared to the expansion method known in the art. For example, the expansion time can be shortened by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or can be shortened by more than 75%. In one embodiment, the cell expansion time is about 3 days, and the time from cell population enrichment to generation of engineered immune cells is about 6 days.

The delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more biomarkers. The presence of one or more biomarkers can be detected by any method known in the art, including (but not limited to) immunohistochemistry and/or fluorescence activated cell sorting (FACS). The one or more biomarkers are selected from the group consisting of: L-selectin (CD62L ⁺ ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof. In certain aspects, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more of L-selectin (CD62L ⁺ ), IL-7Rα, and CD132. Those skilled in the art will recognize that, although the methods of the present invention can increase the relative proportion of immature and undifferentiated T cells or DC cells in a population of collected cells, some mature and differentiated cells may still be present. Therefore, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by calculating the total percentage of immature and undifferentiated cells in a population of cells before and after one or more cells obtained from a donor individual are exposed to hypoxic culture conditions with or without pressure exceeding atmospheric pressure. The methods disclosed herein can increase the percentage of immature and undifferentiated T cells in a T cell population.

The methods described herein further comprise stimulating a cell population such as lymphocytes with one or more T cell stimulants under suitable conditions to produce an activated T cell population. Any combination of one or more suitable T cell stimulants can be used to produce activated T cell populations, including, but not limited to, antibodies or functional fragments thereof targeting T cell stimulatory or co-stimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies (e.g., OKT-3), anti-CD28 antibodies or functional fragments thereof), or any other suitable mitogen (e.g., tetradecylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or natural ligands of T cell stimulatory or co-stimulatory molecules.

Conditions suitable for stimulating or activating an immune cell population as described herein further include temperature, amount of time, and/or the presence of a certain level of CO _2. The stimulation temperature may be about 34° C., about 35° C., about 36° C., about 37° C., or about 38° C., about 34-38° C., about 35-37° C., about 36-38° C., about 36-37° C., or about 37° C.

Another condition for stimulating or activating an immune cell population as described herein may further include a stimulation or activation time. The stimulation time is about 24-72 hours, about 24-36 hours, about 30-42 hours, about 36-48 hours, about 40-52 hours, about 42-54 hours, about 44-56 hours, about 46-58 hours, about 48-60 hours, about 54-66 hours, or about 60-72 hours, about 44-52 hours, about 40-44 hours, about 40-48 hours, about 40-52 hours, or about 40-56 hours. In one embodiment, the stimulation time is about 48 hours or at least about 48 hours.

Other conditions for stimulating or activating immune cell populations as described herein may further include CO _{2 levels. The CO 2 level} used for stimulation is about 1.0-10% CO ₂ , about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0% or about 10.0% CO ₂ , about 3-7% CO ₂ , about 4-6% CO ₂ , about 4.5-5.5% CO _2. In one embodiment, the CO ₂ level used for stimulation is about 5% CO ₂ .

The conditions for stimulating or activating immune cell populations may further include any combination of temperature, stimulation time, and/or in the presence of a certain level of CO _2. For example, the step of stimulating immune cell populations may include stimulating immune cell populations with one or more immune cell stimulants at a temperature of about 36-38° C. and in the presence of a CO _{2 level of about 4.5-5.5% CO 2 for} a period of about 44-52 hours. One or more immune cells of the present application may be administered to an individual for immunization or cell therapy. Thus, one or more immune cells may be collected from an individual in need of immunization or cell therapy. Once collected, one or more immune cells may be treated for any suitable period of time prior to administration to an individual.

The concentration, amount or population of lymphocytes or the resulting products produced by the methods herein is about 1.0-10.0×10 ⁶ cells/ml. In some aspects, the concentration is about 1.0-2.0×10 ⁶ cells/ml, about 1.0-3.0×10 ⁶ cells/ml, about 1.0-4.0×10 ⁶ cells/ml, about 1.0-5.0×10 ⁶ cells/ml, about 1.0-6.0×10 ⁶ cells/ml, about 1.0-7.0×10 ⁶ cells/ml, about 1.0-8.0×10 ⁶ cells/ml, 1.0-9.0×10 ⁶ cells/ml, about 1.0-10.0×10 ⁶ cells/ml, about 1.0-1.2×10 ⁶ cells/ml, about 1.0-1.4×10 ⁶ cells/ml, about 1.0-1.6×10 ⁶ cells/ml, about 1.0-1.8×10 ⁶ cells/ml, about 1.0-2.0×10 ⁶ cells/ml, at least about 1.0×10 ⁶ cells/ml, at least about 1.1×10 ⁶ cells/ml, at least about 1.2×10 ⁶ cells/ml, at least about 1.3×10 ⁶ cells/ml, at least about 1.4×10 ⁶ cells/ml, at least about 1.5×10 ⁶ cells/ml, at least about 1.6×10 ⁶ cells/ml, at least about 1.7×10 ⁶ cells/ml, at least about 1.8×10 ⁶ cells/ml, at least about 1.9×10 ⁶ cells/ml, at least about 2.0×10 ⁶ cells/ml, at least about 4.0×10 ⁶ cells/ml, at least about 6.0×10 ⁶ cells/ml, at least about 8.0×10 ⁶ cells/ml, or at least about 10.0×10 ⁶ cells/ml.

Anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies may be used in conjunction with or independently of the step of stimulating lymphocyte populations by exposing one or more cells obtained from a donor individual to hypoxic culture conditions with or without superatmospheric pressure. Any soluble or immobilized anti-CD2, anti-CD3 and/or anti-CD28 antibodies or functional fragments thereof may be used (e.g., pure OKT3 (anti-CD3), pure 145-2C11 (anti-CD3), pure UCHT1 (anti-CD3), pure L293 (anti-CD28), pure 15E8 (anti-CD28)). In some aspects, the antibodies can be purchased from suppliers known in the art, including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 Pure 1 mg/mL, Part No. 170-076-116), and eBioscience, Inc. In addition, one skilled in the art will understand how to generate anti-CD3 and/or anti-CD28 antibodies by standard methods. In some aspects, the one or more T cell stimulators used in accordance with the step of stimulating lymphocyte populations include antibodies or functional fragments thereof that target T cell stimulatory or co-stimulatory molecules in the presence of T cell cytokines. In one embodiment, the one or more T cell stimulators include anti-CD3 antibodies and IL-2. In one embodiment, the T cell stimulator comprises an anti-CD3 antibody at a concentration of 50 ng/mL. The anti-CD3 antibody concentration is about 20 ng/mL-100 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL. In an alternative aspect, T cell activation is not required.

The methods described herein further comprise transducing a population of activated immune cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, using a single cycle or more of viral transduction to produce a transduced immune cell population. Several recombinant viruses have been used as viral vectors to deliver genetic material to cells. The viral vector that can be used according to the transduction step can be any ecotropic or bitropic viral vector, including (but not limited to) recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. The method further comprises transducing one or more immune cells with a retrovirus. In one aspect, the viral vector used to transduce the activated immune cell population is a MSGV1 γ retroviral vector. In one embodiment, the viral vector used to transduce the activated immune cell population is the PG13-CD19-H3 vector described by Kochenderfer, J. Immunother. 32(7): 689-702 (2009). According to one aspect of this aspect, the viral vector is grown in a suspension culture in a culture medium specifically used for viral vector manufacturing, referred to herein as a viral vector inoculate. Any suitable growth medium and/or supplement for growing viral vectors can be used in the viral vector inoculate according to the methods described herein. According to some aspects, the viral vector inoculate is then added to the serum-free medium described below during the transduction step. In some aspects, one or more immune cells can be transduced via a retrovirus. In one embodiment, the retrovirus comprises a heterologous gene encoding a cell surface receptor. In another embodiment, the cell surface receptor can bind to an antigen on the surface of a target cell, such as a tumor cell. In addition to exposing one or more cells obtained from a donor individual to hypoxic culture conditions with or without pressure exceeding atmospheric pressure, as appropriate, the conditions for transducing an activated immune cell population as described herein may include a specific time, at a specific temperature, and/or in the presence of a specific level of CO _2. The transduction temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C, about 34-38°C, about 35-37°C, about 36-38°C, about 36-37°C. In one embodiment, the transduction temperature is about 37°C. The predetermined temperature for transduction may be about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., or about 39° C., about 34-39° C., about 35-37° C. In one embodiment, the predetermined temperature for transduction may be about 36-38° C., about 36-37° C., or about 37° C. The transduction time is about 12-36 hours, about 12-16 hours, about 12-20 hours, about 12-24 hours, about 12-28 hours, about 12-32 hours, about 20 hours, or at least about 20 hours, about 16-24 hours, about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours. The _CO2 level used for transduction is about 1.0-10% _CO2 , about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% _CO2 , about 3-7% _CO2 , about 4-6% _CO2 , about 4.5-5.5% _CO2 , or about 5% _CO2 .

Transduction of activated immune cell populations as described herein can be performed at a certain temperature and/or in the presence of a specific level of _CO2 for a period of time, in any combination of: a temperature of about 36-38°C, an amount of time of about 16-24 hours, and in the presence of _{a CO2} level of about 4.5-5.5% CO2. Immune cells may be prepared by any of the methods of the present application in combination with: any manufacturing method for preparing T cells for immunotherapy, including (but not limited to) those manufacturing methods described in PCT Publication Nos. WO2015/120096 and WO2017/070395, which are incorporated herein by reference in their entirety for the purpose of describing such methods; any and all methods for preparing Axicabtagene ciloleucel or Yescarta®; any and all methods for preparing Tisagenlecleucel/Kymriah ^TM ; any and all methods for preparing "ready-made" T cells for immunotherapy; and any other method for preparing lymphocytes for administration to humans. The manufacturing method may be adapted to remove circulating tumor cells from cells obtained from a patient.

CAR-T cells can be engineered to express other molecules and can be any of the following exemplary types or other cells available in this technology: first, second, third, fourth, fifth or more CAR-T cells; shield CAR-T cells, motor CAR-T cells, TRUCK T cells, switch receptor CAR-T cells; gene-edited CAR T cells; dual receptor CAR T cells; suicide CAR T cells, drug-induced CAR-T cells, synNotch-induced CAR T cells; and inhibitory CAR T cells. In one embodiment, the T cell is an autologous T cell. In one embodiment, the T cell is an autologous stem cell (for autologous stem cell therapy or ASCT). In one aspect, the T cells are non-autologous T cells.

Cells (such as immune cells or T cells) are genetically modified after isolation or selection using known methods, or activated and expanded in vitro (or differentiated in the case of progenitor cells) before genetic modification. Immune cells (such as T cells) are genetically modified with chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro. Methods for activating and expanding T cells can be found in U.S. Patent Nos. 6,905,874, 6,867,041, and 6,797,514 and PCT Publication No. WO 2012/079000, which are incorporated herein by reference in their entirety. In general, such methods may include contacting PBMCs or isolated T cells with stimulators and co-stimulators (such as anti-CD3 and/or anti-CD28 antibodies) that can be attached to beads or other surfaces in a culture medium with certain interleukins (such as IL-2). The Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells, may be used. T cells may be activated and stimulated to proliferate with suitable feeder cells, antibodies, and/or interleukins as described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012/129514 (incorporated herein by reference in their entirety).

The cell surface receptor expressed by the engineered immune cell can be any antigen or molecule targeted by a CAR, such as an anti-CD19 CAR, a FMC63-28Z CAR, or a FMC63-CD828BBZ CAR (Kochenderfer et al., J Immunother. 2009, 32(7): 689; Locke et al., Blood 2010, 116(20):4099, the subject matter of both of which is incorporated herein by reference. In certain aspects, the predetermined dose of engineered immune cells may be greater than about 1 million to less than about 3 million transduced engineered T cells/kg. In one embodiment, the predetermined dose of engineered T cells may be greater than about 1 million to about 2 million transduced engineered T cells per kilogram of body weight (cells/kg). The predetermined dose of engineered T cells may be greater than about 1 million to about 2 million, at least about 2 million to less than about 3 million transduced engineered T cells per kilogram of body weight. Transduced T cells (cells/kg). In one embodiment, the predetermined dose of engineered T cells can be about 2 million transduced engineered T cells/kg. In another embodiment, the predetermined dose of engineered T cells can be at least about 2 million transduced engineered T cells/kg. Examples of predetermined doses of engineered T cells can be about 2 million, about 2.1 million, about 2.2 million, about 2.3 million, about 2.4 million, about 2.5 million, about 2.6 million, about 2.7 million, about 2.8 million, or about 2.9 million transduced engineered T cells/kg.

The methods described herein include increasing or enriching a population of one or more transduced immune cells over a period of time to produce a population of engineered immune cells. The expansion time can be any suitable time that allows the production of (i) a sufficient number of cells in the population of engineered immune cells to achieve at least one dose for administration to a patient; (ii) a population of engineered immune cells having a favorable ratio of naive cells compared to a typical longer process, or (iii) (i) and (ii). This time will depend on the cell surface receptors expressed by the immune cells, the vector used, the dose required to have a therapeutic effect, and other variables. The predetermined time for expansion can be 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more than 21 days. In one embodiment, the expansion time of the method of the present invention is reduced compared to the expansion methods known in the art. For example, the predetermined time for expansion can be shortened by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or can be shortened by more than 75%. In one example, the expansion time is about 3 days, and the time from lymphocyte population enrichment to the generation of engineered immune cells is about 6 days.

Conditions for expanding the population of transduced immune cells can include temperature and/or the presence of a level of CO _2. In certain aspects, the temperature is about 34° C., about 35° C., about 36° C., about 37° C., or about 38° C., about 35-37° C., about 36-37° C., or about 37° C. The CO ₂ level is 1.0-10% CO ₂ , about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0% CO ₂ , about 4.5-5.5% CO ₂ , about 5% CO ₂ , about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO ₂ .

Each step of the method described herein can be performed in a closed system. The closed system can be a closed bag culture system, using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP cell differentiation bag, Origen Biomedical PermaLife cell culture bag). The cell culture bag used in the closed bag culture system can be coated with a recombinant human fiber binding protein fragment during the transduction step. The recombinant human fiber binding protein fragment can include three functional domains: a central cell binding domain, a heparin binding domain II, and a CS1-sequence. By helping to co-localize target cells and viral vectors, the recombinant human fiber binding protein fragment can be used to increase the gene efficacy of retroviral transduction of immune cells. In one embodiment, the recombinant human fiber binding protein fragment is RETRONECTIN® (Takara Bio, Japan). The concentration of cell culture bag is about 1-60 µg/mL or about 1-40 µg/mL, about 1-20 µg/mL, 20-40 µg/mL, 40-60 µg/mL, about 1 µg/mL, about 2 µg/mL, about 3 µg/mL, about 4 µg/mL, about 5 µg/mL, about 6 µg/mL, about 7 µg/mL, about 8 µg/mL, about 9 µg/mL, about 10 µg/mL, about 11 µg/mL, about 12 µg/mL, about 13 µg/mL, about 14 µg/mL, about 15 µg/mL, about 16 µg/mL, about 17 µg/mL, about 18 µg/mL, about 19 µg/mL, about 20 µg/mL, about 2-5 µg/mL, about 2-10 µg/mL, about 2-20 µg/mL, about 2-25 In one embodiment, the cell culture bag is coated with at least about 10 µg/mL of recombinant human fibronectin fragment. The cell culture bag used in the closed bag culture system can be optionally blocked with human albumin serum (HSA) during the transduction step. In another embodiment, the cell culture bag is not blocked with HSA during the transduction step.

The population of engineered immune cells produced by the above method can be stored at low temperature as appropriate so that the cells can be used later. A method for storing a population of engineered immune cells at low temperature is also provided herein. Such methods may include the steps of washing and concentrating the population of engineered immune cells with a diluent solution. For example, the diluent solution is standard saline, 0.9% saline, PlasmaLyte A (PL), 5% dextrose/0.45% NaCl saline solution (D5), human serum albumin (HSA) or a combination thereof. In addition, after thawing, HSA can be added to the washed and concentrated cells to increase cell viability and cell recovery rate. In another embodiment, the washing solution is normal saline and the washed and concentrated cells are supplemented with HSA (5%). The method may also include the step of producing a cryopreservation mixture, wherein the cryopreservation mixture includes a cell population diluted in a diluting solution and a suitable cryopreservation solution. The cryopreservation solution may be any suitable cryopreservation solution, including but not limited to CryoStor10 (BioLife Solution), mixed with a dilute solution of engineered immune cells in a ratio of 1:1 or 2:1. HSA may be added to provide a final concentration of about 1.0-10%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 1-3% HSA, about 1-4% HSA, about 1-5% HSA, about 1-7% HSA, about 2-4% HSA, about 2-5% HSA, about 2-6% HSA, about 2-7% HSA, or about 2.5% HSA in the cryopreservation mixture. Cryopreservation of a population of engineered immune cells may include washing the cells with 0.9% standard saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStorTM CS10 (the final HSA concentration in the final cryopreservation mixture is 2.5%). In some embodiments, the method also includes a step of freezing the cryopreservation mixture. In addition, the cryopreservation mixture is frozen in a controlled rate freezer using a defined freezing cycle, and the cell concentration is between about 1×10 ⁶ and about 1.5×10 ⁷ cells per milliliter of cryopreservation mixture. The method may also include a step of storing the cryopreservation mixture in vapor phase liquid nitrogen.

The population of engineered immune cells produced by the methods described herein can be cryopreserved at a predetermined dose. The predetermined dose can be a therapeutically effective dose, which can be any therapeutically effective dose as provided below. The predetermined dose of the engineered immune cells can depend on the cell surface receptors expressed by the immune cells (e.g., the affinity and density of the cell surface receptors expressed on the cells), the target cell type, the nature of the disease or pathological condition being treated, or a combination of both.

In one embodiment, the population of engineered T cells can be cryopreserved at a predetermined dose of about 1 million engineered T cells per kilogram of body weight (cells/kg). In a certain embodiment, the population of engineered T cells can be cryopreserved at a predetermined dose of about 500,000 to about 1 million engineered T cells/kg. In a certain embodiment, the population of engineered T cells can be cryopreserved at a predetermined dose of at least about 1 million, at least about 2 million, at least about 3 million, at least about 4 million, at least about 5 million, at least about 6 million, at least about 7 million, at least about 8 million, at least about 9 million, at least about 10 million engineered T cells/kg. In other embodiments, the population of engineered T cells can be less than 1 million cells/kg, 1 million cells/kg, 2 million cells/kg, 3 million cells/kg, 4 million cells/kg, 5 million cells/kg, 6 million cells/kg, 7 million cells/kg, 8 million cells/kg, 9 million cells/kg, 10 million cells/kg, 15 million cells/kg, 16 million cells/kg, 17 million cells/kg, 18 million cells/kg, 19 million cells/kg, 20 million cells/kg, 21 million cells/kg, 22 million cells/kg, 23 million cells/kg, 24 million cells/kg, 25 million cells/kg, 26 million cells/kg, 27 million cells/kg, 28 million cells/kg, 29 million cells/kg, 30 million cells/kg, 31 million cells/kg, 32 million cells/kg, 33 million cells/kg, 34 million cells/kg, 35 million cells/kg, 36 million cells/kg, 37 million cells/kg, 38 million cells/kg, 39 million cells/kg, 40 million cells/kg, 41 million cells/kg, 42 million cells/kg, 43 million cells/kg In some embodiments, the engineered T cells can be cryopreserved at a predetermined dose of about 1 million to about 2 million engineered T cells/kg. A population of engineered T cells can be cryopreserved at a predetermined dose of between about 1 million cells to about 2 million cells/kg, about 1 million cells to about 3 million cells/kg, about 1 million cells to about 4 million cells/kg, about 1 million cells to about 5 million cells/kg, about 1 million cells to about 6 million cells/kg, about 1 million cells to about 7 million cells/kg, about 1 million cells to about 8 million cells/kg, about 1 million cells to about 9 million cells/kg, about 1 million cells to about 10 million cells/kg. The predetermined dose of the engineered T cell population can be calculated based on the individual's body weight. In one example, the engineered T cell population can be cryopreserved in about 0.5-200 mL of cryopreservation medium. In addition, the population of engineered T cells can be cryopreserved in about 0.5 mL, about 1.0 mL, about 5.0 mL, about 10.0 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL, about 10-30 mL, about 10-50 mL, about 10-70 mL, about 10-90 mL, about 50-70 mL, about 50-90 mL, about 50-110 mL, about 50-150 mL, or about 100-200 mL of cryopreservation medium. In certain aspects, the population of engineered T cells can be preferably cryopreserved in about 50-70 mL of cryopreservation medium.

In one embodiment, a serum-free medium without added serum is used to perform at least one of the following: (a) contacting an immune cell population with exogenous IL-2, exogenous IL-7, exogenous IL-15 and/or other interleukins; (b) stimulating an immune cell population; (c) transducing an activated immune cell population; and (d) expanding a population of transduced immune cells. In some embodiments, each of (a) to (d) is performed using a serum-free medium without added serum. As referred to herein, the term "serum-free media" or "serum-free culture medium" means that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, for the purpose of supporting the viability, activation and growth of cultured cells, no serum is added to the culture medium as a separate and distinct component. Any suitable immune cell growth medium can be used to suspend cultured cells according to the methods described herein. For example, the immune cell growth medium may include, but is not limited to, a sterile low glucose solution comprising a suitable amount of buffer, magnesium, calcium, sodium pyruvate and sodium bicarbonate. In one aspect, the T cell growth medium is OPTMIZER™ (Life Technologies). In contrast to typical methods for producing engineered immune cells, the methods described herein can use a medium that is not supplemented with serum (e.g., human or bovine).

The application provides multiple methods for using T cells to treat cancer. In one aspect, the T cells are CD19-targeted CAR-T cells, which can be prepared by any of the methods of the present application in combination with: any manufacturing method steps for preparing T cells for immunotherapy, including (but not limited to) those manufacturing methods described in PCT Publication Nos. WO2015/120096 and WO2017/070395, both of which are incorporated herein by reference for the purpose of describing such methods; any and all methods for preparing Akiramec or Yescarta®; any and all methods for preparing Tesazolidinone/Kymriah ^™ ; any and all methods for preparing "off-the-shelf" T cells for immunotherapy; and any other method for preparing lymphocytes for administration to humans. In some aspects, the manufacturing methods are suitable for removing circulating tumor cells from cells obtained from a patient.

In one aspect, the T cells are CD19 CAR-T cells prepared by the method described in PCT/US2016/057983. In one embodiment, a population of T cells that eliminate circulating tumor cells is prepared from a leukocyte separation product. These cells can be prepared as described in PCT/US2016/057983 and are further described herein as CD19 CAR-T cells. In short, CD19 CAR-T is an autologous CAR T cell product in which an individual's T cells are engineered to express a receptor composed of a single-chain antibody fragment directed against CD19 linked to CD28 and CD3ζ activation domains, which eliminate cells expressing CD19. After CAR binds to CD19 ⁺ target cells, the CD3ζ domain activates downstream signaling cascades, leading to T cell activation, proliferation, and acquisition of effector functions, such as cytotoxicity. The intracellular signaling domain of CD28 provides a co-stimulatory signal that works with the primary CD3ζ signal to enhance T cell function, including interleukin (IL)-2 production. In summary, these signals can stimulate the proliferation of CAR T cells and direct killing of target cells. In addition, activated T cells can secrete interleukins, trending factors, and other molecules that can recruit and activate additional anti-tumor immune cells. The anti-CD19 CAR in CD19 CAR-T cells can include FMC63-28Z.

Since circulating tumor cells exist in some cancers, the manufacturing of CD19 CAR-T includes CD4 ⁺ and CD8 ⁺ T cell enrichment steps. The T cell enrichment or separation step can reduce circulating tumor cells expressing CD19 in the leukocyte apheresis material and can be associated with the activation, expansion and clearance of anti-CD19 CAR T cells during manufacturing.

The methods described herein can enhance the therapeutic outcome or efficacy of an immune or cell therapy, which can be a recipient T cell therapy selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), allogeneic T cell transplantation, non-T cell transplantation, and any combination thereof. The recipient T cell therapy broadly includes any method of selecting, enriching in vitro, and administering to a patient autologous or allogeneic T cells that recognize and bind to tumor cells. TIL immunotherapy is a type of donor T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, enriched ex vivo, and administered to the patient. TIL cells can be autologous or allogeneic. Autologous cell therapy is a donor T cell therapy that involves isolating T cells capable of targeting tumor cells from a patient, enriching the T cells ex vivo, and administering the T cells back to the same patient. Allogeneic T cell transplants can include transplanting naturally occurring T cells that have been expanded ex vivo or genetically engineered T cells. As described in detail above, engineered autologous cell therapy is a donor-transfer T cell therapy in which a patient's own lymphocytes are isolated, genetically engineered to express a tumor-targeting molecule, expanded ex vivo, and returned to the patient. Non-T cell transplants may include autologous or allogeneic therapy with non-T cells such as, but not limited to, natural killer (NK) cells.

The immunocell therapy of this application is engineered autologous cell therapy (eACT™). According to this aspect, the method may include collecting immune cells from a donor. The isolated immune cells can then be contacted with an exogenous activating agent (e.g., a cytokine), expanded, and engineered to express chimeric antigen receptors ("engineered CAR T cells") or T cell receptors ("engineered TCR T cells"). In some aspects, the engineered immune cells treat tumors in an individual. For example, one or more immune cells are transduced with a retrovirus containing a heterologous gene encoding a cell surface receptor. In one embodiment, the cell surface receptor is capable of binding to an antigen on the surface of a target cell, such as a tumor cell. In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor. In another embodiment, one or more immune cells may be engineered to express a chimeric antigen receptor. The chimeric antigen receptor may comprise a binding molecule for a tumor antigen. The binding molecule may be an antibody or an antigen binding molecule thereof. For example, the antigen binding molecule may be selected from scFv, Fab, Fab', Fv, F(ab')2 and dAb and any fragment or combination thereof. The chimeric antigen receptor may further comprise a hinge region. The hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8 α. In one embodiment, the hinge region is derived from the hinge region of IgG4. The chimeric antigen receptor may also include a transmembrane domain. The transmembrane domain may be a transmembrane domain of any transmembrane molecule that serves as an accessory receptor on an immune cell or a transmembrane domain of a member of the immunoglobulin superfamily. In a certain embodiment, the transmembrane domain is derived from the transmembrane domain of CD28, CD28T, CD8 α, CD4 or CD19. In another embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain. In another embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain. The chimeric antigen receptor may further include one or more co-stimulatory signal transduction regions. For example, the synergistic stimulatory signaling region can be CD28, CD28T, OX-40, 41BB, CD27, inducible T cell co-stimulator (ICOS), CD3 γ, CD3 δ, CD3 ε, CD247, Ig α (CD79a) or Fcγ receptor. In another embodiment, the synergistic stimulatory signaling region is a CD28 signaling region. In another embodiment, the synergistic stimulatory signaling region is a CD28T signaling region. In an additional embodiment, the chimeric antigen receptor further comprises a CD3ζ signaling domain.

In some aspects, the tumor antigen is selected from 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin/m, b-catenin/mutant), BCMA (B cell maturation antigen), Bcr-abl (break point cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exon 7/8), CAMEL (antigen recognized by CTL on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (apoptotic protease-8), CDC27m (cell division cycle 27 mutation), CDK4/m (cytokine dependent kinase 4 mutation), CEA (carcinoembryonic antigen), CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2,3,4 (erythrocytic leukemia viral oncogene homolog-2,-3,4), ELF2M (elongation factor 2 mutation), ETV6-AML1 (Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetyl glucosaminidase V), Gp100 (glycoprotein 100kD), HAGE (helicose antigen), HER-2/neu (human epidermal receptor-2/neural; also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2 mutation), HST-2 (human epidermal tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit-2), KIAA0205, KDR (kinase insert domain receptor), kappa-light chain, LAGE (L antigen), LDLR/FUT (low-density lipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-L fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, murine CMV-infected cells, MART-1/Melan-A (melanoma antigen recognized by T cells-1/melanoma antigen A), MC1R (melanocortin receptor 1), myosin/m (myosin mutation), MUC1 (mucin 1), MUM-1, -2, -3 (common mutations in melanoma 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (protein of 190KD bcr-abl), Pml/RARa (promyelocytic leukemia/retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejection tumor 1 or 3), SSX1, -2, -3, 4 (Synovial sarcoma X1, -2, -3, -4), TAA (tumor associated antigen), TAG-72 (tumor associated glycoprotein 72), TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate isomerase mutation), TRP-1 (tyrosinase-related protein 1 or gp75), TRP-2 (tyrosinase-related protein 2), TRP-2/INT2 (TRP-2/intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms' tumor gene) and any combination thereof. In one embodiment, the tumor antigen is CD19.

T cell therapy involves administering to a patient engineered T cells that express a T cell receptor ("engineered TCR T cell"). The T cell receptor (TCR) may contain a binding molecule for a tumor antigen. In some aspects, the tumor antigen is selected from the group consisting of 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7/8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIA A0205, KDR, kappa-light chain, LAGE, LDLR/FUT, LeY, L1CAM, MAGE, MAGE-A1, mesothelin, murine CMV-infected cells, MART-1/Melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NKG2D ligand, NY-BR-1, NY-ESO-1, carcinoembryonic antigen, P15, p190 minor bcr-abl, Pml/RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF-R2, WT1, and any combination thereof.

"CD19-guided genetically modified autologous T cell immunotherapy" refers to a suspension of chimeric antigen receptor (CAR) positive immune cells. One example of this type of immunotherapy is clear CAR-T therapy, which uses CAR-T cells that are free of circulating tumor cells and enriched for CD4+/CD8+ T cells. Another example is Axicel (also known as Axi-cel™, YESCARTA ^® ). See Kochenderfer et al., (J Immunother 2009;32:689 702). Other non-limiting examples include JCAR017, JCAR015, JCAR014, Kymriah (tisagenlecleucel), Uppsala U. anti-CD19 CAR (NCT02132624), and UCART19 (Celectis). See Sadelain et al. Nature Rev. Cancer Vol. 3 (2003); Ruella et al., Curr Hematol Malig Rep., Springer, NY (2016) and Sadelain et al. Cancer Discovery (April 2013). To prepare CD19-directed genetically modified autologous T cell immunotherapy, the patient's own T cells can be harvested and genetically modified ex vivo by retroviral transduction to express a chimeric antigen receptor (CAR) comprising a mouse anti-CD19 single chain variable fragment (scFv) linked to CD28 and CD3-ζ synergistic stimulatory domains. In some embodiments, the CAR comprises a mouse anti-CD19 single chain variable fragment (scFv) linked to 4-1BB and CD3-ζ synergistic stimulatory domains. Anti-CD19 CAR T cells can be expanded and infused back into the patient, where they can recognize and eliminate target cells expressing CD19.

In one aspect, the TCR comprises a binding molecule for a viral oncogene. In one embodiment, the viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (EBV) and human T-lymphotropic virus (HTLV). In other embodiments, the TCR comprises a binding molecule for a testicular, placental or fetal tumor antigen. In one embodiment, the testicular, placental or fetal tumor antigen is selected from the group consisting of NY-ESO-1, synovial sarcoma X-breakpoint 2 (SSX2), melanoma antigen (MAGE) and any combination thereof. In another embodiment, the TCR comprises a binding molecule for a lineage-specific antigen. In additional embodiments, the lineage-specific antigen is selected from the group consisting of: melanoma antigen recognized by T cells 1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof. In one embodiment, the T cell therapy comprises administering to the patient an engineered CAR T cell expressing a chimeric antigen receptor that binds to CD19 and further comprises a CD28 co-stimulatory domain and a CD3-ζ signaling region. In additional embodiments, the T cell therapy comprises administering to the patient KTE-C19. In one embodiment, the antigenic portion also includes (but is not limited to) Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, type 2, or type 8 (HSV1, HSV2, or HSV8) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL. gM, UL20, UL32, US43, UL45, UL49A), cellular macrophage virus (CMV) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL. gM or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoprotein gp120, gp41 or p24), influenza virus antigens (such as hemagglutinin (HA) or neuraminic acid sidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) viral antigens (such as L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) viral antigens or varicella-zoster virus antigens. In such aspects, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on target virus-infected cells. In other aspects, the antigen portion is associated with cells with abnormal immune or inflammatory functions. Such antigenic portions may include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendritic neuroglia glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen associated with or related to pathogenic autoimmune processes.

The methods disclosed herein may involve T cell therapy comprising transferring one or more T cells to a patient. T cells may be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells, such as engineered CAR+ T cells or engineered TCR+ T cells, may be at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ cells, or at least about 10 ¹⁰ cells. In another aspect, the therapeutically effective amount of T cells, e.g., engineered CAR+ T cells or engineered TCR+ T cells, is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In one embodiment, the therapeutically effective amount of T cells, such as engineered CAR+ T cells or engineered TCR+ T cells, is about 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, about 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 6 cells/kg, about 9×10 ⁶ cells/kg, about 1×10 ⁷ cells/kg, about 2×10 ⁷ cells/kg, about 3×10 ⁷ cells/kg, about 4×10 ⁷ cells/kg, about 5×10 ⁷ cells/kg, about 6×10 ⁶ cells/kg, about ⁷ cells/kg, about 7×10 ⁷ cells/kg, about 8×10 ⁷ cells/kg, or about 9×10 ⁷ cells/kg. In one embodiment, the amount of CD19 CAR-T cells is 2×10 ⁶ cells/kg, and for individuals ≥100 kg, the maximum dose is 2×10 ⁸ cells. In another embodiment, the amount of CD19 CAR-T cells is 0.5×10 ⁶ cells/kg, and for individuals ≥100 kg, the maximum dose is 0.5×10 ⁸ cells.

Patients may be pretreated or lymphodepleted prior to administration of T cell therapy. Patients may be pretreated according to any method known in the art, including, but not limited to, treatment with one or more chemotherapy drugs and/or radiotherapy. In some aspects, pretreatment may include any treatment that reduces the number of endogenous lymphocytes, removes the interleukin groove, increases the serum level of one or more homeostatic interleukins or proinflammatory factors in the body, enhances the effector function of T cells administered after conditioning, enhances antigen presenting cell activation and/or availability, or any combination thereof. Pretreatment may include increasing the serum level of one or more interleukins in an individual. Such methods further include administering a chemotherapeutic agent. The chemotherapeutic agent may be a lymphodepleting (pretreatment) chemotherapeutic agent. Beneficial pretreatment treatment regimens, along with associated beneficial biomarkers, are described in U.S. Patent No. 9,855,298, which is hereby incorporated by reference herein. These describe, for example, methods of conditioning a patient in need of T cell therapy, comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (between 200 mg/m ² per day and 2000 mg/m ² per day) and a prescribed dose of fludarabine (between 20 mg/m ² per day and 900 mg/m ² per day). One such dosing regimen involves treating a patient comprising administering to the patient about 500 mg/m ² of cyclophosphamide per day and about 60 mg/m ² of fludarabine per day for three days prior to administering to the patient a therapeutically effective amount of engineered T cells. In one aspect, the conditioning regimen comprises cyclophosphamide 500 mg/m ² + fludarabine 30 mg/m ² for 3 days. It can be administered on days -4, -3 and -2 or on days -5, -4 and -3 (day 0 is the day the cells are administered). In one embodiment, the conditioning regimen comprises 200 mg/m ² , 250 mg/m ² , 300 mg/m ² , 400v, 500 mg/m ² cyclophosphamide daily for 2, 3 or 4 days and 20 mg/m ² , 25 mg/m ² or 30 mg/m ² fludarabine daily for 2, 3 or 4 days. In one embodiment, conditioning chemotherapy (30 mg/m ² of fludarabine per day and 500 mg/m ² of cyclophosphamide per day) is administered on days -5, -4 and -3 prior to intravenous infusion of a suspension of CD19 CAR-T cells after leukapheresis. In some embodiments, the intravenous infusion time is between 15 and 120 minutes. In one embodiment, the intravenous infusion time is between 1 and 240 minutes. In some embodiments, the intravenous infusion time is up to 30 minutes. In some embodiments, the intravenous infusion time is up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or up to 100 minutes. In some embodiments, the infusion volume is between 50 and 100 mL. In some embodiments, the infusion volume is between 20 and 100 ml. In some embodiments, the infusion volume is about 30, 35, 40, 45, 50, 55, 60 or about 65 ml. In some embodiments, the infusion volume is about 68 mL. In some embodiments, the suspension is frozen and used within 6, 5, 4, 3, 2, 1 hours of thawing. In some embodiments, the suspension is not yet frozen. In some embodiments, the immunotherapy is infused from an infusion bag. In some embodiments, the infusion bag is agitated during the infusion. In some embodiments, the immunotherapy is administered within 3 hours after thawing. In some embodiments, the suspension further comprises albumin. In some embodiments, the albumin is present in an amount of about 2-3% (v/v). In some embodiments, the albumin is present in an amount of about 2.5% (v/v). In some embodiments, the albumin is present in an amount of about 1%, 2%, 3%, 4% or 5% (v/v). In some embodiments, the albumin is human albumin. In some embodiments, the suspension further comprises DMSO. In some embodiments, the DMSO is present in an amount of about 4-6% (v/v). In some embodiments, the DMSO is present in an amount of about 5% (v/v). In some embodiments, the DMSO is present in an amount of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% (v/v).

The methods disclosed herein can be used to treat cancer in an individual, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate a patient's tumor, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain aspects, the methods can induce a complete response. In other aspects, the methods can induce a partial response.

Cancers that can be treated include tumors that are not vascularized, not substantially vascularized, or vascularized. Cancers can also include solid or non-solid tumors.

In one embodiment, the method can be used to treat B cell malignancies that carry high levels of circulating CD19-expressing tumor cells and would be indicated for a diverse patient population with high unmet need.

Exemplary Treatments for MCL

In some embodiments, the malignant disease may be mantle cell lymphoma (MCL). MCL is an aggressive subtype of non-Hodgkin lymphoma (NHL). MCL accounts for approximately 6% of all new cases of NHL in the United States (US) and 5% to 7% of malignant lymphomas in Western Europe. The estimated annual incidence of MCL in the United States and Europe is approximately 1 to 2 per 100,000 people. MCL is more likely to affect men than women, and the median age at diagnosis is 68 years. In some embodiments, r/r MCL is r/r to treatment with allogeneic stem cell transplantation (allo-SCT). Allogeneic-SCT itself can provide durable remission in approximately 25% of patients with relapsed or refractory (r/r) MCL if the patient's disease is chemosensitive prior to transplantation, but allogeneic-SCT also causes treatment-related mortality of up to 40%.

In some embodiments, r/r MCL is r/r to treatment with bortezomib, lenalidomide, and temsirolimus, which by themselves result in an ORR in the range of 22% to 32%. Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib and acalabrutinib result in an ORR of 68% and 81%, respectively, in r/r MCL patients. However, the majority of patients progress following BTK inhibitor treatment and respond poorly to salvage therapy, with ORRs ranging from 20% to 42%, median duration of response (DOR) ranging from 3 to 5.4 months, and median OS ranging from 2.5 to 9 months. In some embodiments, the present invention provides CAR T cell intervention for the treatment of cancers with poor prognostic factors, such as high Ki67 tumor proliferation index expression (≥30% or ≥50%) and mutant TP53. In some embodiments, the cancer is MCL. In some embodiments, the MCL morphology is typical, pleomorphic, or blastoid. In some embodiments, the Ki-67 index may be between 5% and 80%. In some embodiments, the Ki-67 index is about 38%. In some embodiments, high-risk patients have Ki-67 ≥50% and/or TP53 mutations by next-generation sequencing. In some embodiments, the patient is ≥18 years old. In some embodiments, MCL is pathologically confirmed by documentation of cyclin D1 overexpression and/or the presence of t(11:14).

In some embodiments, CAR T cell intervention comprises T cells expanded from a T cell population that is depleted of circulating lymphoma cells and enriched for CD4+/CD8+ T cells by positive selection of mononuclear cells from a leukocyte separation sample, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing an anti-CD19 CAR construct. In some embodiments, the CAR construct is FMC63-28Z CAR. CAR T cells generated using this method may be referred to as KTE-X19. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments, the dose of CAR-positive T cells is 2×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1.6×10 ⁶ anti-CD19 CAR T cells/kg, 1.8×10 ⁶ anti-CD19 CAR T cells/kg, or 1.9×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the CD19 CAR construct contains a CD3ζ T cell activation domain and a CD28 signaling domain.

In some embodiments, CAR T cells are administered as a single infusion on day 0 following conditioning therapy with 25 mg/m ² of fludarabine per day on days -5, -4, and -3 and 900 mg/m ² of cyclophosphamide per day on day -2 following leukapheresis. In some embodiments, conditioning therapy comprises 300 mg/m ² of cyclophosphamide per day and 30 mg/m ² of fludarabine per day for 3 days. In some embodiments, conditioning chemotherapy comprises 30 mg/m ² of fludarabine per day and 500 mg/m ² of cyclophosphamide per day on days -5, -4, and -3. In some embodiments, the patient may also have received acetaminophen and diphenhydramine or another H1-antihistamine about 30 to 60 minutes prior to infusion of anti-CD19 CAR T cells. In some embodiments, the patient receives one or more additional doses of anti-CD19 CAR T cells.

In some embodiments, the MCL cancer is relapsed/refractory MCL (r/r MCL). In some embodiments, the patient has received one or more prior treatments. In some embodiments, the patient has received 1-5 prior treatments. In some embodiments, prior treatments may include autologous SCT, anti-CD20 antibodies, chemotherapy containing anthracycline or bendamustine, and/or bruton's tyrosine kinase inhibitors (BTKi). In some embodiments, BTKi is ibrutinib (Ibr). In some embodiments, BTKi is acalabrutinib (Acala). In some embodiments, the present invention provides that patients with MCL previously treated with ibrutinib respond more significantly to anti-CD19 CAR T cell therapy than patients previously treated with acalabrutinib. Thus, the present invention provides a method of treating r/r MCL with anti-CD19 CAR T cell therapy, wherein the patient has been previously treated with ibrutinib or acalabrutinib and the cancer is preferably relapsed/refractory to ibrutinib or acalabrutinib. In some embodiments, the BTKi is tirabrutinib (ONO-4059), zanubrutinib (BGB-3111), CGI-1746, or spebrutinib (AVL-292, CC-292).

In some embodiments, the present invention provides that for patients previously treated with Ibr, Acala, or both, the median (range) peak CAR T cell content is 95.9 (0.4-2589.5), 13.7 (0.2-182.4), or 115.9 (17.2-1753.6), respectively. In some embodiments, the ORR/CR rate for anti-CD19 CAR T cell therapy in MCL patients is 94%/65% in patients previously treated with Ibr, 80%/40% in patients previously treated with Acala, and 100%/100% in patients previously treated with two BTKi. In some embodiments, the 12-month survival rate in patients previously treated with Ibr, Acala, or both is 81%, 80%, or 100%, respectively. In some embodiments, CAR T cell expansion is associated with ORR/CR rates in patients previously treated with Ibr and/or Acala. Therefore, in one embodiment, the patient is treated with both Ibr and Acala. In one embodiment, the present invention provides a method for predicting ORR/CR in MCL patients previously treated with Ibr and/or Acala by measuring peak CAR T cell levels and comparing them to a reference standard. In one embodiment, the present invention provides a method for predicting sustained response based on measurements of CAR T cell peak levels/baseline tumor burden (CEN and INV). In one embodiment, the higher the ratio, the higher the likelihood of sustained response at/to 12 months. In one embodiment, a rate between 0.00001 and 0.005 predicts no response at / to 12 months. In one embodiment, a rate between 0.006 and 0.3 predicts relapse at / to 12 months. In one embodiment, a rate between 0.4 and 1 predicts sustained response at / to 12 months. In one embodiment, the rate can be determined by one of ordinary skill from an average population.

In some embodiments, the additional inclusion criteria include the criteria listed in Example 2. In some embodiments, the additional exclusion criteria include the criteria listed in Example 2.

In some embodiments, the patient may have received transitional therapy (after leukapheresis and prior to chemotherapy) of dexamethasone (e.g., 20-40 mg PO or IV daily or equivalent for 1-4 days), methylprednisolone, ibrutinib (e.g., 560 mg PO daily), and/or acalabrutinib (e.g., 100 mg PO twice daily) after leukapheresis, and completed, e.g., within 5 days or less prior to conditioning chemotherapy. In some embodiments, such patients may already have a high disease burden. In some embodiments, the transitional therapy is selected from an immunomodulator, R-CHOP, bendamustine, an alkylating agent, and/or a platinum-based agent.

In some embodiments, the present invention provides that all MCL patients who responded to CAR T cell infusion achieved T cell expansion, while no expansion was observed in non-responding patients. In some embodiments, the response is an objective response (complete response + partial response). The present invention provides that the CAR T cell content is correlated with the ORR in the first 28 days, wherein in responders, compared with non-responders, the area under the curve (AUC _{0-28 )} and peak content from day 0 to day ₂₈ are higher by >200 times, indicating that the higher the expansion, the greater and possibly deeper the response, also indicated by > ⁸⁰ times higher peak/AUC CAR T cell content in minimal residual disease (MRD, ^10-5 sensitivity) negative patients compared to MRD positive patients (at week 4). Therefore, the present invention provides a method for predicting a patient's response to CAR T cell therapy for MCL and MRD, comprising measuring peak/AUC CAR T cell levels and comparing them to a reference standard. In some embodiments, peak CAR T cell expansion is observed between days 8 and 15 after CAR T cell administration. In some embodiments, CAR T cell levels are measured by qPCR. In some embodiments, peak CAR T cell levels, _{AUC 0-28} , and/or MRD are monitored by next generation sequencing. In some examples, the number of CAR T cells is measured as the number of cells per microliter of blood. In some examples, the number of CAR T cells is measured by the number _of CAR gene copies per microgram of host DNA. In some examples, the number of CAR T cells is measured as described in Kochenderfer JN et al. J. Clin. Oncol. 2015;33:540-549. In one embodiment, the CAR T cell content is measured as described in Locke FL et al. Mol Ther. 2017;25(1):285-295.

In some embodiments, the present invention provides that there is a difference between T cell expansion in responders and non-responders. In some embodiments, the present invention provides that the median peak anti-CD19 CAR T cell level in responders (complete remission and partial remission responders) was 102.4 cells/μL (range: 0.2 to 2589.5 cells/μL; n=51) and in non-responders was 12.0 cells/μL (range: 0.2 to 1364.0 cells/μL, n=8). In some embodiments, the present invention provides that the median AUC on days 0-28 (AUC _{0 - 28} ) was 1487.0 cells/μl·day (range: 3.8 to 2.77×10 ⁴ cells/μl·day; n=51) in patients with objective responses and 169.5 cells/μl·day (range: 1.8 to 1.17 10×10 ⁴ cells/μl·day; n=8) in non-responders. The median peak anti-CD19 CAR T cells (24.7 cells/μL) and AUC _{0 - 28} levels (360.4 cells/μL·day) in patients who received neither corticosteroids nor tocilizumab (n=18) were similar to those in patients who received corticosteroids alone (n=2) (peak: 24.2 cells/μL; AUC _{0 -} 28: 367.8 cells/μL·day). In patients who received tocilizumab alone (n=10), the mean peak anti-CD19 CAR T cells were 86.5 cells/μL and the AUC _{0 - 28} levels were 86.5 cells/μL. In patients who received corticosteroids with tocilizumab (n=37), the mean peak anti-CD19 CAR T cell value was 74.1 cells/μL and AUC _{0 - 28} was 1188.9 cells/μL·day. In patients who received corticosteroids with tocilizumab ( _n =37), the mean peak was 167.2 cells/μL and AUC 0 - 28 was 1996.0 cells/μL·day. In patients ≥65 years of age (n=39), the median peak anti-CD19 CAR T cell value was 74.1 cells/μL and 112.5 cells/μL in patients <65 years of age (n=28). The median anti-CD19 CAR T cell AUC _{0 - 28} value was 876.5 cells/μL·day in patients ≥65 years of age and 1640.2 cells/μL·day in patients <65 years of age. There was no significant effect on _{AUC 0-28} and C _max of T cells. Therefore, the _present invention provides a method for predicting response in MCL, comprising measuring T cell expansion after anti-CD19 CAR T treatment and comparing the level with a reference standard.

In some embodiments, the present invention provides that CAR T cell expansion in patients with grade ≥ 3 MCL exceeds that in those patients with grade ≤ 3 CRS and NE events. Therefore, the present invention provides a method for predicting grade ≥ 3 CRS and NE events, comprising measuring CAR T cell expansion after CAR T cell treatment and comparing the levels to a reference value, wherein the higher the CAR T cell expansion, the higher the probability of grade ≥ 3 CRS and NE events.

In some embodiments, the interleukin level is measured by protein or mRNA level and is protein or mRNA level (one of them). In some embodiments, the interleukin level is measured as described in Locke FL et al. Mol Ther. 2017; 25(1): 285-295.

In some embodiments, the present invention provides that the peak levels of serum GM-CSF and IL-6 (reached about 8 days after administration of CAR T cells) in MCL patients are positively correlated with level ≥3 CRS and level ≥3 NE. Therefore, the present invention provides a method for predicting level ≥3 CRS and level ≥3 NE, which comprises measuring the peak levels of GM-CSF and IL-6 after administration of CAR T cells and comparing them with reference levels, wherein the higher the peak levels of these interleukins, the higher the probability of level ≥3 CRS and NE.

In some embodiments, the present invention provides that serum ferritin is positively correlated with grade ≥ 3 CRS in MCL patients. Therefore, the present invention provides a method for predicting grade ≥ 3 CRS, comprising measuring the peak level of serum ferritin after CAR T cell administration and comparing it with a reference level, wherein the higher the peak level of ferritin, the higher the probability of grade ≥ 3 CRS.

In some embodiments, the present invention provides that serum IL-2 and IFN-γ are positively correlated with level ≥3 NE in MCL patients. Therefore, the present invention provides a method for predicting level ≥3 CRS, which comprises measuring the peak levels of serum IL-2 and IFN-γ after administration of CAR T cells and comparing them with reference levels, wherein the higher the peak levels of IL-2 and IFN-γ, the higher the probability of level ≥3 NE.

In some embodiments, the present invention provides that the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and vascular cell adhesion molecule (VCAM) in MCL patients are positively correlated with grade ≥3 NE. Therefore, the present invention provides a method for predicting grade ≥3 CRS, comprising measuring the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or vascular cell adhesion molecule (VCAM) after CAR T cell administration and comparing it to a reference level, wherein the higher the cerebrospinal fluid levels of C-reactive protein, ferritin, IL-6, IL-8 and/or vascular cell adhesion molecule (VCAM), the higher the probability of grade ≥3 NE. In some embodiments, one or more adverse events are managed according to Table 13 and/or Table 14.

In some embodiments, the present invention provides that interleukins whose peak serum levels are positively correlated with level ≥3 CRS include IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and perforin. In some embodiments, the present invention provides that interleukins whose peak serum levels are correlated with level ≥3 NE include IL-2, IL-1 Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. In some embodiments, the present invention provides that interleukins associated with level ≥3 CRS and NE include IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B. In some embodiments, the serum level of cytokines reaches a peak within 7 days of CAR T cell administration. Therefore, the present invention provides a method for predicting level ≥3 CRS after CAR T cell administration, comprising measuring the peak serum level of IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B and/or perforin after anti-CD19 CAR T treatment and comparing the level with a reference standard. Therefore, the present invention also provides a method for predicting grade ≥3 CRS and grade ≥3 NE in MCL, which comprises measuring the peak serum levels of IL-6, TNFα, GM-CSF, IL-10, MIP-1b and granzyme B after anti-CD19 CAR T treatment and comparing the levels with reference standards.

In some embodiments, the present invention provides that there is a trend of increased peak levels of proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα, sPD-L1 and VCAM-1) cytokines in MCL patients with mutant TP53 relative to wild-type TP53. Therefore, in some embodiments, the present invention provides a method for improving the response to CAR T cell therapy in MCL, comprising manipulating the levels of proliferative and/or inflammatory cytokines after administration of CAR T cells.

In some embodiments, the present invention provides that for patients who are MRD negative one month after CAR T cell administration, the peak levels of IFN-γ and IL-6 are increased, and IL-2 has an increasing trend, relative to patients who are MRD positive at one month. Therefore, the present invention provides a method for predicting whether a patient in MCL is MRD negative, comprising measuring the peak serum levels of IFN-γ, IL-6 and/or IL-2 after anti-CD19 CAR T treatment and comparing the levels to a reference standard.

In some embodiments, the present invention provides that T cell product phenotypes vary among MCL types. In some embodiments, the present invention provides that in the anti-CD19 CAR T products manufactured, the median (range) CD4+/CD8+ T cell ratios in patients with typical, blastoid, and pleomorphic MCL are 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. Product T cell phenotypes (median [range]) included less differentiated CCR7+ T cells (typical 40.0% [2.6-88.8]; blastoid 35.3% [14.3-73.4]; pleomorphic 80.8% [57.3-88.8]) and effector and effector memory CCR7- T cells (typical 59.9% [11.1-97.4]; blastoid 64.8% [26.6-85.7]; pleomorphic 19.2% [11.1 - 42.7]). In some embodiments, the invention provides that the 12-month survival rate in patients with typical, blastoid, or pleomorphic MCL is 86.7%, 67.9%, or 100%, respectively. Thus, the present invention provides a method for improving the treatment of classical, blastoid or pleomorphic MCL by manipulating the phenotype of T cells administered to the patient.

Exemplary Treatments for B-Cell ALL

B-ALL cells often express CD19, and CAR T-cell therapy targeting CD19 is a treatment approach in R/R B-ALL. Pehlivan K.C. et al. Curr Hematol Malig Rep. 2018;13(5):396-406. Anti-CD19 CAR T-cell therapy containing CD3ζ and CD28 co-stimulatory domains developed by the National Cancer Institute (Kochenderfer JN et al. J Immunother. 2009;32(7):689-702; Kochenderfer JN et al. Blood. 2010;116(19):3875-3886) showed an overall remission rate of 70% after a median follow-up period of 10 months in a phase 1 trial in children with R/R B-ALL and adults ≤30 years of age. Lee DW et al. Lancet. 2015;385(9967):517-528. A similar CAR construct evaluated in a phase 1 trial in adults with R/R B-ALL provided an 83% complete remission (CR) rate and a median OS of 12.9 months at a median follow-up of 29 months. Park JH et al. N Engl J Med. 2018;378(5):449-459. In these studies, CAR T cells were prepared from leukocyte fractionated samples that were not enriched for CD4+/CD8+ T cells.

In some embodiments, the invention is directed to a T cell product, wherein T cells are expanded from a T cell population that is depleted of circulating lymphoma cells and enriched for CD4+/CD8+ T cells by positive selection of mononuclear cells from a leukocyte separation sample, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing an anti-CD19 CAR construct. In some embodiments, such T cell products can be used to treat ALL, CLL, AML. In some embodiments, the CAR construct is FMC63-28Z CAR. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments, the dose of CAR-positive T cells is 2×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the dose of CAR-positive T cells is 1.6×10 ⁶ anti-CD19 CAR T cells/kg, 1.8×10 ⁶ anti-CD19 CAR T cells/kg, or 1.9×10 ⁶ anti-CD19 CAR T cells/kg. In some embodiments, the CD19 CAR construct contains a CD3ζ T cell activation domain and a CD28 signaling domain. In some embodiments, the T cell product is KTE-X19. In some embodiments, the present invention provides that the anti-CAR T cell products prepared as described in the previous paragraphs can be used in B cell ALL and B cell NHL. In one embodiment, the T cell product has the characteristics of the products of Table 23. In some embodiments, the product characteristics can be selected from the percentage of specific subsets (primitive, central memory, effector and effector memory) T cells, the percentage of CD4+ cells, the percentage of CD8+ cells and the CD4/CD8 ratio. In some embodiments, the product characteristic is the IFNγ production level (pg/mL) in a co-culture of target CD19-expressing cancer cells (e.g., Toledo) cells mixed with an anti-CD19 CAR T cell product at a 1:1 ratio. In one embodiment, IFNγ can be measured in the cell culture medium using a qualified ELISA 24 hours after incubation. In some embodiments, one or more of these product characteristics are superior to those of anti-CAR T cells prepared by leukapheresis that are not enriched for CD4+/CD8+ positive cells. In some embodiments, superior product characteristics can be selected from an increase in the percentage of cells with a naive phenotype (CD45RA+CCR7+), a decrease in the percentage of cells with a differentiated phenotype (CCR7-), a decrease in the level of cells producing IFNγ, and an increase in the level of CD8+ cells. In some embodiments, the anti-CD19 T cell product comprises _TCM , central memory T cells (CD45RA-CCR7+); T _EFF , effector T cells (CD45RA+CCR7-); T _EM , effector memory T cells (CD45RA-CCR7-); and/or _TN , naive-like T cells (CD45RA+CCR7+). In some embodiments, the product comprises _TN naive-like T cells, meaning that the T cells are CD45RA+CCR7+ and comprise stem cell-like memory cells. In some embodiments, the T cell product is KTE-X19. In some embodiments, KTE-X19 has ≥ 190 pg/mL IFN-γ production. In some embodiments, KTE-X19 has ≥ 90% CD3+ cells. In some other embodiments, the percentage of NK cells in KTE-X19 is 0.1% (range 0.0%-2.8%). In some other embodiments, the percentage of CD3 ^- cell impurities in KTE-X19 is 0.5% (range 0.3%-3.9%).

In some embodiments, the cancer is relapsed/refractory B-cell ALL. In some embodiments, the patient is ≤21 years old. In some embodiments, the patient is ≤21 years old, weighs ≥10 kg, and has B-cell ALL that is primary refractory, relapsed within 18 months of initial diagnosis, R/R after ≥2 lines of systemic therapy, or R/R after allogeneic stem cell transplantation at least 100 days prior to enrollment. In one embodiment, the cancer is an indolent lymphoma or leukemia. In one embodiment, the cancer is an aggressive B-cell lymphoma, which includes diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), mantle cell lymphoma and its blastoid variants, and many types, subtypes and variants of B-lymphoblastic lymphoma. DLBCL can be DLBCL NOS, T-cell/tissue-rich large B-cell lymphoma, CNS primary DLBCL, primary skin DLBCL, EBV-positive DLBCL of the leg type in the elderly. Other large B-cell lymphomas include primary septal (thymic) LBCL, DLBCL associated with chronic inflammation, lymphomatoid granulomas, ALK-positive LBCL, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, and primary effusion lymphoma. Other types of lymphoma include unclassifiable B-cell lymphoma with features intermediate between DLBCL and Burkitt's lymphoma; unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin's lymphoma; splenic marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma of MALT, nodal marginal zone B-cell lymphoma, hairy cell leukemia, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), and primary effusion lymphoma. The cancer may be at any stage, from stage 1 to stage 4.

ALL is a common childhood malignancy, accounting for approximately 80% of childhood leukemias and approximately 25% of all childhood cancers. Approximately 20% of pediatric patients do not achieve long-term remission after initial treatment, and the 5-year OS rate is approximately 55%. Hunger SP et al., N Engl J Med. 2015;373:1541-1552; Sun W et al., Leukemia. 2018;32:2316-2325; Rheingold SR et al. J Clin Oncol. 2019;37(Suppl, Abstract):10008 and Oskarsson T et al., Haematologica. 2016;101:68-76. Poor outcomes are seen in patients who have an early relapse after initial treatment or who have primary refractory disease; those who have R/R disease after stem cell transplantation; and those who have multiple relapses. Sun W, Leukemia. 2018;32:2316-2325; Rheingold SR et al, J Clin Oncol. 2019;37(Suppl, Abstract):10008; Oskarsson T et al, Haematologica. 2016;101:68-76; Nguyen K et al, Leukemia. 2008;22:2142-2150; Crotta A et al, Curr Med Res Opin. 2018;34:435-440; Schrappe M et al, N Engl J Med. 2012;366:1371-1381. Patients who relapse within 18 months of initial diagnosis typically have a 5-year OS rate of 21%-28%. Rheingold SR et al., J Clin Oncol. 2019;37(Suppl, Abstract):10008; Nguyen K et al., Leukemia. 2008;22:2142-2150. The likelihood of achieving remission and the duration of EFS decrease with each subsequent line of salvage therapy. Sun W et al., Leukemia. 2018;32:2316-2325. Pediatric and adolescent patients with R/R ALL continue to have poor outcomes after treatment with the novel therapies blinatumomab and inotuzumab ozogamicin, with 1-year OS rates of approximately 36%, highlighting the need for more effective treatment options. von Stackelberg A et al., J Clin Oncol. 2016;34:4381-4389. 10; Bhojwani D et al., Leukemia. 2019;33:884-892.

In some embodiments, the cancer is B-cell NHL and key inclusion criteria include age <18 years, weight ≥10 kg, histologically confirmed diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS), primary septal large B-cell lymphoma, Burkitt's lymphoma (BL), Burkitt-like lymphoma, or unclassified B-cell lymphoma intermediate between DLBCL and BL, with ≥1 measurable lesion. In one embodiment, the disease may have been primary refractory to NHL treatment, R/R after ≥2 lines of systemic therapy, or R/R after autologous or allogeneic stem cell transplantation ≥100 days prior to inclusion. Patients with acute graft-versus-host disease or chronic graft-versus-host disease requiring treatment within 4 weeks of enrollment may not be eligible.

In some embodiments, such B cell ALL and/or B cell NHL patients receive conditioning chemotherapy as follows: 25 mg/m ² fludarabine per day on days -4, -3, and -2, and 900 mg/m ² cyclophosphamide per day on day -2, followed by a single infusion of CD4+/CD8+ enriched anti-CD19 CAR T cells (prepared as described just above) at a target dose of 1×10 ⁶ anti-CD19 CAR T cells/kg on day 0.

In some embodiments, the present invention provides for the successful treatment of B cell ALL using CD4+/CD8+ enriched cancer cell depleted anti-CD19 CAR T cells, wherein the patient is ≥18 years old, has R/R B cell ALL, defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≤12 months after initial remission, relapsed or refractory after ≥2 prior lines of systemic therapy, or relapsed after allogeneic stem cell transplantation (SCT). In some embodiments, the patient is required to have ≥5% bone marrow blasts, Eastern Coast Collaborative on Cancer performance status of 0 or 1, and adequate renal, liver, and heart function. For patients who have previously received blinatumomab, CD19 expression ≥ 90% of leukemic blasts is required. Patients with Philadelphia chromosome positive (Ph+) disease, concomitant extramedullary disease, central nervous system (CNS)-2 disease (with <5 white blood cells/cubic millimeter of cerebrospinal fluid [CSF] blasts) without neurological changes, and patients with Down syndrome are eligible. CNS-3 disease (with ≥5 white blood cells/cubic millimeter of CSF blasts) and a history of CNS disorders not associated with neurological changes are excluded. In some embodiments, additional inclusion and exclusion criteria are described in Example 9.

In some embodiments, the patient may have primary refractory cancer. In some embodiments, the patient may have cancer that relapsed after SCT. In some embodiments, the patient may have previously received blinatumomab, which may be the last therapy used prior to anti-CD19 CAR T cell therapy. In some embodiments, the patient baseline characteristics are any of the patient baseline characteristics described in Table 18 .

In some embodiments, 2×10 ⁶ , 1×10 ⁶ or 0.5×10 ⁶ CAR T cells/kg are administered to these B cell ALL patients. In some embodiments, 0.5×10 6 CAR T cells/kg are administered in a total volume of 40 mL of formulation. In another embodiment, 0.5×10 ⁶ CAR T cells/ ^kg are administered in a total volume of 68 mL of formulation. In some embodiments, the CAR T cell product is formulated into a total volume of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900 or 1000 mL. In some embodiments, the 40 mL formulation is intended to maintain cell density and cell viability during the freeze/thaw process. In some embodiments, treatment is associated with adverse events. In some embodiments, one or more adverse events are managed according to any of Tables 13 , 14 , 16 , or a combination thereof. In some embodiments, one or more adverse events are managed according to the original management criteria of Table 16. In some embodiments, one or more adverse events are managed according to the revised management criteria of Table 16. In some embodiments, vasopressors may be administered to treat CRS. In some embodiments, signs and symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia, and hypotension. In some embodiments, signs and symptoms associated with a neurological event include encephalopathy, seizures, altered mental status, speech disorders, tremors, and confusion.

In some embodiments, the patient may have a high disease burden at baseline, defined by local review as having >25% leukemic blasts in the bone marrow or ≥1,000 blasts/mm3 in the peripheral circulation. In some embodiments, the patient may receive transition chemotherapy after leukapheresis and prior to conditioning chemotherapy. In some embodiments, transition chemotherapy follows one of the predefined transition chemotherapy regimens of Table 17 .

In some embodiments, the conditioning chemotherapy/lymphodepleting regimen is administered after clearance from transitional chemotherapy for ≥7 days or 5 half-lives (if shorter). In some embodiments, the conditioning chemotherapy/lymphodepleting regimen consists of 25 mg/m ² fludarabine intravenously (IV) daily on days -4, -3, and -2 and 900 mg/m ² cyclophosphamide IV daily on day -2. On day 0, a single infusion of anti-CD19 CAR T cells may be administered. In some embodiments, additional infusions of anti-CD19 CAR T cells may be administered at a later time. In some embodiments, patients who achieve a complete response to the initial infusion may receive a second infusion of anti-CD19 CAR T cells if remission evolves >3 months later, provided that CD19 expression has been preserved and there are no suspected neutralizing antibodies against the CAR.

In some embodiments, droplet digital polymerase chain reaction can be used to measure the presence, expansion and persistence of transduced anti-CD19 CAR+ T cells in the blood. In some embodiments, the procedure is as described in Locke FL et al. Mol Ther. 2017;25(1):285-295. In some embodiments, the present invention provides a method of treatment, wherein the CAR T cell content is as described in Table 22. In some embodiments, the present invention provides that the CAR T cells are undetectable at the time of relapse. The median peak CAR T cell content can be highest at 1×10 ⁶ CAR T cells/kg and can be similar between patients receiving original versus revised AE management. In some embodiments, patients who achieve CR/CRi have a greater median peak increase than non-responders, as do patients with undetectable MRD versus patients with detectable MRD. Higher median peak increases are also observed in patients with grade ≥3 NE versus patients with grade ≤2 NE. Some patients who relapse may have detectable CD19 positive cells at the time of relapse, or may have undetectable CD19 positive cells. In some embodiments, undetectable MRD, defined as <1 leukemic cell per 10,000 viable cells, can be assessed using flow cytometry (NeoGenomics, Fort Myers, FL) as described in: Borowitz MJ, Wood BL, Devidas M, et al., Blood. 2015;126(8):964-971; Bruggemann M. et al. Blood Adv. 2017;1(25):2456-2466; or Gupta S. et al. Leukemia. 2018;32(6):1370-1379.

In some embodiments, the present invention provides that peak levels of some interleukins, chemokines, and proinflammatory markers occur by day 7. In some embodiments, some of these tend to be higher in patients given 2×10 ⁶ CAR T cells/kg compared to 1×10 ⁶ CAR T cells/kg (IL-15, CRP, SAA, CXCL10, IFNγ), or tend to be lower in patients with revised AE management compared to patients with original AE management (IL-6, ferritin, IL-1RA, IFNγ, IL-8, CXCL10, MCP-1). In some embodiments, the levels of these proteins/biomarkers vary as described in Figures 9, 10, and 11). Therefore, in some embodiments, the present invention provides methods of using these protein levels as biomarkers of level ≥3 and/or level 0-2 CRS. In some embodiments, the present invention provides methods of using these protein levels as biomarkers of level ≥3 and/or level 0-2 CRS according to the values in Figure 11.

In some embodiments, the present invention provides that the peak IL-15 serum level is lower in patients with grade ≥3 CRS. In some embodiments, the present invention provides that the median peak levels of several proinflammatory markers tend to be higher in patients with grade ≥3 CRS and patients with grade ≥3 NE (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, granzyme B, as described in Figure 11. Therefore, in some embodiments, the present invention provides a method for predicting whether a patient will have grade ≥3 CRS by measuring the peak level of serum IL-15 and comparing it to a reference standard. In some embodiments, the present invention provides a method for predicting whether a patient will have grade ≥3 CRS and/or grade ≥3 In some embodiments, the present invention provides a method for improving anti-CD19 CAR T cell therapy by administering an agent that reduces the level of one or more of these biomarkers.

Reference levels/standards can be established by any method known to those of ordinary skill in the art. It is used to identify thresholds or groups of values (e.g., quartiles) from which comparisons can be made to determine in which group the measured value of each individual (interleukin level, CAR T cell number, etc.) decreases or exceeds or falls below which threshold. As is typical in this technology, these groups are established from comparisons of different selected groups. Depending on where the measured value decreases, a large number of treatment characteristics can be predicted, such as objective response, CRS level, NE level, and similar characteristics.

In certain embodiments, the cancer may be selected from a tumor derived from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic cancer, adrenocortical carcinoma, AIDS-related cancers, anal cancer, coccygeal cancer, astrocytoma, atypical teratoid/rhabdoid tumor, central nervous system tumor, B cell leukemia, lymphoma or other B cell Malignant diseases, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibromyoma, brain stem neurofibroma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disease, colon cancer, large Colorectal cancer, cranio-pharyngioma, cutaneous T-cell lymphoma, embryonal tumor, central nervous system tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, sensitive neuroembryonic tumor, Ewing's sarcoma family of tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, malignant fibroblastoma and osteosarcoma of bones, gallbladder cancer, stomach (gastric ) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, neuroglioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, swallowing cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma (kaposi sarcoma), kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibroblastoma and osteosarcoma of bone, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma carcinoma), mesothelioma, occult primary metastatic squamous cell carcinoma, midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myelodysplasia/myeloproliferative neoplasia, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma, myeloproliferative disorders, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous tissue of bone cell tumor, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pinealoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell abscess/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome syndrome), small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cervical cancer, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral and renal pelvic cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms Tumor. In certain embodiments, the cancer is treated with KTE-X19.

In one embodiment, the method can be used to treat a tumor, wherein the tumor is a lymphoma or a leukemia. Lymphomas and leukemias are blood cancers that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of multipotent hematopoietic stem cell found in the bone marrow. This stem cell gives rise to myeloid progenitor cells and lymphoid progenitor cells, which in turn give rise to the various types of white blood cells found in the body. White blood cells produced from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells generated from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient. In certain embodiments, tumors are treated with KTE-X19.

In general, lymphomas can be divided into at least two subgroups: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of cancers that originate from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B cell lymphomas account for 80-85% of reported cases. In 2013, it was estimated that approximately 69,740 new cases of NHL and more than 19,000 deaths related to the disease occurred. Non-Hodgkin's lymphoma is the most common hematologic malignancy and the seventh leading new cancer site in men and women, and accounts for 4% of all new cancer cases and 3% of cancer-related deaths. In certain embodiments, lymphoma is treated with KTE-X19.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. In the United States, approximately 22,000 new cases of DLBCL are diagnosed each year. It is classified as an aggressive lymphoma and most patients are cured with conventional chemotherapy (NCCN Guidelines NHL 2014). First-line treatment for DLBCL typically consists of an anthracycline-containing regimen with rituximab, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prespasadena), which has an objective response rate of approximately 80% and a complete response rate of approximately 50%, with approximately one-third of patients having refractory disease to initial therapy or relapsing after R-CHOP. For those patients who relapse after responding to first-line therapy, approximately 40-60% of patients can achieve a second response with additional chemotherapy. The standard of care for second-line therapy for patients eligible for autologous stem cell transplantation (ASCT) includes rituximab and combination chemotherapy such as R-ICE (rituximab, ifosfamide, carboplatin and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine and cisplatin), each of which has an objective response rate of approximately 63% and a complete response rate of approximately 26%. Patients who respond to second-line therapy and are considered adequate candidates for transplantation receive consolidation with high-dose chemotherapy and ASCT, which is curative in approximately half of transplanted patients. Patients who fail ASCT have a very poor prognosis and no curative options. Primary septal large B-cell lymphoma (PMBCL) has different clinical, pathological, and molecular features than DLBCL. PMBCL is thought to arise from thymic (medullary) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL is usually identified in the younger adult population in the 40s, slightly more often in women. Gene expression profiling analysis suggests that the pathways dysregulated in PMBCL overlap with Hodgkin's lymphoma. Initial therapy for PMBCL typically includes an anthracycline-containing regimen with rituximab, such as dose-adjusted infusions of ethtoposide, doxorubicin, and cyclophosphamide with vincristine, prazolone, and rituximab (DA-EPOCH-R), with or without regional radiation therapy. Follicular lymphoma (FL, a B-cell lymphoma) is the most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHL. Some patients with FL will histologically transform (TFL) to DLBCL, which is more aggressive and associated with a poorer outcome. Histologic transformation to DLBCL occurs at an annual rate of approximately 3% over 15 years, with the risk of transformation continuing to decline in the following years. The biologic mechanism of histologic transformation is unknown. Initial treatment of TFL is influenced by prior therapy for follicular lymphoma but typically includes an anthracycline-containing regimen with rituximab to eliminate the aggressive component of the disease. Treatment options for relapsed/refractory PMBCL and TFL are similar to those in DLBCL. Given the low incidence of these diseases, large prospective randomized studies have not been performed in these patient populations. Patients with disease that is refractory to chemotherapy have a prognosis similar to or worse than that of patients with refractory DLBCL. For example, individuals with refractory aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have a huge unmet medical need and warrant further investigation with novel treatments in these groups. In certain embodiments, DLBCL is treated with KTE-X19.

The CAR T cell therapy of the present invention can be administered as a first-line treatment or a second-line or subsequent-line treatment. In some embodiments, CAR T cell therapy is administered as a third-line, fourth-line, fifth-line, etc. The prior therapy of these lines can be any prior anticancer therapy, including (but not limited to) Bruton's tyrosine kinase inhibitor (BTKi), checkpoint inhibitors (e.g., anti-PD1 antibodies, pembrolizumab (Keytruda), Cemiplimab (Libtayo), nivolumab (Opdivo); anti-PD-L1 antibodies, atezolizumab (Tecentriq), avelumab (Avelu mab, Bavencio), Durvalumab, Imfinzi; anti-CTLA-4 antibody, ipilimumab, Yervoy), anti-CD19 antibody (e.g., blinatumomab), anti-CD52 antibody (e.g., alentuzumab); allogeneic stem cell transplantation, anti-CD20 antibody (e.g., rituximab), systemic chemotherapy, rituximab, anthracycline, ofatumumab, and combinations thereof. Prior therapy may also be used in combination with the CD19 CAR T therapy of this application. In one aspect, eligible patients may have disease that is refractory to the most recent therapy or has relapsed within 1 year after autologous hematopoietic stem cell transplantation (HSCT/ASCT). CAR T cell therapy may be administered to patients who have or are suspected of having cancer that is refractory and/or relapsed to one or more lines of prior therapy. The cancer may be refractory to one line of therapy (i.e., primary refractory) or refractory to one or more lines of therapy. The cancer may relapse twelve months after initial remission, relapse or be refractory after two or more lines of prior therapy, or relapse after HSCT/ASCT. In some embodiments, the cancer is refractory to ibrutinib or acalabrutinib. In some embodiments, the cancer is NHL, and the disease must have been primary refractory, R/R after two or more lines of systemic therapy, or R/R after ≥100 days of autologous or allogeneic stem cell transplantation prior to enrollment in CAR T cell therapy, and immunosuppressive drug treatment has been stopped for ≥4 weeks. In certain embodiments, the CAR T cell therapy is KTE-X19.

Thus, the method can be used to treat lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL/CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B-cell lymphoma), splenic diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia/lymphoma, Burkitt's lymphoma, and lymphoblastic lymphoma. In some embodiments, the lymphoma or leukemia is selected from B-cell chronic lymphocytic leukemia/small cell lymphoma, B-cell prolymphocytic leukemia, lymphocytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasm (e.g., plasma cell myeloma (i.e., multiple myeloma) or plasma cell tumor), extranodal marginal zone B-cell lymphoma (e.g. MALT lymphoma), marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicular center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granuloma, primary septal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-related multicentric Castleman disease, Burkitt's lymphoma/ Leukemia, T-cell prolymphocytic leukemia, T-cell large granulocytic lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia/lymphoma, extranodal NK/T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides/Sezary syndrome syndrome, primary cutaneous pleomorphic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, pleomorphic large cell lymphoma, B-lymphoblastic leukemia/lymphoma, B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities, T-lymphoblastic leukemia/lymphoma, and Hodgkin's lymphoma. In some aspects, the cancer is refractory to one or more prior treatments, and/or the cancer has relapsed after one or more prior treatments. In certain embodiments, the leukemia or lymphoma is treated with KTE-X19.

In one embodiment, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary septal (thymic) large B-cell lymphoma. In one embodiment, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is refractory to one or more of the following treatments or the cancer relapses after one or more of the following treatments: chemotherapy, radiation therapy, immunotherapy (including T cell therapy and/or treatment with antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof. In one embodiment, the cancer is refractory diffuse large B-cell lymphoma. In certain embodiments, the cancer is treated with KTE-X19.

In some embodiments, the CAR T cell therapy is KTE-X19 and the cancer is selected from MCL, ALL, CLL, and SLL. In some embodiments, the CAR T cell therapy is KTE-X19 and the cancer is NHL. In some embodiments, the cancer is selected from diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS), primary septal large B-cell lymphoma, Burkitt's lymphoma (BL), Burkitt-like lymphoma, or unclassified B-cell lymphoma intermediate between DLBCL and BL. In some embodiments, the cancer is relapsed/refractory. In some embodiments, KTE-X19 treatment is administered as a first line, second line, or after 1 or more lines of prior therapy. In some embodiments, the patient is a pediatric patient, an adolescent patient, an adult patient, less than 65 years old, over 65 years old, or any other age group.

In some embodiments, the compositions comprising the immune cells disclosed herein can be administered in conjunction with a number of additional therapeutic agents. In one embodiment, the additional therapeutic agent is administered concurrently with T cell therapy. In one embodiment, the additional therapeutic agent is administered before, during, and/or after T cell therapy. In one embodiment, one or more additional therapeutic agents are administered prophylactically. In one aspect, the compositions comprising immune cells are administered in conjunction with an agent for managing adverse events (many of which are described elsewhere in this application, including in the Examples section). These agents may manage one or more of the signs and symptoms of adverse reactions such as fever, hypotension, tachycardia, hypoxia, and chills, including cardiac arrhythmias (including atrial tremors and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizures, encephalopathy, headache, tremors, vertigo, aphasia, delirium, insomnia anxiety, systemic anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

Examples of such agents include, but are not limited to, tosilimab, steroids (e.g., methylprednisolone), rabbit anti-thymocyte globulin. In some aspects, for non-neutropenic fever, vancomycin and aztreonam (1 gm each IV twice daily) may be administered. In some aspects, the method further comprises administering a non-sedative anti-epileptic drug for the prevention of epilepsy; administering at least one of erythropoietin, darbepoetin alfa, platelet transfusions, filgrastim, or pegfilgrastim; and/or administering tosilimab, siltuximab. In one aspect, the agent is a member of the CSF family, such as GM-CSF (granulocyte-macrophage colony stimulating factor, also known as CSF2). GM-CSF can be produced by a large number of hematopoietic and non-hematopoietic cell types upon stimulation, and it can activate/'activate' bone marrow populations to produce inflammatory mediators, such as TNF and interleukin 1β (IL1β). In some embodiments, the GM-CSF inhibitor is an antibody that binds to and neutralizes circulating GM-CSF. In some embodiments, the antibody is selected from Lenzilumab; namilumab (AMG203); GSK3196165/MOR103/Otilimab (GSK/MorphoSys), KB002 and KB003 (KaloBios), MT203 (Micromet and Nycomed) and MORAb-022/gimsilumab (Morphotek). In some embodiments, the antibody is a biosimilar thereof. In some embodiments, the antagonist is E21R, which is a modified form of GM-CSF that antagonizes the function of GM-CSF. In some embodiments, the inhibitor/antagonist is a small molecule. In one embodiment, the CSF family member is M-CSF (also known as macrophage colony stimulating factor or CSF1). Non-limiting examples of agents that inhibit or antagonize CSF1 include small molecules, antibodies, chimeric antigen receptors, fusion proteins, and other agents. In one embodiment, the CSF1 inhibitor or antagonist is an anti-CSF1 antibody. In one embodiment, the anti-CSF1 antibody is selected from antibodies manufactured by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110/lacnotuzumab), or a biosimilar version of any of the foregoing. In some embodiments, the inhibitor or antagonist inactivates the activity of GM-CSF-R-α (also known as CSF2R) or CSF1R receptor. In some embodiments, the inhibitor is selected from Mavrilimumab (formerly CAM-3001), a fully human GM-CSF receptor alpha monoclonal antibody currently under development by MedImmune, Inc.; cabiralizumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly), Emactuzumab (also known as RG7155 or RO5509554); FPA008, a humanized mAb (Five Prime/BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041/AFS98/TG3003 (ElsaLys Bio, In some embodiments, the inhibitor or antagonist is expressed in CAR-T cells. In some embodiments, the inhibitor is a small molecule (e.g., heteroarylamide, quinolinone series, pyridopyrimidine series); BLZ945 (Novartis), PLX7486, ARRY-382, Pexidrtinib (also known as PLX3397), or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine; GW 2580 (CAS 870483-87-7), KI20227 (CAS 623142-96-1), AC708, Ambit Siosciences; or Cannarile et al. Journal for ImmunoTherapy of Cancer 2017, 5:53 and US20180371093 (incorporated herein by reference for disclosed inhibitors). Other neutralizing antibodies to GM-CSF or its receptors have been described in the art, including, for example, "GM-CSF as a target in inflammatory/autoimmune disease: current evidence and future therapeutic potential" Hamilton, J. A. Expert Rev. Clin. Immunol., 2015; and "Targeting GM-CSF in inflammatory diseases" Wicks, I. P., Roberts, A. W. Nat. Rev. Rheumatol., 2016. In other embodiments, the agent is an anti-IL6 or anti-IL-6 receptor blocker, including tocilizumab and siltuximab.

In one embodiment, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and methyl trimer Cyanamides, including altretamine, triethylmelamine, triethylphosphatamide, triethylthiophosphatamide and trihydroxymethylmelamine; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, isocyclophosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, actinomycin D dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, ropterin), trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOLTM, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer; chlorambucil; gemcitabine; 6-thioguanine; oxalopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; VP-16; isocyclic phosphamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives, such as TargretinTM (besarotin), PanretinTM (alitretinoin); ONTAKTM (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some aspects, the compositions comprising the immune effector cells expressing CAR and/or TCR disclosed herein can be combined with the administration of anti-hormonal agents for regulating or inhibiting the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitors 4 (5)-imidazoles, 4-hydroxytamoxifen, trioxifene, raloxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Combinations of chemotherapeutic agents, including but not limited to CHOP, cyclophosphamide (Cytoxan®), doxorubicin (hydroxycrane), vincristine (Oncovin®), and prazosin, may also be administered when appropriate.

The (chemical) therapeutic agent can be administered at the same time as or within a week after the administration of the engineered cells or nucleic acids. In other aspects, the (chemical) therapeutic agent is administered 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cells or nucleic acids. In some aspects, the (chemical) therapeutic agent is administered at least 1 month before the administration of the cells or nucleic acids. In some aspects, the method further comprises the administration of two or more chemical therapeutic agents.

A variety of additional therapeutic agents may be used in conjunction/combination with the compositions or medicaments/treatments described herein. For example, additional treatments that may be useful include PD-1 inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche), tocilizumab (with and without corticosteroids), inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R (anti-CSF1 antibodies are selected from antibodies manufactured by Roche (e.g., RG7155), Pfizer (PD-0360324), Novartis (MCS110/lanotuzumab); limumab (formerly CAM-3001), a current fully human GM-CSF receptor alpha monoclonal antibody, developed by MedImmune, Inc.; Cabiratumumab (Five Prime Therapeutics); LY3022855 (IMC-CS4) (Eli Lilly), Ematitumomab (also known as RG7155 or RO5509554); FPA008, a humanized mAb (Five Prime/BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041/AFS98/TG3003 (ElsaLys Bio, Transgene), SNDX-6352 (Syndax). In some embodiments, the inhibitor or antagonist is expressed in CAR-T cells. In some embodiments, the inhibitor is a small molecule (e.g., heteroarylamide, quinolinone series, pyridopyrimidine series); BLZ945 (Novartis), PLX7486, ARRY-382, pecitinib (also known as PLX3397), or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-06-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine; GW 2580 (CAS 870483-87-7), KI20227 (CAS 623142-96-1), AC708, Ambit Siosciences; or any CSF1R inhibitor listed in Cannarile et al. Journal for ImmunoTherapy of Cancer 2017, 5:53 and US20180371093 (incorporated herein by reference for disclosed inhibitors). Other neutralizing antibodies to GM-CSF or its receptors have been described in the art. Additional therapeutic agents suitable for use in combination with the compositions or medicaments/treatments and methods disclosed herein include, but are not limited to, ibrutinib (IMBRUVICA®), ofavumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab entamoxetine (trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofavumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib anib), afatinib, lapatinib, neratinib, lenalidomide, axitinib, masitinib, pazopanib, sunitinib, sorafenib, tocilizumab, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, azopanib), regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toseranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurine, ruxolitinib, pacritinib, cobimetinib, selumetinib selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin, mTOR inhibitors (such as everolimus and temsirolimus), hedgehog inhibitors (such as sonidegib and vismodegib), CDK inhibitors (such as CDK inhibitor (palbociclib)).

Compositions or medicaments/treatments comprising immune cells may be administered with or in combination with anti-inflammatory agents. Anti-inflammatory agents or drugs may include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, corticosteroids, hydrocortisone, hydrocortisone, methylprednisolone, prednisone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialic acid. Exemplary analgesics include acetaminophen, oxycodone, propoxyphene hydrochloride, tramadol. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.); interleukin inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®); cytokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant antibodies. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auranofin) and intramuscular) and minocycline.

The compositions or agents/treatments described herein may be administered in conjunction with an interleukin and/or interleukin modulator as another therapeutic agent. Examples of interleukins are lymphocyte mediators, mononuclear globulin, and traditional polypeptide hormones. Interleukins include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as filtrate stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance (mullerian-inhibiting substance); substance); mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet growth factor; transformation growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO, Epogen ^® , Procrit ^® ); bone inducing factor; interferons, such as interferon-α, interferon-β and interferon-γ; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term interleukin includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence interleukins. In one embodiment, the compositions described herein are administered in combination with a steroid or corticosteroid.

Corticosteroid treatment can be used to treat adverse events. Corticosteroids (or any other steroid and any other treatment for adverse events) can be used prophylactically before any symptoms of adverse events are detected and/or after the adverse events are detected. It can be administered one or more days before T cell administration, on the day of T cell administration (before, after and/or during T cell administration), and/or after T cell administration. It can be administered before, during or after conditioning therapy. Any corticosteroid may be suitable for this purpose. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, the two can be administered in combination. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include, but are not limited to, alclomethasone, algestone, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17 valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasone, clocortolone (e.g., clocortolone pivalate), clobetasone (e.g., clobetasone valerate ... pivalate), cloprednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortivazol, deflazacort, desonide, desoximethasone, dexamethasone (e.g., dexamethasone 21-phosphate, dexamethasone acetate, dexamethasone sodium phosphate), diflorasone (e.g., diflorasone diacetate), diacetate), diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, fludrocortisone (e.g., fludrocortisone acetate), flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, fluocortin, fluocortolone, fluorometholone (e.g., fluorometholone acetate), acetate), fluperolone (e.g., fluperolone acetate), fluprednidene, flupredni solone, flurandrenolide, fluticasone (e.g., fluticasone propionate), formocortal, halcinonide, halobetasol, halometasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21-butyrate, hydrocortisone acetopropionate, hydrocortisone acetate, hydrocortisone propionate, hydrocortisone butyrate, hydrocortisone cyclopentyl propionate, hydrocortisone hemisuccinate, hydrocortisone propionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate), mazipredone, medrysone, meprednisone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g., mometasone furoate), paramethasone (e.g., paramethasone acetate), acetate), prednicarbate, prednisolone (e.g., prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemisuccinate, prednisolone-21 (β-D-glucuronide), beta-D-glucuronide), prednisolone metasulphobenzoate, prednisolone steaglate, prednisolone tebutate, prednisolone tetrahydrophthalate), prednisolone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone (e.g., triamcinolone acetonide, triamcinolone acetonide, benetonide), triamcinolone hexacetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are discussed in detail, for example, in Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th edition 1980) and Remington: The Science and Practice of Pharmacy, 22nd edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) and any other editions (incorporated herein by reference). In some embodiments, the glucocorticoid is selected from the following: cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone and prednisolone. In one embodiment, the glucocorticoid is dexamethasone. In other embodiments, the steroid is a halocorticoid. Any other steroid can be used in the methods provided herein.

One or more corticosteroids may be administered at any dose and frequency of administration, which may be adjusted based on the severity/grade of the adverse events (e.g., CRS and NE). Tables 13, 14, and 16 provide examples of dosing regimens for the management of CRS and NE. In another embodiment, corticosteroid administration comprises oral or IV dexamethasone 10 mg, 1-4 times per day. Another embodiment, sometimes referred to as "high-dose" corticosteroids, comprises IV administration of 1 g of methylprednisolone per day, alone or in combination with dexamethasone. In some embodiments, one or more corticosteroids are administered at a dose of 1-2 mg/kg per day.

Corticosteroids may be administered in any amount effective to improve one or more symptoms associated with an adverse event, such as CRS or neurotoxicity. Corticosteroids, such as glucocorticoids, can be administered, for example, to a 70 kg adult individual in an amount between or about 0.1 and 100 mg, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to 40 mg, 0.1 to 30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg, 0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg, or 1 to 10 mg per dose. Typically, a corticosteroid, such as a glucocorticoid, is administered to an average adult subject in an amount of between or about 0.4 and 20 mg per dose, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg.

In some embodiments, the corticosteroid can be administered to an average adult subject, typically weighing about 70 kg to 75 kg, for example, at or about the following dosages: 0.001 mg/kg (subject), 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg, 0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, 0.08 .15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg, or 1.4 mg/kg.

In general, the dose of corticosteroid administered depends on the specific corticosteroid because there are differences in potency between different corticosteroids. It is generally understood that the potency of drugs varies, so the dose can be varied to obtain an equivalent effect. Various glucocorticoids and routes of administration are known to be equivalent in potency. Information related to the administration of equivalent steroids (in a non-chronotherapy manner) can be found in British National Formulary (BNF) 37, March 1999.

In some embodiments, the adverse event/reaction may be selected from one or more of the following: Adverse events / reactions Immune system disorders Blood and lymphatic system disorders Interleukin-1 Release Syndrome ^{Coagulopathya Hypogammaglobulinemia} Heart disease Infection and infection ^Tachycardiab Infection-Pathogen Unspecified ^bradycardiac Viral infection Nonventricular ^arrhythmias Bacterial infection Gastrointestinal diseases Metabolic and nutritional disorders Nausea Decreased appetite constipate Musculoskeletal ^pain Diarrhea ^Motor dysfunction Abdominal ^pain Psychiatric disorders Oral ^pain Neurological disorders ^Vomiting Brain ^disease Dysphagia Tremor Fever ^{Headache Fatigue Aphasia} Chills ^{Dizziness Edema Neurosis} Dry mouth Insomnia ^{Pain Delirium} Immune system disorders Anxiety Interleukin-1 Release Syndrome Kidney and urinary disorders ^{Hypogammaglobulinemia} Renal ^{insufficiency} Infection and infection ^Decreased urine output Infection-Pathogen Unspecified Hypoxia Viral infection ^Cough Bacterial infection ^Difficulty breathing Metabolic and nutritional disorders Pleural effusion Decreased appetite Skin and subcutaneous tissue disorders Musculoskeletal ^pain Skin ^{rashx Motor} dysfunction Vascular disorders Psychiatric disorders ^Hypotension Neurological disorders High blood pressure Brain ^{disease Thrombosis} Tremor Bleeding ^{Headache Aphasia Dizziness Neurosis} Insomnia ^Delirium Anxiety

Other adverse reactions include: Gastrointestinal disorders: dry mouth; Infections and infectious diseases: fungal infections; Metabolic and nutritional disorders: dehydration; Nervous system disorders: ataxia, epilepsy, increased intracranial pressure; Respiratory tract, thoracic and diaphragmatic disorders: respiratory failure, pulmonary edema; Skin and subcutaneous tissue disorders: rash; Vascular disorders: bleeding.

In one embodiment, interleukin release syndrome symptoms include, but are not limited to, fever, chills, fatigue, anorexia, myalgia, joint pain, nausea, vomiting, headache, rash, diarrhea, tachypnea, hypoxemia, tachycardia, hypotension, increased pulse pressure, early increase in cardiac output, late decrease in cardiac output, hallucinations, seizures, gait changes, seizures, and death. In one embodiment, the method for grading CRS is described in Neelapu et al., Nat Rev Clin Oncol. 15(1):47-62 (2018) and Lee et al., Blood 2014; 124:188-195. In one embodiment, neurotoxicity/neurological events can be graded by the method described in: Lee et al., Blood 2014; 124: 188-195.

In some embodiments, the adverse event is managed with tocilizumab (or another anti-IL6/IL6R agent/antagonist), corticosteroid therapy, or an anti-epileptic drug for the prevention of toxicity. In some embodiments, the adverse event is managed by one or more agents selected from the group consisting of GM-CSF, CSF1, inhibitors of GM-CSFR or CSF1R, anti-thymocyte globulin, lanzilumab, malimumab, interleukins, and anti-inflammatory agents.

In some embodiments, the present invention provides methods for preventing the development of adverse reactions to T cell therapy of the present invention or reducing its severity. In some embodiments, cell therapy is administered with one or more agents that prevent adverse events, delay the onset of adverse events, reduce the symptoms of adverse events, and treat adverse events, including interleukin release syndrome and neurotoxicity. In one embodiment, the agent has been described above. In other embodiments, the agent is described below. In some embodiments, the agent is administered before, after, or simultaneously with the administration of cells by one of the methods and dosages described elsewhere in this specification. In one embodiment, the agent is administered to an individual who is susceptible to the disease but has not yet been diagnosed with the disease.

In this regard, the disclosed methods may comprise administering a "prophylactically effective amount" of tocilizumab, corticosteroid therapy, or an anti-epileptic drug for the prevention of toxicity. In some embodiments, the methods comprise administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, lanzilumab, malimumab, an interleukin, and/or an anti-inflammatory agent. The pharmacological and/or physiological effects may be preventive, i.e., the effects completely or partially prevent the disease or its symptoms. A "prophylactically effective amount" may refer to an amount that is effective to achieve the desired preventive outcome (e.g., to prevent an adverse reaction) at the desired dosage and within the desired time period.

In some embodiments, the method comprises managing an adverse reaction in any subject. In some embodiments, the adverse reaction is selected from the group consisting of interleukin release syndrome (CRS), neurotoxicity, allergic reaction, severe infection, hemocytopenia and hypogammaglobulinemia. In some embodiments, the signs and symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia and chills, including arrhythmia (including atrial tremor and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), epilepsy, encephalopathy, headache, tremor, vertigo, aphasia, delirium, insomnia anxiety, systemic anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia and anemia. In some embodiments, patients have been identified and selected based on one or more of the biomarkers of adverse events. In some embodiments, patients have been identified and selected simply by clinical manifestations (e.g., the presence and level of toxic symptoms). In some embodiments, adverse events are managed by any of the regimens of Tables 13, 14, 16, and 17.

In some embodiments, the method comprises preventing or reducing the severity of CRS in chimeric receptor therapy. In some embodiments, the engineered CAR T cells are deactivated after administration to the patient. In some embodiments, the method comprises identifying CRS based on clinical manifestations. In some embodiments, the method comprises assessing and treating other causes of fever, hypoxia, and hypotension. Patients experiencing ≥ Grade 2 CRS (e.g., hypotension, unresponsiveness to fluids, or hypoxia, requiring supplemental oxygen) should be monitored using continuous cardiac telemetry and a pulse oximetry analyzer. In some embodiments, for patients experiencing severe CRS, consider performing an echocardiogram to assess cardiac function. For severe or life-threatening CRS, intensive supportive care may be considered. In some embodiments, the method comprises monitoring the patient for signs and symptoms of CRS at least once a day for 7 days after infusion in a certified healthcare facility. In some embodiments, the method comprises monitoring the patient for signs or symptoms of CRS for 4 weeks after infusion. In some embodiments, the method comprises advising the patient to seek immediate medical care at any time if signs or symptoms of CRS occur. In some embodiments, the method comprises treatment with supportive care, tosilimab, or tosilimab and corticosteroids as indicated at the first sign of CRS.

In some embodiments, the method comprises monitoring a patient for signs and symptoms of neurotoxicity. In some embodiments, the method comprises ruling out other causes of neurological symptoms. Patients experiencing ≥ Grade 2 neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. For severe or life-threatening neurotoxicity, provide intensive supportive care. In some embodiments, the symptoms of neurotoxicity are selected from encephalopathy, headache, tremor, vertigo, aphasia, delirium, insomnia, and anxiety.

In some embodiments, cell therapy is administered before, during/concurrently with, and/or after administration of one or more agents (e.g., steroids) or treatments (e.g., debulking) that treat and or prevent (preventive) one or more symptoms of an adverse event. A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive outcome at the desired dosage and for the desired time period. In one embodiment, a prophylactically effective amount is used in an individual before or at an early stage of a disease. In one embodiment, a prophylactically effective amount will be less than a therapeutically effective amount. In one embodiment, adverse event treatment or prevention is administered to any patient who will receive, is receiving, or has received cell therapy. In some embodiments, the method of managing adverse events comprises monitoring the patient for signs and symptoms of neurotoxicity at least once a day for 7 days after infusion in a certified healthcare facility. In some embodiments, the method comprises monitoring the patient for signs or symptoms of neurotoxicity and/or CRS for 4 weeks after infusion.

In some embodiments, the present invention provides two methods of managing adverse events in individuals receiving CAR T cell therapy with steroids and anti-IL6/anti-IL-6R antibodies. In one embodiment, the present invention provides a method of managing adverse events, wherein if there is no improvement after 3 days, corticosteroid therapy is initiated for management of all symptoms of grade 1 CRS, and for all grade ≥1 neurologic events. In one embodiment, if there is no improvement after 3 days, tocilizumab is initiated for all symptoms of grade 1 CRS, and for all grade ≥2 neurologic events. In one embodiment, the present invention provides a method of reducing total steroid exposure in a patient receiving adverse event management after CAR T cell administration, the method comprising starting corticosteroid therapy for management of all conditions of grade 1 CRS and for all grade ≥1 neurological events if there is no improvement after 3 days, and/or starting tocilizumab for all conditions of grade 1 CRS and for all grade ≥2 neurological events if there is no improvement after 3 days. In one embodiment, corticosteroids and tocilizumab are administered in a regimen selected from the examples section. In one embodiment, the present invention provides that early steroid use does not cause an increased risk of serious infection, reduce CAR T cell expansion, or reduce tumor response.

In one embodiment, the present invention supports the safety of levetiracetam prophylaxis in CAR T cell cancer treatment. In one embodiment, the cancer is NHL. In one embodiment, the cancer is R/R LBCL and the patient receives KTE-X19. Therefore, in one embodiment, the present invention provides a method for managing adverse events in patients treated with CAR T cells, comprising administering a prophylactic dose of an anti-epileptic drug to the patient. In some embodiments, the patient receives levetiracetam (e.g., 750 mg twice daily orally or intravenously), starting on day 0 of CAR T cell treatment (after conditioning) and at the onset of grade ≥2 neurotoxicity (if a neurological event occurs after discontinuation of prophylactic levetiracetam). In one embodiment, if the patient does not experience any grade ≥ 2 neurotoxicity, levetiracetam is tapered as clinically indicated and discontinued. In one embodiment, levetiracetam prophylaxis is combined with any other adverse event management regimen.

In one embodiment, patients may receive levetiracetam (750 mg orally or intravenously twice daily), starting on day 0. At the onset of a grade ≥2 neurologic event, the levetiracetam dose is escalated to 1000 mg twice daily. If the patient does not experience any grade ≥2 neurologic events, levetiracetam is tapered and discontinued as clinically indicated. Patients also receive tosilimab on day 2 (8 mg/kg IV [not to exceed 800 mg] over 1 hour). Further tosilimab may be recommended at the onset of grade 2 CRS in patients with comorbidities or older age or in the setting of grade ≥3 CRS (tosilimab ± corticosteroids). For patients who experienced a grade ≥2 neurologic event, tosilimab was initiated, and corticosteroids were added for patients with comorbidities or older age, or if any grade ≥3 neurologic event occurred and symptoms worsened despite tosilimab.

In one embodiment, the present invention provides that prophylactic steroid use appears to reduce the rate of severe CRS and NE to a similar level as early steroid administration. Therefore, the present invention provides a method for adverse event management in CAR T cell therapy, wherein patients receive 10 mg dexamethasone PO on day 0 (before infusion), day 1, and day 2. Steroids can also be administered, starting for grade 1 CRS at the time of grade 1 NE and when no improvement is observed after 3 days of supportive care. For grade ≥ 1 CRS, if no improvement is observed after 24 hours of supportive care, tocilizumab can also be administered. In one embodiment, the present invention provides for the management of adverse events of CAR T cell therapy with antibodies that neutralize and/or clear GM-CSF to prevent treatment-related CRS and/or NE in treated patients. In one embodiment, the antibody is lanzikumab.

In some embodiments, the adverse event is managed by administering an agent that is an antagonist or inhibitor of IL-6 or IL-6 receptor (IL-6R). In some embodiments, the agent is an antibody that neutralizes IL-6 activity, such as an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the agent is or comprises tosilimab (alizumab) or sarilumab, an anti-IL-6R antibody. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Patent No. 8,562,991. In some cases, the agent targeting IL-6 is an anti-TL-6 antibody, such as sirukumab, elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101 or olokizumab (CDP6038) and combinations thereof. In some embodiments, the agent can neutralize IL-6 activity by inhibiting ligand-receptor interactions. In some embodiments, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutant protein, such as the mutant protein described in U.S. Patent No. 5,591,827. In some embodiments, the agent that acts as an antagonist or inhibitor of IL-6/IL-6R is a small molecule, a protein or a peptide or a nucleic acid.

In some embodiments, other agents useful for managing adverse reactions and their symptoms include antagonists or inhibitors of interleukin receptors or interleukins. In some embodiments, the interleukin or receptor is IL-10, TL-6, TL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFα, TNFR1, such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-β receptor (TGF-βI, II or III), IFN-γ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra/IL-1RP) or IL-10 receptor (IL-10R), IL-1 and IL-1Rα/IL-1β. In some embodiments, the agent comprises siltuximab, cerulein, onokimab (CDP6038), estumomab, ALD518/BMS-945429, sulucumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the agent is an antagonist or inhibitor of an interleukin such as transforming growth factor β (TGF-β), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNF α, IL-1, interferon γ (IFN-γ), or monocytic protein-1 (MCP-1). In some embodiments, it is an agent that targets (e.g., inhibits or acts as an antagonist) an interleukin receptor such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGF-β receptor (TGF-β I, II or III), IFN-γ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNF α receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra/IL-1RP) or IL-10 receptor (IL-10R), and combinations thereof. In some embodiments, the agent is administered by one of the methods and dosages described elsewhere in this specification, before, after, or simultaneously with administration to the cells.

In some embodiments, the agent is administered at or about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kg to 6 mg/kg, 2 mg/kg to 4 mg/kg, or 6 mg/kg to 8 mg/kg, each including endpoints, or the agent is administered at or about 2 mg/kg, 4 mg/kg, 6 mg/kg, or 8 mg/kg. In some embodiments, the agent is administered at or about 10 mg/kg. In some embodiments, the agent is administered by intravenous infusion. In one embodiment, the agent is tocilizumab. In some embodiments, the agent (e.g., particularly tocilizumab) is administered prior to, subsequent to, or simultaneously with administration to the cells by one of the methods and dosages described elsewhere in this specification.

In some embodiments, the method comprises identifying CRS based on clinical manifestations. In some embodiments, the method comprises assessing and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, it can be managed according to the recommendations in Protocol A, which can also be used in combination with other treatments of the present invention, including neutralization or reduction of the CSF/CSFR1 axis. Patients experiencing ≥ Grade 2 CRS (e.g., hypotension, unresponsiveness to fluids, or hypoxia, requiring supplemental oxygen) should be monitored with continuous cardiac telemetry and pulse oximetry. In some embodiments, for patients experiencing severe CRS, consider performing an echocardiogram to assess cardiac function. For severe or life-threatening CRS, supportive care may be considered. In some embodiments, a biosimilar or equivalent of tosilimab may be used in place of tosilimab in the methods disclosed herein. In other embodiments, another anti-IL6R may be used in place of tosilimab.

In some embodiments, adverse events are managed according to the following regimen (Regime A): CRS Level (a) Tocilizumab Corticosteroids Grade 1 : Symptoms requiring symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgia, malaise). N/A N/A Grade 2 Symptoms requiring and responsive to moderate intervention. Oxygen requirement less than 40% _FiO2 or hypotension responsive to fluids or low doses of one vasopressor or Grade 2 organ toxicity ( b ) . Administer 8 mg/kg tosilimab (c) (not to exceed 800 mg) IV over 1 hour. Repeat tosilimab every 8 hours as needed if unresponsive to IV fluids or increased supplemental oxygen. Limit to a maximum of 3 doses in a 24-hour period; if there is no clinical improvement in signs and symptoms of CRS, give a maximum of 4 doses total. If there is no improvement within 24 hours of starting tocilizumab, manage according to Grade 3. Grade 3 Symptoms requiring and responding to aggressive intervention. Oxygen requirement greater than or equal to 40% _FiO2 or hypotension requiring high-dose or multiple vasopressors or Grade 3 organ toxicity or Grade 4 transaminase elevations. According to Level 2 Administer 1 mg/kg methylprednisolone IV twice daily or equivalent dexamethasone (e.g., 10 mg IV every 6 hours). Continue corticosteroids until events are Grade 1 or less, then taper over 3 days. If no improvement, manage as for Grade 4. Grade 4 Life-threatening symptoms. Respiratory support, continuous veno-venous hemodialysis (CVVHD), or grade 4 organ toxicity (excluding elevated transaminases) required. According to Level 2 Give 1000 mg methylprazosin IV daily for 3 days; if improved, manage as above. If no improvement or if worsening, consider another immunosuppressant. (a) Lee DW et al . (2014). Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 Jul 10 ; 124 (2):188-195 . (b) See Table 2 for management of neurotoxicity . (c) See ACEMTRA ® ( tosilimab ) prescribing information , https://www.gene.com/download/pdf/actemra_prescribing.pdf ( last accessed Oct 18 , 2017 ) . Initial US approval for this indication was in 2010. Neurotoxicity

In some embodiments, the method comprises monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method comprises excluding other causes of neurological symptoms. Patients experiencing ≥ Grade 2 neurotoxicity should be monitored using continuous cardiac telemetry and pulse oximetry. For severe or life-threatening neurotoxicity, provide intensive supportive care. For any ≥ Grade 2 neurotoxicity, consider non-sedative anti-epileptic drugs (e.g., levetiracetam) for the prevention of seizures. The following treatments may be used in combination with other treatments of the present invention, including neutralization or reduction of the CSF/CSFR1 axis.

In some embodiments, adverse events are managed according to the following regimen (Regime B): Graded Assessment Concurrent CRS No concurrent CRS Level 2 Administer tocilizumab according to the table above (Schedule A) to manage Grade 2 CRS. If there is no improvement within 24 hours of starting tocilizumab, administer 10 mg dexamethasone IV every 6 hours (if not already taking other steroids). Continue dexamethasone until the event is Grade 1 or less, then taper over 3 days. Administer 10 mg dexamethasone IV every 6 hours. Continue dexamethasone until the event is Grade 1 or less, then taper over 3 days. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 3 Tosilimab was administered according to (Schedule A) to manage grade 2 CRS. In addition, 10 mg of dexamethasone was administered IV with the first dose of tosilimab, and the dose was repeated every 6 hours. Dexamethasone was continued until the event was grade 1 or less, then tapered over 3 days. Administer 10 mg dexamethasone IV every 6 hours. Continue dexamethasone until the event is Grade 1 or less, then taper over 3 days. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 4 Administer tocilizumab according to (Regimen A) to manage Grade 2 CRS. Administer 1000 mg methylprednisolone IV daily with the first dose of tocilizumab and continue 1000 mg methylprednisolone IV daily for 2 additional days; if improved, manage as above. Give 1000 mg of methylprazosin IV daily for 3 days; if improved, manage as above. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures.

Additional Safety Management Strategies Using Corticosteroids

Administration of corticosteroids and/or tosilimab at Grade 1 may be considered prophylactic. Supportive care may be provided at all CRS and NE severity grades in all regimens. In one embodiment of the regimen for the management of adverse events related to CRS, tosilimab and/or corticosteroids are administered as follows: Grade 1 CRS: no tosilimab; no corticosteroids; Grade 2 CRS: tosilimab (only in the case of comorbidities or older age); and/or corticosteroids (only in the case of comorbidities or older age); Grade 3 CRS: tosilimab; and/or corticosteroids; Grade 4 CRS: tosilimab; and/or corticosteroids. In another embodiment of the regimen for the management of adverse events related to CRS, tosilimab and/or corticosteroids are administered as follows: Grade 1 CRS: tosilimab (if no improvement after 3 days); and/or corticosteroids (if no improvement after 3 days); Grade 2 CRS: tosilimab; and/or corticosteroids; Grade 3 CRS: tosilimab; and/or corticosteroids; Grade 4 CRS: tosilimab; and/or corticosteroids, high dose.

In one embodiment of a regimen for the management of adverse events associated with NE, tosilimab and/or corticosteroids are administered as follows: Grade 1 NE: no tosilimab; no corticosteroids; Grade 2 NE: no tosilimab; no corticosteroids; Grade 3 NE: tosilimab; and/or corticosteroids (standard dose only if no improvement on tosilimab); Grade 4 NE: tosilimab; and/or corticosteroids. In another embodiment of the regimen for managing adverse events associated with NE, tosilimumab and/or corticosteroids are administered as follows: Grade 1 NE: no tosilimumab; and/or corticosteroids; Grade 2 NE: tosilimumab; and/or corticosteroids; Grade 3 NE: tosilimumab; and/or corticosteroids; high dose; Grade 4 NE: tosilimumab; and/or corticosteroids, high dose. In one embodiment, corticosteroid treatment is initiated when CRS grade ≥2 and tosilimumab is initiated when CRS grade ≥2. In one embodiment, corticosteroid treatment is initiated when CRS grade ≥1 and tosilimumab is initiated when CRS grade ≥1. In one embodiment, corticosteroid treatment is initiated at NE grade ≥3 and tosilimab is initiated at CRS grade ≥3. In one embodiment, corticosteroid treatment is initiated at CRS grade ≥1 and tosilimab is initiated at CRS grade ≥2. In some embodiments, prophylactic use of tosilimab administered on day 2 reduces the rate of grade ≥3 CRS. One or more corticosteroids may be administered in any dose and frequency of administration, which may be adjusted based on the severity/grade of the adverse event (e.g., CRS and NE). Tables 1 and 2 provide examples of dosing regimens for the management of CRS and NE, respectively. In another embodiment, corticosteroid administration comprises oral or IV dexamethasone 10 mg, 1-4 times a day. Another embodiment, sometimes referred to as a "high-dose" corticosteroid, comprises 1 g of methylprednisolone administered IV per day, alone or in combination with dexamethasone. In some embodiments, one or more corticosteroids are administered in a dose of 1-2 mg/kg per day. In general, the dose of corticosteroid administered depends on the specific corticosteroid, as there are differences in potency between different corticosteroids. It is generally understood that the potency of drugs varies, so the dose can be varied in order to obtain the same effect. The equivalence of various glucocorticoids and routes of administration is well known in terms of potency. Information related to the administration of equivalent steroids (in a non-chronotherapy manner) can be found in British National Formulary (BNF) 37, March 1999. The application also provides for the administration and administration of cells prepared by the methods of the application, such as an infusion bag for CD19-guided genetically modified autologous T cell immunotherapy, containing about 68 mL of a suspension of chimeric antigen receptor (CAR)-positive T cells for infusion. In some embodiments, the CAR T cells are formulated into about 40 mL for infusion. In some embodiments, the CAR T cell product is formulated into a total volume of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 500, 700, 800, 900, 1000 mL. In one embodiment, the administration and administration of cells prepared by the method of the present application, such as CD19-guided genetically modified autologous T cell immunotherapy, contains about 40 mL of a suspension of 1×10 ⁶ CAR-T positive cells. The target dose may be between about 1×10 ⁶ and about 2×10 ⁶ CAR-positive live T cells per kilogram of body weight, with a maximum of 2×10 ⁸ CAR-positive live T cells.

In some embodiments, the dosage form comprises a cell suspension for infusion in a disposable patient-specific infusion bag; the route of administration is intravenous; the entire contents of each disposable patient-specific infusion bag are infused within 30 minutes by gravity or a peristaltic pump. In one embodiment, the dosing regimen is a single infusion consisting of 2.0×10 ⁶ anti-CD19 CAR T cells/kg body weight (±20%), with a maximum dose of 2×10 ⁸ anti-CD19 CAR T cells (individual ≥100 kg). In some embodiments, the T cells constituting the dose are CD19 CAR-T cells.

In some embodiments, the CD19-directed T cell immunotherapy is KTE-X19, which is prepared as described elsewhere in this application. In one embodiment, KTE-X19 can be used to treat MCL, ALL, CLL, SLL, and any other B cell malignancies. In some embodiments, the CD19-directed genetically modified autologous T cell immunotherapy is Axi-cel™ ( ^YESCARTA® , axicabtagene ciloleucel), prepared by one of the methods of this application. The amounts, dosing regimens, methods of administration, individuals, cancers within the scope of these methods are described elsewhere in this application, alone or in combination with another chemotherapeutic agent, with or without pretreatment, and for any of the patients described elsewhere in the application.

The following examples are provided to illustrate various aspects of the present application. Therefore, the specific aspects discussed should not be understood as limiting the scope of the present application. For example, although the following examples are directed to T cells transduced with anti-CD19 chimeric antigen receptors (CARs), those skilled in the art should understand that the methods described herein can be applied to immune cells transduced with any CAR. It will be apparent to those skilled in the art that a variety of equivalents, variations, and modifications can be made without departing from the scope of the application, and it should be understood that such equivalent aspects will be included herein. In addition, all references cited in the application are incorporated herein by reference in their entirety, as if fully set forth herein.

The patents and scientific literature mentioned herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts, genome database sequences, and scientific literature cited herein are incorporated by reference.

Other features and advantages of the present invention will be apparent from the drawings and the following detailed description including examples. Example Example 1

In this study, patients with R/R MCL who had received 1-5 prior lines of therapy including a Brudeon's tyrosine kinase inhibitor (BTKi) were treated with autologous anti-CD19 CAR T cells.

Eligible R/R MCL patients (age ≥18 years) had an ECOG score of 0-1 and ≤5 prior therapies, including chemotherapy, anti-CD20 antibodies, and BTK inhibitors (BTKi). Patients underwent leukapheresis and chemotherapy (cyclophosphamide 300 mg/ ^m2 /d and fludarabine 30 mg/ ^m2 /d for 3 days), followed by infusion of CD19 CAR-T at a target dose of 2× ¹⁰⁶ CAR T cells/kg. Patients could have received transitional therapy with dexamethasone, ibrutinib, or acalabrutinib after leukapheresis and before chemotherapy. The primary endpoint was objective response rate (ORR [complete response (CR) + partial response (PR)]) according to Lugano classification. Interim efficacy endpoints were investigator-assessed using the modified IWG malignant lymphoma response criteria. Key secondary endpoints were duration of response (DOR), progression-free survival (PFS), OS, frequency of adverse events (AEs), blood CAR T cell levels, and serum interleukin levels.

Twenty-eight patients received CD19 CAR-T cells with ≥ 1 year follow-up (median 13.2 months [range 11.5-18.5]). Forty-three percent of patients had an ECOG score of 1, 21% had blastoid morphology, 82% had stage IV disease, 50% had intermediate/high-risk MIPI, 86% received a median of 4 (range 1-5) prior lines of therapy, and 57% were refractory to the last prior line of therapy. In 20/28 patients, the median Ki-67 index was 38% (range 5%-80%). Eight patients received transitional therapy; all had disease beyond the transition period. The ORR was 86% (95% CI, 67%-96%), with a CR rate of 57% (95% CI, 37%-76%). 75% of responders maintained responses and 64% of treated patients had ongoing responses. The 12-month estimates of DOR, PFS, and OS were 83% (95% CI, 60%-93%), 71% (95% CI, 50%-84%), and 86% (95% CI, 66%-94%), respectively, and the median was not reached. Grade ≥3 AEs (≥20% of patients) were anemia (54%), thrombocytopenia (39%), neutropenia (36%), neutrophil count decreased (32%), leukocyte count decreased (29%), encephalopathy (25%), and hypertension (21%). Grade 3/4 interleukin release syndrome (CRS) assessed by Lee DW et al., Blood 2014;124:188 was reported in 18% of patients and manifested as hypotension (14%), hypoxia (14%), and fever (11%). Grade 3/4 neurologic events (NE) were reported in 46% of patients and included encephalopathy (25%), confusional state (14%), and aphasia (11%). No grade 5 CRS or NE occurred. All CRS events and the majority of NE (15/17 patients) were reversible. The median time to onset and resolution of CRS was 2 days (range 1-7) and 13 days (range 4-60), respectively. The median time to onset of NE was 6 days (range 1-15) and the median time to resolution was 20 days (range 9-99). The median CAR T cell levels as measured by peak and area under the curve were 99 cells/μL (range 0.4-2589) and 1542 cells/μL (range 5.5-27239), respectively. Peak CAR T cell expansion was observed between days 8 and 15 and declined over time. Example 2

This example provides an additional analysis of the above study. Eligible patients were aged ≥18 years, had MCL confirmed on pathology by cyclin D1 overexpression and/or the presence of t(11:14), and were relapsed/refractory to 1-5 prior regimens for MCL. Prior therapy must have included chemotherapy containing anthracyclines or bendamustine, an anti-CD20 monoclonal antibody, and ibrutinib or acalabrutinib. All patients received prior BTKi. Although patients had to have prior BTKi therapy, it was not required to be the last line of therapy prior to study entry, and patients were not required to be refractory to BTKi therapy. Eligible patients had an absolute lymphocyte count ≥100 cells/μL. Patients who underwent autologous SCT within 6 weeks of CD19 CAR-T infusion or had prior CD19-targeted therapy or allogeneic SCT were excluded.

Additional inclusion criteria included: at least 1 measurable lesion. Previously irradiated lesions were considered measurable only if progression was documented after completion of radiation therapy; if the only measurable disease was nodal disease, at least 1 lymph node should be ≥ 2 cm; brain magnetic resonance imaging (MRI) showed no evidence of central nervous system (CNS) lymphoma; patients must have been free of any prior systemic therapy or BTKi at the time of planned leukapheresis. (ibrutinib or acalabrutinib) for at least 2 weeks or 5 half-lives (whichever is shorter), except for systemic suppressive/stimulatory immune checkpoint therapy; at least 3 half-lives must have passed since any previous systemic suppressive/stimulatory immune checkpoint molecule therapy (e.g., ipalimumab, nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists) when the patient is scheduled for leukapheresis; toxicity caused by previous therapy must have stabilized and recovered to ≤ Grade 1 (except clinically irrelevant toxicity, such as alopecia); Eastern Cooperative on Cancer (ECOG) performance status of 0 or 1; absolute neutrophil count (ANC) ≥ 1 000/μl; platelet count ≥75 000/μl; absolute lymphocyte count ≥100/μl; adequate renal, hepatic, pulmonary, and cardiac function, defined as: creatinine clearance (as estimated by Cockcroft Gault) ≥60 cc/min; serum alanine aminotransferase/aspartate aminotransferase ≤2.5 upper limit of normal (ULN); total bilirubin ≤1.5 mg/dl, Gilbert's syndrome syndrome; ejection fraction ≥ 50% as determined by echocardiography (ECHO), no evidence of pericardial effusion, and no clinically relevant electrocardiographic (ECG) findings; no clinically relevant pleural effusion; baseline airway oxygen saturation > 92%; and women of childbearing age must have had a negative serum or urine pregnancy test. Women who were surgically sterilized or at least 2 years postmenopausal were considered infertile.

Additional exclusion criteria include: history of malignant disease other than non-melanoma skin cancer or carcinoma in situ (e.g., cervical, bladder, breast), unless disease-free for at least 3 years; history of allogeneic stem cell transplantation; prior CAR therapy or other genetically modified T-cell therapy; history of severe rapid-onset allergic reaction to aminoglycosides; presence of fungal, bacterial, viral, or other infection that is uncontrolled or requires management with intravenous (IV) antimicrobials. Simple urinary tract infection (UTI) and uncomplicated bacterial pharyngitis are allowed if responsive to aggressive therapy and after consultation with a medical monitor; history of human immunodeficiency virus (HIV) infection or acute or chronic active hepatitis B or C infection. Patients with a history of hepatitis infection must have cleared their infection as determined by standard serological and genetic testing; any indwelling lines or drains (e.g., percutaneous nephrostomy tube, indwelling Foley catheter, biliary drain, or pleural/peritoneal/pericardial catheter) are permitted. Ommaya reservoirs and dedicated central venous access catheters such as Port-a-Cath or Hickman catheters are permitted. catheter); patients with detectable cerebrospinal fluid malignancy or brain metastasis or a history of: CNS lymphoma, cerebrospinal fluid malignancy or brain metastasis; history or presence of CNS disorders such as epilepsy, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, cerebral edema, posterior reversible encephalopathy syndrome, or any autoimmune disease involving the CNS; history of myocardial infarction, cardiac angioplasty or stenting, unstable angina, active arrhythmia, or other clinically relevant heart disease within 12 months of enrollment; patients with atrial or ventricular lymphoma involving the heart Patients: History of symptomatic deep venous embolism or pulmonary embolism within the last 6 months of enrollment; Ongoing or impending oncological emergency (e.g., tumor mass effect, tumor lysis syndrome) that may require acute treatment; Primary immunodeficiency; Any medical condition that may interfere with safety or efficacy assessment of study treatment; History of severe rapid-onset hypersensitivity reaction to any agent used in this study; Live vaccines ≤ 6 weeks before the planned start of the conditioning regimen; Women of childbearing age who are pregnant or breastfeeding because preparative chemotherapy may have hazardous effects on the fetus or infant; From the time of consent until completion of CD19 Patients of both sexes who are unwilling to implement birth control 6 months after CAR-T cell therapy; patients who, in the judgment of the investigator, are unlikely to complete all protocol-required study visits or procedures, including follow-up visits, or comply with study participation requirements; and a history of autoimmune diseases (e.g., Crohn's disease, rheumatoid arthritis, systemic lupus) that have caused end-organ damage or required systemic immunosuppression/systemic disease-modifying agents within the last 2 years.

All patients underwent leukapheresis to obtain cells for CD19 CAR-T cell therapy manufacturing. The manufacturing method was modified from that of akilostat to remove circulating lymphoma cells by positive enrichment of CD4 ⁺ /CD8 ⁺ cells. Conditioning chemotherapy with fludarabine (30 mg/ ^m2 per day) and cyclophosphamide (500 mg/ ^m2 per day) was administered on days -5, -4, and -3, followed by a single intravenous infusion of 2× ¹⁰⁶ CAR T cells/kg of CD19 CAR-T cells on day 0. The doses were obtained from studies of akilobase in large B-cell lymphoma and CD19 CAR-T cells in acute lymphoblastic leukemia. Neelapu SS et al. The New England journal of medicine 2017;377:2531; Locke FL et al. Mol Ther 2017;25:285; Shah BD et al. Journal of Clinical Oncology 2019;37:(suppl; abstract 7006); and Lee DW et al. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 2017;28:1008PD, all of which are incorporated herein by reference in their entirety. After leukapheresis and before conditioning therapy, patients with high disease burden received transitional therapy with dexamethasone or equivalent corticosteroids, ibrutinib, or acalabrutinib, at the investigator's discretion, followed by repeated baseline positron emission tomography-computed tomography (PET-CT) scans. The goal of transitional therapy was not curative but to keep patients stable during the manufacturing period. Hospitalization until day 7 after CD19 CAR-T cell infusion was required.

The primary endpoint was objective response rate (ORR [complete response (CR) + partial response] (PR)) as assessed by the Independent Radiology Review Committee (IRRC) using the Lugano classification. Cheson et al., J Clin Oncol 2014;32:3059-68. Bone marrow assessment in addition to PET-CT was required to confirm CR. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), OS, investigator-assessed ORR according to Cheson et al., J Clin Oncol 2007;25:579-86, incidence of adverse events (AEs), levels of CAR T cells in blood and serum interleukins, and changes in European Quality of Life-5 Dimensions with 5 levels per dimension (EQ-5D-5L) scores over time. CAR T cell presence, expansion, and persistence, as well as serum interleukins, and their association with clinical outcomes were assessed as previously reported. Kochenderfer JN et al J Clin Oncol 2017;35:1803-13; Locke FL et al Mol Ther 2017;25:285-95, all of which are incorporated herein by reference in their entirety.

Changes in EQ-5D-5L scores from baseline to month 6 were assessed. Interleukin release syndrome (CRS) was graded according to Lee et al. Blood 2014; 124: 188, which is incorporated herein by reference in its entirety. The severity of AEs, including neurological events and CRS symptoms, was graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03. Minimal residual disease (MRD; 10 ^{- 5} sensitivity) was an exploratory analysis assessed in cryopreserved peripheral blood mononuclear cells at baseline and months 1, 3, and 6 and analyzed by next-generation sequencing using clonoSEQ analysis (Adaptive Biotechnologies, Seattle, WA).

For all patients, positron emission tomography to computed tomography (PET-CT) scans of disease-specific sites were required at baseline, 4 weeks after infusion, and at regular intervals during the post-treatment period. A bone marrow aspirate/biopsy was required to confirm a complete response in patients with bone marrow disease involvement at baseline and in patients with indeterminate bone marrow involvement at baseline, or if a baseline bone marrow biopsy was not performed or was unavailable. Patients with symptoms of CNS malignancies underwent a lumbar puncture for examination of cerebrospinal fluid (CSF) at screening. When appropriate, a lumbar puncture was also performed for patients with new-onset grade ≥2 neurotoxicity after anti-CD19 CAR T-cell infusion. In addition, for the patients who signed the consent form, lumbar puncture was performed at baseline before anti-CD19 CAR T cell infusion and after anti-CD19 CAR T cell infusion (day 5 ± 3 days) for collection of CSF; samples were submitted to a central laboratory and analyzed for changes in interleukin levels.

The primary analysis of efficacy was performed after 60 patients were enrolled, treated, and assessed for response 6 months after the Week 4 disease assessment, as required by the protocol. This analysis had ≥96% power to distinguish active therapy with a true response rate of 50% from therapy with a response rate ≤25% with a one-sided alpha level of 0.025. The exact binomial test was used to analyze ORR. All efficacy endpoints, including time-to-event endpoints, as assessed using Kaplan-Meier estimates, were analyzed in the above 60 efficacy-evaluable patients. Safety analyses were performed in all treated patients (n=68). The association between efficacy and CAR T cells and interleukin levels was measured using the Wilcoxon rank-sum test; P values were adjusted using Holm's procedure. Full analysis set (N=74): consisted of all enrolled/leukapheresis patients and was used to summarize patient disposition. Safety analysis set (n=68): defined as all patients treated with any dose of anti-CD19 CAR T cells. This analysis set was used to summarize demographics and baseline characteristics and all safety analyses. Inferential analysis (efficacy evaluable) set (n=60): consisted of the first 60 patients treated with CD19 CAR-T cells. This analysis set was used for hypothesis testing of the primary endpoint of objective response rate at the primary analysis and all other efficacy analyses. The hypothesis for the primary endpoint was that the ORR to CD19 CAR-T cells using central assessment would exceed the pre-specified historical control rate of 25% using the exact binomial test at a one-sided significance level of 0.025. This hypothesis was tested in the inferential analysis set. The historical control rate for ORR was determined a priori based on 2 retrospective studies that were public at the time of study protocol development. In these 2 studies, the efficacy of salvage therapy was evaluated in patients with relapsed/refractory MCL who had evolved after treatment with BTKi (a prior therapy required for study eligibility). These studies showed that the ORR to salvage therapy was approximately 25% in patients with relapsed/refractory MCL who had ≥3 prior lines of therapy before receiving BTKi. Wang M et al. Lancet 2018;391:659; Martin P et al. Blood 2016;127:1559, both of which are incorporated herein by reference in their entirety.

Seventy-four patients were enrolled; CD19 CAR-T cells were manufactured for 71 and administered to 68. The primary efficacy analysis, performed after 60 patients were treated, showed an ORR of 93% (67% complete response). At a median follow-up of 12.3 months (range 7.0-32.3), 57% of patients remained in remission and the median duration of response was not reached. The estimated 12-month progression-free survival and overall survival rates were 61% and 83%, respectively. Common grade ≥3 adverse events were cytopenias (94%) and infections (32%). Grade ≥3 interleukin release syndrome and neurological events occurred in 15% and 31% of patients, respectively; neither was fatal. Two grade 5 infectious adverse events occurred.

CD19 CAR-T cells were manufactured for 71 patients (96%) and administered to 68 (92%). The median time from leukapheresis to delivery of CD19 CAR-T cells to the study site was 16 days (range 11-128). One patient with manufactured CD19 CAR-T cells was treated with bendamustine-rituximab due to rapid PD after leukapheresis, which made the patient ineligible for the study. After subsequently developing PD, the patient's raw product was shipped from the manufacturing facility 127 days after the initial leukapheresis date and arrived at the treatment site 1 day later. Three patients with manufacturing issues did not proceed with additional apheresis due to AEs (n=1; deep venous embolism), death due to progressive disease (PD; n=1), or withdrawal of consent (n=1). Two additional patients discontinued before conditioning chemotherapy due to death due to PD. One patient with persistent atrial fibrillation (exclusion criterion) after conditioning chemotherapy was considered ineligible for CD19 CAR-T cell infusion. The median follow-up period for efficacy-evaluable patients was 12.3 months (range, 7.0-32.3); 28 patients had a follow-up period of ≥24 months.

The median age was 65 years (range, 38-79) and 57 (84%) patients were male. (Table 1) 65% had an ECOG performance status of 0 and 35% had an ECOG performance status of 1. Patients had high-risk features at baseline, including stage IV disease (85%), blastoid or pleomorphic morphology (31%), Ki-67 proliferation index ≥30% (40/49 [82%]) (Wang ML et al. The Lancet Oncology 2016;17:48), and TP53 mutations (6/36 [17%]). Eighty-one percent of patients had received ≥3 prior lines of therapy (median, 3 [range, 1-5]).

Table 1. Baseline Patient Characteristics Features N=68 Age, median ( range ) , years 65 (38-79) ≥65 years old, n (%) 39 (57) Male, n (%) 57 (84) ECOG performance status score, n (%) 0 44 (65) 1 24 (35) Stage IV disease, n (%) 58 (85) Bone marrow involvement, n (%) 37 (54) Spleen involvement, n (%) 23 (34) Extranodal disease, n (%) ^* 38 (56) Large mass (≥10 cm) , n (%) 7 (10) Simplified MIPI , n (%) ^† Low risk 28 (41) Medium risk 29 (43) High risk 9 (13) Missing twenty three) MCL morphology, n (%) typical 40 (59) Polymorphism 17 (25) Mother cell-like 4 (6) Other/Unknown ^†† 11 (16) Ki -67 proliferation index , median ( range ) , % ^§ 65 (1-95) ≥30%, n/n (%) 40/49 (82) ≥50%, n/n (%) 34/49 (69) TP53 mutation, n (%) 6/36 (17%) CD19 status, n/n (%) Positive 47/51 (92) Negative 4/51 (8) Number of previous treatments , median ( range ) 3 (1-5) ≥3 lines of prior therapy, n (%) 55 (81) Previous treatment, ^¶ n (%) Anti-CD20 68 (100) BTK 68 (100) Ibrutinib 58 (85) Acalabrutinib 16 (24) Both 6 (9) Anthracycline or bendamustine 67 (99) Anthracycline 49 (72) Bendamustine 37 (54) Autologous SCT 29 (43) Bortezomib 24 (35) Lenalidomide 19 (28) Other research drugs 11 (16) Venetoclax 6 (9) Relapsed / refractory subgroup , n (%) Relapse after autologous SCT 29 (43) The last type is refractory to previous treatment 27 (40) Relapse after last prior treatment 12 (18) Ibrutinib-refractory, n (%) 38 (56) Acalabrutinib-refractory, n (%) 8 (12) ^* Bone marrow and spleen involvement were excluded. ^† At diagnosis. ^†† One patient was reported by the investigator to have kappa light chain-restricted MCL at diagnosis. Morphology was reported as unknown in 10 patients. ^§Ki -67 data were available for 49 patients at diagnosis. ^¶ All treatments given during induction plus consolidation/maintenance and/or continuous complete response counted as 1 regimen. BTKi, Bruden's tyrosine kinase inhibitor; ECOG, Eastern Cooperative Oncology Group; MCL, mantle cell lymphoma; MIPI, International Prognostic Index for Mantle Cell Lymphoma; SCT, stem-cell transplantation.

All patients progressed on BTKi (ibrutinib n=58; acalabrutinib n=16; both n=6), and 43% had prior autologous SCT ( Table 2 ). The median time from the end of the last transition period excluding BTKi therapy to CD19 CAR-T cell infusion was 88 days (range 25-1047). Forty percent of patients were refractory to the last therapy, including 3 patients with confirmed ibrutinib intolerance who progressed after the last therapy. Twenty-five patients (37%) received transition therapy with ibrutinib (n=14), acalabrutinib (n=5), dexamethasone (n=12), and/or methylprednisolone (n=2). Post-transition scans showed that most patients had a tumor burden above the median at screening.

Table 2. Transitional treatment Features N = 68 Any transitional treatment, n (%) 25 (37) Ibrutinib 14 (21) Acalabrutinib 5 (7) Dexamethasone 12 (18) Prussin Methyl twenty three) BTKi and steroids , n (%) 6 (9) Ibrutinib + steroids 4 (6) Acalabrutinib + steroids twenty three) BTKi, Bruton's tyrosine kinase inhibitor.

Among the 60 patients treated with CD19 CAR-T as specified in the protocol, the IRRC-assessed ORR was 93% (95% CI, 84-98) at a minimum follow-up period of 7 months, including a 67% CR rate and a 27% PR rate. A high degree of agreement (95%) was observed between the IRRC-assessed and investigator-assessed ORRs ( Table 3 ).

Table 3. Response according to Cheson BD et al. J Clin Oncol 2007;25:57 based on investigator assessment in efficacy-evaluable patients and according to Lugano classification (2014) by IRRC review in intention-to-treat patients. n (%) Investigator-assessed power evaluable N = 60 IRRC assessed intention to treat N = 74 Objective response rate 53 (88) 63 (85) Complete response 42 (70) 44 (59) Partial response 11 (18) 19 (26) Stable disease 5 (8) 3 (4) Disease progression twenty three) twenty three) Not assessed ^* 0 (0) 6 (8) Concordance rate with IRRC -assessed ORR , % ^† 95 N/A Kappa coefficient (95% CI) 0.7 (0.4-1.0) N/A Concordance rate with IRRC -estimated CR rate , % ^† 90 N/A Kappa coefficient (95% CI) 0.8 (0.6-0.9) N/A ^* Not assessed at the time of analysis. ^† Concordance rate is the percentage of individuals for whom the IRRC-assessed reading agreed with the investigator-assessed reading. CR, complete response; IRRC, independent radiology review committee; N/A, not available; ORR, objective response rate.

The IRRC-assessed ORR for all enrolled patients (n=74) was 85% (95% CI, 75-92), including a 59% CR rate. The ORR was consistent across key subgroups, including age, relapsed/refractory subgroups, number of prior therapies, MCL morphology, disease stage, extranodal disease, bone marrow involvement, simplified MIPI, CD19 positivity, tumor burden, serum lactate dehydrogenase level, TP53 mutation status, Ki-67 index, use of tocilizumab or steroids for management of AEs, and use of transition therapy. The median time to initial response was 1.0 month (range, 0.8-3.1), and the median time to CR was 3.0 months (range, 0.9-9.3). Of the 42 patients who initially achieved a PR or SD, 24 patients (57%), including 21 with an initial response of PR and 3 with an initial response of SD, subsequently converted to a CR after a median of 2.2 months (range, 1.8-8.3) after the initial response; 18 of these 24 patients remain in remission. MRD analysis was performed in 29/60 patients (48%); 24/29 patients (83% [19 CR; 5 PR]) were MRD-negative at Week 4, and 15/19 patients (79%) with available data remained MRD-negative at Month 6. MRD could not be assessed in all patients because formalin-fixed paraffin-embedded tumor section samples were not available for calibration, which is required for this approach and used to establish dominant rearranged IgH (VDJ or DJ), IgK, or IgL receptor gene sequences in the blood that are tracked over time. Two patients who evolved after responding to CD19 CAR-T cells received a second infusion approximately 1 and 2.6 years after the initial infusion; these patients are being analyzed on an ongoing basis.

After a median follow-up of 12.3 months, the median DOR had not been reached (median (95% CI); not reached (8.6, NE). The median progression-free survival (95% CI) had not been reached (9.2, NE). The median overall survival (95% CI) had not been reached (24.0, NE). Fifty-seven percent of all patients and 78% of CR patients remained in remission. However, the initial 28 patients treated had a median follow-up of 27.0 months (range; 25.3-32.3), with 43% continuing to remit without additional therapy. Durable response rates were consistent across key covariates, including age, MCL morphology, relapsed/refractory subgroup, Ki-67 index, disease stage, extranodal disease, bone marrow involvement, simplified MIPI, TP53 mutation, CD19 positivity, transition therapy, tumor burden, and use of tocilizumab or steroids. Three patients with CD19- tumors at baseline achieved CR and remained in durable response as of the data cutoff. Median PFS and OS were not reached, with estimated 12-month rates of 61% (95% CI, 2.0-3.0), respectively. CI, 45-74) and 83% (95% CI, 71-91). Although the sample size was limited, subgroup analysis of PFS showed that the 6-month PFS rate was consistent among patients with blastoid or pleomorphic morphology, TP53 mutation, or Ki-67 index ≥50%. At the time of this analysis, 76% of all patients were still alive. Among patients with response, 14 had PD. One patient with PR underwent allogeneic SCT.

This study showed an ORR of 93% in 60 protocol-specified patients with relapsed/refractory MCL, all of whom had relapsed following or were refractory to BTKi therapy. This ORR included a 67% CR rate after a single infusion. The median DOR had not been reached after a median follow-up of 12.3 months; 57% of all patients and 78% of CR patients remained in response. Twenty-eight (28) patients treated with CD19 CAR-T cells had a longer median follow-up of 27 months (range 25.3-32.3), and 43% continued to respond without additional therapy. Response rates, including durable responses, were generally similar among key subgroups including patients with high-risk characteristics. Patients with Ki-67 ≥ 50% and those with blastoid/pleomorphic morphology or TP53 mutations had high ORR and 6-month PFS rates similar to the overall population, suggesting that CD19 CAR-T cell therapy benefits patients with generally worse prognoses.

All patients who responded after CAR T cell infusion achieved T cell expansion. This expansion was not observed in non-responding patients, suggesting that responses may be associated with adequate CAR T cell expansion. Similar to previous studies, CAR T cell levels correlated with ORR in the first 28 days, suggesting that higher expansion leads to better and potentially deeper responses, as indicated by >80-fold higher peak/AUC CAR T cell levels in MRD-negative patients compared to MRD-positive patients. Response rates were also similar regardless of whether transition therapy was administered, and the majority of patients with a post-transition scan (87%) had an increase in SPD compared to the pre-transition scan.

All treated patients experienced ≥1 AE of any grade, of which 99% had grade ≥3 AEs (Table 2). The most common AEs of any grade were fever (94%), neutropenia (87%), thrombocytopenia (74%), and anemia (68%). The most common grade ≥3 AEs were neutropenia (85%), thrombocytopenia (51%), anemia (50%), and infection (32%). Twenty-six percent of patients had grade ≥3 hematopoiesis >90 days after CD19 CAR-T cells, including neutropenia (16%), thrombocytopenia (16%), and anemia (12%). CRS occurred in 91% of patients ( Table 4 ). No patient died from CRS. The majority of conditions were grade 1/2 (76%), with grade ≥3 CRS occurring in 15% of patients. The most common grade ≥3 CRS symptoms were hypotension (22%), hypoxia (18%), and fever (11%). For management of CRS, 59% of patients received tocilizumab, 22% received steroids, and 16% required vasopressors. The median time to onset of any grade and grade ≥3 CRS after infusion was 2 days (range 1-13) and 4 days (range 1-9), respectively; all events resolved within a median of 11 days.

Table 4. Adverse events, interleukin-1 release syndrome, and neurological events N=68 n (%)* Any level Level 1 Level 2 Level 3 Level 4 Level 5 Any adverse events 68 (100) 0 (0) 1 (1) 11 (16) 52 (76) twenty three) Fever 64 (94) 14 (21) 41 (60) 9 (13) 0 0 Neutropenia 59 (87) 0 (0) 1 (1) 11 (16) 47 (69) 0 (0) Thrombocytopenia 50 (74) 9 (13) 6 (9) 11 (16) 24 (35) 0 (0) Anemia 46 (68) 0 12 (18) 34 (50) 0 0 Low blood pressure 35 (51) 4 (6) 16 (24) 13 (19) twenty three) 0 Chills 28 (41) 17 (25) 11 (16) 0 0 0 Hypoxia 26 (38) twenty three) 10 (15) 8 (12) 6 (9) 0 cough 25 (37) 14 (21) 11 (16) 0 0 0 Hypophosphatemia 25 (37) twenty three) 8 (12) 15 (22) 0 0 Fatigue 24 (35) 10 (15) 13 (19) 1 (1) 0 0 headache 24 (35) 15 (22) 8 (12) 1 (1) 0 0 Tremor 24 (35) 19 (28) 5 (7) 0 0 0 Hypoalbuminemia 23 (34) 5 (7) 17 (25) 1 (1) 0 0 Hyponatremia 22 (32) 15 (22) 0 7 (10) 0 0 Nausea 22 (32) 11 (16) 10 (15) 1 (1) 0 0 Increased alanine transaminase 21 (31) 13 (19) twenty three) 5 (7) 1 (1) 0 Brain disease 21 (31) 5 (7) 3 (4) 7 (10) 6 (9) 0 Hypokalemia 21 (31) 12 (18) 4 (6) 3 (4) twenty three) 0 Tachycardia 21 (31) 14 (21) 7 (10) 0 0 0 CRS ^† 62 (91) 20 (29) 32 (47) 8 (12) twenty three) 0 Symptoms Fever 62 (100) 15 (24) 40 (65) 7 (11) 0 0 Low blood pressure 35 (56) 4 (6) 16 (26) 14 (23) 1 (2) 0 Hypoxia 23 (37) 1 (2) 10 (16) 8 (13) 4 (6) 0 Chills 21 (34) 12 (19) 9 (15) 0 0 0 Tachycardia 16 (26) 11 (18) 5 (8) 0 0 0 headache 15 (24) 7 (11) 8 (13) 0 0 0 Increased alanine transaminase 10 (16) 5 (8) 1 (2) 3 (5) 1 (2) 0 Increased aspartate aminotransferase 9 (15) 4 (6) 0 (0) 5 (8) 0 0 Fatigue 9 (15) 6 (10) twenty three) 1 (2) 0 0 Nausea 9 (15) 5 (8) 4 (6) 0 0 0 Any neurological event 43 (63) 13 (19) 9 (13) 15 (22) 6 (9) 0 Tremor 24 (35) 19 (28) 5 (7) 0 0 0 Brain disease 21 (31) 5 (7) 3 (4) 7 (10) 6 (9) 0 Confusion 14 (21) 3 (4) 3 (4) 8 (12) 0 0 Aphasia 10 (15) 3 (4) 4 (6) 3 (4) 0 0 * Includes adverse events occurring in ≥30% of patients, and symptoms of CRS and neurologic events occurring in ≥15% of patients. ^† Percentages in the CRS column were calculated for the 62 patients who experienced CRS.

Sixty-three percent of patients experienced NE ( Table 4 ). No patient died from NE. Grade 1/2 NE occurred in 32% of patients and Grade ≥3 NE occurred in 31%. Common Grade ≥3 NE were encephalopathy (19%), confusional state (12%), and aphasia (4%). One patient developed Grade 4 cerebral edema and fully recovered with invasive multicenter therapy including ventriculostomy. Tocilizumab and steroids were used to treat NE in 26% and 38% of patients, respectively. The median time to onset of any grade and Grade ≥3 NE was 7 days (range 1-32) and 8 days (range 5-24), respectively. The median duration of NE was 12 days, with complete resolution of the event in 37/43 patients (86%). As of this analysis, 4 patients had ongoing events, including grade 1 tremor (n=3), grade 2 concentration impairment (n=1), and grade 1 sensory dullness (n=1). Severe AEs occurred in 68% of patients ( Table 5 ).

Table 5. Serious adverse events occurring in at least 3 patients N = 68 Serious adverse events, n (%) Any level Level 1 Level 2 Level 3 Level 4 Level 5 Either 46 (68) twenty three) 7 (10) 20 (29) 13 (19) twenty three) Brain disease 15 (22) twenty three) 1 (1) 6 (9) 6 (9) 0 Fever 15 (22) 7 (10) 5 (7) 3 (4) 0 0 Low blood pressure 11 (16) 0 3 (4) 6 (9) twenty three) 0 Hypoxia 8 (12) 0 0 4 (6) 4 (6) 0 Acute kidney injury 5 (7) 0 0 1 (1) 4 (6) 0 Confusion 5 (7) 0 0 5 (7) 0 0 pneumonia 5 (7) 0 0 5 (7) 0 0 Anemia 4 (6) 0 0 4 (6) 0 0 respiratory failure 4 (6) 0 0 0 4 (6) 0 Sepsis 4 (6) 0 0 1 (1) 3 (4) 0 Aphasia 3 (4) 0 0 3 (4) 0 0 Pleural effusion 3 (4) 0 1 (1) 1 (1) 1 (1) 0 Tachycardia 3 (4) 0 3 (4) 0 0 0

Thirty-two percent of patients experienced grade ≥ 3 infections. The most common was pneumonia (9%) ( Table 6 ).

Table 6. Infections occurring in at least 2 patients N = 68 Infection, n (%) Any level Level 1 Level 2 Level 3 Level 4 Level 5 Either 38 (56) 1 (1) 15 (22) 17 (25) 4 (6) twenty two) ^* Upper respiratory tract infection 9 (13) 0 (0) 8 (12) 1 (1) 0 (0) 0 (0) pneumonia 7 (10) 0 (0) 1 (1) 6 (9) 0 (0) 0 (0) Sinusitis 5 (7) 0 (0) 5 (7) 0 (0) 0 (0) 0 (0) Sepsis 4 (6) 0 (0) 0 (0) 1 (1) 3 (4) 0 (0) Oral candidiasis 4 (6) 0 (0) 4 (6) 0 (0) 0 (0) 0 (0) Herpes zoster 3 (4) 0 (0) 3 (4) 0 (0) 0 (0) 0 (0) influenza 3 (4) 0 (0) 3 (4) 0 (0) 0 (0) 0 (0) Staphylococcal bacteremia 3 (4) 0 (0) 0 (0) twenty three) 0 (0) 1 (1) Cell giant virus infection twenty three) 0 (0) twenty three) 0 (0) 0 (0) 0 (0) Fungal skin infections twenty three) 1 (1) 1 (1) 0 (0) 0 (0) 0 (0) Cellulitis twenty three) 0 (0) twenty three) 0 (0) 0 (0) 0 (0) Bronchitis twenty three) 0 (0) 1 (1) 1 (1) 0 (0) 0 (0) Nasopharyngitis twenty three) twenty three) 0 (0) 0 (0) 0 (0) 0 (0) Tooth infection twenty three) 0 (0) 0 (0) twenty three) 0 (0) 0 (0) ^* One patient died of staphylococcal bacteremia. One patient died of organizing pneumonia (in addition to organizing pneumonia, acute renal injury with previously undiagnosed pulmonary embolism was found in the setting of infection and during autopsy).

Two cases of grade 2 cytomegalovirus infection occurred (3%). Grade 3 hypogammaglobulinemia and grade 3 tumor lysis syndrome occurred in one patient each (1%). Twenty-two patients (32%) received intravenous immunoglobulin therapy. No cases of replication-competent retroviruses, EBV-associated lymphoproliferation, hemophagocytic lymphohistiocytosis, or CD19 CAR-T cell-associated secondary cancers were reported. EQ-5D scores showed a decrease in patient-reported health-related quality of life from baseline at week 4, but by month 3, improvements were observed in mobility, self-care, daily activities, and global health (EQ-5D visual analog scale), with global health returning to baseline or better in most patients by month 6 ( Table 7 ).

Table 7. Summary of EQ-5D by clinic visit EQ-5D Filter Week 4 3rd Month 6th month Activity , n/n (%) Patients reporting no problems 53/62 (85) 25/51 (49) 37/54 (69) 30/40 (75) Patients who have deteriorated since screening N/A 21/51 (41) 13/54 (24) 8/40 (20) Self-care, n/n (%) Patients reporting no problems 59/62 (95) 35/52 (67) 45/54 (83) 37/40 (93) Patients who have deteriorated since screening N/A 16/52 (31) 9/54 (17) 3/40 (8) Daily activities, n/n (%) Patients reporting no problems 53/65 (82) 22/51 (43) 38/55 (69) 30/41 (73) Patients who have deteriorated since screening N/A 25/51 (49) 13/55 (24) 8/41 (20) Pain / discomfort , n/n (%) Patients reporting no problems 43/65 (66) 34/54 (63) 33/55 (60) 28/42 (67) Patients who have deteriorated since screening N/A 9/54 (17) 13/55 (24) 5/42 (12) Anxiety / depression, n/n (%) Patients reporting no problems 49/65 (75) 36/54 (67) 38/55 (69) 26/42 (62) Patients who have deteriorated since screening N/A 11/54 (20) 12/55 (22) 10/42 (24) EQ-5D VAS ^* n 65 52 55 42 Mean (SD) 82.0 (15.4) 74.5 (15.6) 80.1 (15.6) 84.8 (17.5) Median(range) 85 (75 - 95) 78 (60 - 89) 83 (70 - 92) 90 (80 - 95) VAS decrease ≥10 since screening, n/n (%) N/A 26/52 (50) 16/55 (29) 5/42 (12) ^* The EQ-5D visual analog scale (VAS) assesses overall health on a scale from 0 to 100, where higher scores indicate better health. EQ-5D, European 5-dimensional quality of life; N/A, not available; SD, standard deviation

Sixteen patients (24%) who received CD19 CAR-T cells died, primarily from PD (n=14 [21%]). Two patients had grade 5 AEs (3%), including 1 patient with organizing pneumonia associated with conditioning chemotherapy and 1 patient with staphylococcal bacteremia associated with conditioning chemotherapy and CD19 CAR-T cell therapy.

The median time to peak anti-CD19 CAR T cell levels after CD19 CAR-T cell infusion was 15 days (range, 8-31), and some patients with evaluable samples at data cutoff had detectable cells at 24 months (6/10 [60%]) in the presence of normal median B cell levels. CAR T cell persistence in the blood over time, as measured by qPCR, showed a decrease over time in patients with sustained responses and in those who relapsed.

Rapid expansion, regression to baseline, and clearance over time are consistent with the known mechanism of action of anti-CD19 CAR T cells with CD28 and CD3ζ co-stimulatory domains. All 4 patients who did not respond to CD19 CAR-T cell therapy had detectable B cells at baseline; none experienced B cell aplasia at any point in the study. Although not associated with baseline tumor burden, expansion correlated with response (P=0.0036), with the area under the curve (AUC) and peak values >200-fold higher in responders versus nonresponders, with similar trends in MRD-negative versus MRD-positive patients at Week 4. For both CRS and NE, the expansion was greater in patients with grade ≥3 relative to those with grade ≤2 events, and the highest peak and AUC were noted in patients who received tocilizumab ± steroids after CD19 CAR-T cell infusion. The median time to peak for the interleukins evaluated was 8 days; most resolved to baseline levels by 28 days. Serum granulocyte-macrophage colony-stimulating factor and interleukin (IL)-6 were associated with grade ≥3 CRS and NE. Serum ferritin was associated only with grade ≥3 CRS, while serum IL-2 and interferon-γ were associated only with grade ≥3 NE. In addition, cerebrospinal fluid interleukin analysis showed higher levels of C-reactive protein, ferritin, IL-6, IL-8, and vascular cell adhesion molecule 1 in patients with grade ≥3 NE. Induction of anti-CAR antibodies was not observed in any patient.

Rates of grade ≥3 CRS and NE were similar to those previously reported in aggressive NHL with anti-CD19 CART cell therapy. Neelapu SS et al The New England journal of medicine 2017;377:2531; Schuster SJ et al The New England journal of medicine 2019;380:45. There were no deaths due to CRS or NE, and most symptoms developed early in treatment and were generally reversible, with no long-term clinical sequelae impairing activities of daily living. Given that these toxicities were observed commensurate with the rise and peak levels of CAR T cells in the blood, the observed correlation between peak serum interleukins and grade ≥3 CRS and/or grade ≥3 neurologic events suggests a role for CD19 CAR-T cells in these toxicities. The correlation of peak levels of CAR and myeloid cell-associated serum interleukins, chemokines, and effector molecules with toxicity is consistent with previously published data using similar CAR constructs in the NHL setting. ^{10 , 13} There was one case of grade 4 cerebral edema, but the patient fully recovered and remains in CR with no unresolved neurological sequelae at 24 months of follow-up. Patient-reported outcomes similarly demonstrated no deficits in long-term quality of life after CD19 CAR-T cell therapy. Example 3

This example provides an additional analysis of the above study. Eligible R/R MCL patients (age ≥18 years) had an ECOG score of 0-1 and ≤5 prior therapies, including chemotherapy, anti-CD20 antibody, and BTKi. Patients underwent leukapheresis and chemotherapy (cyclophosphamide 300 mg/m ² /d and fludarabine 30 mg/m ² /d on days -5, -4, -3 for 3 days), followed by a single infusion of CD19 CAR-T cells at a target dose of 2×10 ⁶ CAR T cells/kg by a single IV infusion on day 0. The CD19 CAR construct contains the CD3ζ T cell activation domain and the CD28 signaling domain. The manufacturing method removes circulating leukemic cells expressing CD19 from leukapheresis products. Sabatino M et al., Blood 2016;128:1227.

Some patients received transitional therapy with dexamethasone (20-40 mg or equivalent PO or IV daily for 1-4 days), ibrutinib (560 mg PO daily), or acalabrutinib (100 mg PO twice daily), administered after leukapheresis and completed ≤5 days before starting conditioning chemotherapy; PET-CT was required after the transition period. The primary endpoint was objective response rate (ORR [complete response (CR) + partial response]). Key secondary endpoints were duration of response (DOR), progression-free survival (PFS), OS, frequency of adverse events (AEs), levels of CAR T cells in the blood, and levels of interleukins in the serum. Efficacy and safety analyses included all patients who received CD19 CAR-T cell therapy.

Key inclusion criteria included R/R MCL, defined as disease progression after or failure to demonstrate CR or PR to the last regimen; one to five prior lines of therapy, which must include chemotherapy containing anthracyclines or bendamustine and anti-CD20 monoclonal antibody therapy and ibrutinib or acalabrutinib; ≥ 1 measurable lesion; age ≥ 18 years; ECOG of 0 or 1; and adequate bone marrow, kidney, liver, lung, and heart function. Key exclusion criteria included prior autologous stem cell transplantation (auto-SCT); prior CD19-targeted therapy; prior CAR T-cell therapy; clinically relevant infection; and history of CNS involvement or current CNS involvement in MCL or other CNS disorders.

A total of 68 patients received CD19 CAR-T cell therapy. Updated safety (68 patients) and efficacy (60 patients) results are presented here, with a median follow-up of 12.3 months [range, 7.0-32.3]. A total of 28 patients (47%) had a follow-up of ≥24 months. The median time to initial response was 1.0 month [range, 0.8-3.1] and the median time to complete response was 3.0 months [range, 0.9-9.3]. A total of 24 patients (40%) converted from PR/SD to CR, including 21 (35%) from PR to CR and 3 patients (5%) from SD to CR.

The median age was 65 years (range, 38-79) and 39 (57%) patients were male. One hundred percent (100%) of patients had an ECOG score of 0/1, 25% had blastoid morphology, 85% had stage IV disease, 56% had intermediate/high risk MIPI, 81% received 3 or more prior therapies, with a median of 3 (range 1-5) prior therapies, 99% received prior anthracycline or bendamustine, 100% received prior anti-CD20 monoclonal antibody, and 100% received prior BTKi (85% ibrutinib, 24% acalabrutinib, and 9% both). Forty-three (43%) patients relapsed after autologous SCT, 56% were refractory to ibrutinib, and 12% were refractory to acalabrutinib. In 34/49 patients with available data, Ki-67 index was ≥50%. Twenty-five patients (37%) received transition therapy (21% ibrutinib, 7% acalabrutinib, 18% dexamethasone, 3% methylprednisolone, 9% BTKi and steroids, 6% ibrutinib and steroids, 3% acalabrutinib and steroids); 23/25 patients had post-transition PET-CT to document measurable disease before CD19 CAR-T cell infusion (20/23 SPD ^mm2 increase since screening; 3/23 SPD ^mm2 slightly decreased since screening).

High ORR was observed in both efficacy-evaluable and ITT patients. ORR was 95% consistent; CR was 90% consistent. The ORR assessed by the investigators in 60 efficacy-evaluable patients was 88% (95% CI, 77%-95%), with a CR rate of 70% (95% CI, 57%-81%) and a PR rate of 18% (95% CI, 10%-30%). The ORR in 60 efficacy-evaluable patients assessed by IRRC was 93% (95% CI, 84%-98%), with a CR rate of 67% (95% CI, 53% - 78%) and a PR rate of 27% (95% CI, 16%-40%). ORRs were consistent across key subgroups (age, MCL morphology, Ki-67 index, disease stage, simplified MIPI, use of steroids to manage AEs, use of tocilizumab, and use of transitional therapy). The investigator-assessed ORR in ITT patients was 80% (95% CI, 69%-88%), with a CR rate of 59% (95% CI, 47%-71%) and a PR rate of 20% (95% CI, 12%-31%). The ORR in ITT patients assessed by IRRC was 85% (95% CI, 75%-92%), with a CR rate of 59% (95% CI, 47%-71%) and a PR rate of 26% (95% CI, 16%-37%).

Median DOR was not reached after a median follow-up of 12.3 months. Fifty-seven percent (57%) of all patients and 78% of CR patients remained in remission. The first 28 patients treated had a median follow-up of 27.0 months (range 25.3-32.3), of which 43% remained in remission without additional therapy. Median PFS and median OS were not reached after a median follow-up of 12.3 months. The 12-month PFS rate (95% CI) was 61% (45%-74%). The 12-month OS rate (95% CI) was 83% (71%-91%).

More than 35% of patients had treatment-emergent adverse events (0% grade 1; 1% grade 2; 16% grade 3; 76% grade 4 and 3% grade 5). The most common grade ≥3 AEs (≥20% of patients) were neutropenia (69%, grade 4), thrombocytopenia (35%, grade 4), anemia (50%, grade 3), and hypophosphatemia (22%, grade 3). No patients died from interleukin release syndrome (CRS). Grade ≥3 CRS assessed by Lee DW et al., Blood. 2014, 124:188 was reported in 15% of patients. The most common symptoms of any grade CRS were hypotension (51%), hypoxia (34%), and fever (91%). Adverse event management included tocilizumab (59%) and corticosteroids (22%). The median time to onset was 2 days (range 1-13), the median duration was 11 days, and 62/62 (100%) patients with any grade CRS had a resolved event.

Neurological events (NE) of any grade were reported in 63% of patients (31% had grade ≥3 NE) and included encephalopathy (31%), confusional state (21%), and tremors (35%). No patients died from neurological events. One patient had grade 4 cerebral edema, which completely resolved with invasive multicenter therapy including ventriculostomy and IV rabbit antithymocyte globulin (ATG). All CRS events and the majority of NE (37/43 patients) were reversible. The median time to onset and duration of NE were 7 days (range 1-32) and 12 days, respectively.

Higher peak levels of CAR T cells were associated with responders compared with nonresponders (objective response). At week 4, higher peak levels of CAR T cells were associated with negative MRD compared with positive MRD. The median time to peak anti-CD19 CAR T cell levels after CD19 CAR-T cell infusion was 15 days (range 8-31). Anti-CD19 CAR T cells were detectable at 24 months in the majority of patients (6/10 [60%]) with evaluable samples. Expansion correlated with response and MRD status. Expansion was greater in patients with grade ≥3 relative to ≤2 CRS and neurologic events.

Several correlations were observed between peak serum biomarker levels and toxicity. Analytes associated with grade ≥3 CRS included IL-15, IL-2 Rα, IL-6, TNFα, GM-CSF, ferritin, IL-10, IL-8, MIP-1a, MIP-1b, granzyme A, granzyme B, and perforin. Analytes associated with grade ≥3 neurologic events included IL-2, IL-1 Ra, IL-6, TNFα, GM-CSF, IL-12p40, IFN-γ, IL-10, MCP-4, MIP-1b, and granzyme B. And analytes associated with both grade ≥3 CRS and neurologic events included IL-6, TNFα, GM-CSF, IL-10, MIP-1b, and granzyme B.

The CD19 CAR-T cell therapy described herein, administered as a single infusion, demonstrated a high rate of durable responses in R/R MCL. In patients who had previously failed BTKi, 93% ORR, including a 67% CR rate, was the highest reported rate of disease control. Of the initial 28 patients treated, 43% remained in remission after a follow-up period of ≥24 months. The safety profile was consistent with that reported in previous studies of anti-CD19 CAR T cell therapy in aggressive NHL. There were no deaths due to CRS or neurologic events; most symptoms developed early in treatment and were generally reversible. Efficacy, reliable and rapid manufacturing, and manageable toxicities establish a role for the CD19 CAR-T cell therapy described herein in the treatment of patients with R/R MCL with unmet medical needs. Example 4

This example provides additional analysis of the clinical study described above. Eligible patients were aged ≥18 years, had MCL confirmed pathologically by overexpression of cyclin D1 and/or the presence of t(11:14), and were relapsed/refractory to 1-5 prior regimens for MCL. Prior therapy must have included chemotherapy containing anthracyclines or bendamustine, an anti-CD20 monoclonal antibody, and ibrutinib or acalabrutinib. All patients received prior BTKi. Although patients had to have prior BTKi therapy, it was not required to be the last line of therapy prior to study entry, and patients were not required to be refractory to BTKi therapy. Eligible patients had an absolute lymphocyte count ≥100 cells/μL. Patients who underwent autologous SCT or had prior CD19-targeted therapy or allogeneic SCT within 6 weeks of CD19 CAR-T infusion were excluded. All patients underwent leukapheresis to obtain cells for CD19 CAR-T cell therapy manufacturing. Patients received transitional therapy as appropriate, which included dexamethasone (20-40 mg or equivalent PO or IV daily for 1-4 days), ibrutinib (560 mg oral (PO) daily), or acalabrutinib (100 mg PO twice daily). The manufacturing process was modified from that of akilotrimazole to remove circulating lymphoma cells by positive enrichment of CD4 ⁺ /CD8 ⁺ cells. This product is referred to herein as "CAR T cells." This product can also be identified as KTE-X19. Conditioning chemotherapy with fludarabine (30 mg/m ² per day) and cyclophosphamide (500 mg/m ² per day) was administered on days -5, -4, and -3, followed by a single intravenous infusion of 2×10 ⁶ CAR T cells/kg of CD19 CAR-T cells on day 0. More details on the patient's treatment can be found in Example 2.

The objectives of this study were twofold. First, to compare the pharmacological profile of the CAR T product in lower- and higher-risk patients, defined by TP53 (tumor protein p53) gene mutation status and Ki-67 tumor proliferation marker, in the clinical trial ZUMA-2. Patients with high-risk MCL features, including tumor protein p53 gene (TP53) mutations and high Ki-67 proliferation markers, typically have a poor prognosis under current standard therapy. Cheah CY et al., J Clin Oncol. 2016;34:1256-1269. Lower-risk patients in this analysis had a Ki-67 proliferation index <50% (by central assessment) or had wild-type TP53; higher-risk patients had Ki-67 ≥50% or had TP53 mutations by next-generation sequencing. In the primary efficacy analysis of ZUMA-2 (N=60), the ORR was 93% (67% CR) after a median follow-up period of 12.3 months. 57% and 78% of all CR patients had a durable response. In ZUMA-2, ORRs were generally comparable between lower-risk and higher-risk patients, including those with a Ki-67 proliferation index < or ≥50% and those with unmutated versus mutated TP53. Wang M et al., New Engl J Med. 2020;382:1331-1342.

The secondary objective was to characterize the pharmacodynamic profile in patients who achieved early (Day 28) minimal residual disease (MRD)-negative status and in patients with Grade 4 neurotoxicity. In a previous analysis of the ZUMA-2 results, CAR T cell levels in the blood, measured by peak and area under the curve (AUC) from Day 0 to Day 28, were associated with ORR (including undetectable MRD) and Grade ≥3 CRS and neurologic events. Wang M et al., New Engl J Med. 2020;382:1331-1342. In that analysis, CRS and neurologic events were primarily reversible (N=68 patients treated): 15% had Grade ≥3 CRS; 31% had Grade ≥3 neurologic events; and two had Grade 5 AEs (one of which was CAR T product-related). MRD was assessed by next-generation sequencing ( ^10-5 sensitivity) as previously reported. ^Wang M et al., New Engl J Med. 2020;382:1331-1342.

This update reports pharmacology data for all 68 patients treated with CAR T cells in ZUMA-2. Product properties, CAR T cell levels in blood, and interleukin levels in serum and their association with clinical outcomes were analyzed by using previously described methods. Locke FL et al., Mol Ther. 2017;25:285-295. Correlations between subgroup outcomes and CAR T cell and interleukin levels were measured using the Wilcoxon rank-sum test. P values were not adjusted for multiple testing.

CAR T cell product properties were generally comparable across prognostic groups defined by the Ki-67 proliferation marker and TP53 mutation status, with a tendency toward a more differentiated phenotype in the high Ki-67 subgroup and a tendency toward a CD4-based phenotype in patients with TP53 mutations ( Table 8 ).

Table 8 Median ( range ) Patients treateda ⁽ n = 65) Ki-67 proliferation marker TP53 ＜50% (n = 14) ≥50% (n = 34) Mutation (n = 6) No mutation (n = 30) CD4/CD8 Ratio 0.7 (0.04, 3.7) 0.8 (0.4, 1.7) 0.7 (0.04, 3.7) 1.2 (0.7, 3.7) 0.7 (0.04, 1.9) Naive T cells, % 24.5 (0.3, 80.7) 30.4 (11.0, 57.0) 20.1 (0.3, 68.8) 23.0 (11.8, 46.5) 25.2 (0.3, 78.1) Central memory T cells, % 12.8 (2.3, 51.6) 10.1 (8.4, 45.0) 12.0 (2.3, 51.6) 13.2 (6.0, 51.6) 10.2 (2.3, 45.0) Effector memory T cells, % 24.5 (0.8, 70.3) 19.4 (6.3, 56.1) 29.1 (5.8, 70.3) 25.9 (7.0, 38.2) 29.4 (2.2, 70.3 Effector T cells, % 28.7 (2.8, 65.2) 23.7 (11.5, 49.30) 32.4 (2.8, 65.2) 29.1 (2.8, 44.7) 29.1 (8.4, 54.5) ^aProduct characterization data were available for 65 of the 68 patients treated. Product characterization data were available for the following patients: Ki-67 data were available for 48 of the 49 patients and TP53 mutation data were available for all 36 patients. TP53, tumor protein p53 gene

CAR T cell expansion was also comparable in groups with different prognostic factors defined by Ki-67 proliferation index and TP53 mutation status. Peak levels and AUC of CAR T cells in the blood after administration were comparable in patients with wild-type versus mutant TP53 or Ki-67 proliferation index <50% versus ≥50%, which is consistent with comparable efficacy in these subgroups. The primary endpoint of objective response rate (ORR) in patients is shown in Table 9. The median time to response was 28 days (range: 24 to 92 days), with a median follow-up period of 12.3 months. 28 patients had a potential follow-up period of ≥24 months and 12 of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

The ORR was 100% vs. 94% in patients with a Ki-67 proliferation index <50% vs. ≥50%, and the CR rate was 64% vs. 78% in patients with a Ki-67 proliferation index <50% vs. ≥50%. Table 9. The number of patients for whom data on the Ki-67 proliferation index were available was 49.

Table 9 ORR (95% CI) , % CR rate (95% CI) , % Ki-67 PI ＜ 50% 100 (77 - 100) 64 (35 - 87) Ki-67 PI ≥ 50% 94 (79 - 99) 78 (60 - 91)

In patients with wild-type vs mutant TP53, the ORR was 100% in both groups, and the CR rate was 67% vs 100% in wild-type vs mutant TP53. Table 10. The number of patients for whom data for TP53 were available was 36. All six patients with TP53 mutations and all 30 patients without mutations responded. Of the six patients with TP53 mutations, three had grade ≥3 neurotoxicity and two had grade ≥3 CRS.

Table 10 ORR (95% CI) , % CR Rate (95% CI) ， % TP53 mutation 100 (54 - 100) 100 (54 - 100) TP53 no mutation 100 (88 - 100) 67 (47 - 83)

Up to 44 biomarkers were measured in serum before treatment, on day 0, and at multiple time points after CAR T cell infusion until day 28, including IL (interleukin); INF-γ (interferon gamma), MCP-1 (monocytic cytokine-1), IL-2Rα (IL-2 receptor α), sPD-L1 (soluble planned death-ligand 1), and sVCAM (soluble vascular cell adhesion molecule). The pharmacodynamic profiles of the two prognostic groups with Ki-67 proliferation index <50% vs. ≥50% were comparable in terms of proliferative (IL-15, IL-2), inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1), immunoregulatory (IFN-γ, IL-10), chemokines (IL-8 and MCP-1), and effector cytokines (granzyme B). In addition, proliferative (IL-15, IL-2) and inflammatory (IL-6, IL-2Rα, sPD-L1, and VCAM-1) cytokines tended to be increased in patients with mutant TP53 relative to those with wild-type TP53. Figures 1A-1F.

Peak levels of selected interleukins in serum were also increased in patients who achieved MRD-negative status. MRD was analyzed in 29 of 68 patients (43%); 24 of these patients (83% [19 patients had a complete response and 5 patients had a partial response]) were MRD-negative one month after CAR T cell administration. One month after CAR T cell administration, median peak levels of interferon (IFN)-γ and interleukin (IL)-6 were increased in MRD-negative patients (n = 24/29) relative to MRD-positive patients (n = 5/29) and IL-2 tended to increase. Peak levels of interleukins in serum were reached within 7 days of treatment. Consistent trends were seen for PD-L1 and granzyme B. Increased peak CAR T cell counts measured within 14 days of treatment were also seen in patients who were MRD-negative at 1 month. Figures 2A-2I.

Six patients experienced grade 4 neurologic events, including one with cerebral edema. Three patients also had grade 4 CRS. Patients with grade 4 neurologic events showed increased peak levels of proinflammatory serum biomarkers (e.g., IFNγ, MCP-1, TNF-α, IL-2, and IL-6) compared to patients without neurologic events.

Brain edema resolved completely after aggressive multicenter therapy. Wang M et al., New Engl J Med. 2020;382:1331-1342. This patient had the highest expansion of CAR T cells and peak serum levels of IL-2; multiple interleukins were elevated several-fold in this patient compared with medians in other studies/ZUMA-2 patients. Table 11 .

Table 11 Patients with cerebral edema Other ZUMA-2 patients (n=67), median (IQR) Baseline (Day 0) Peak (after administration of CAR T cells) Baseline (Day 0) Peak (after administration of CAR T cells) CAR T cell content, cells/μl 0 431.3 0 83.1 (17.2 - 264.3) ^a IFN-γ, pg/mL 7.5 584.4 7.5 (7.5 - 17.7) 411.2 (144.8 - 1876) MCP-1, pg/mL 462.6 1500 882.9 (557.2 - 1164.8) 1084.3 (804.2 - 1500) TNFα, pg/mL 1.9 10.4 5.7 (3.2 - 10.6) 9.5 (5.5 - 23.2) sVCAM-1, ng/mL 527.5 1659.7 1195.9 (791.7 - 2533.1) 1900.7 (1032.4 - 3646.7) IL-2, ng/mL 0.9 16.7 0.9 (0.9 - 0.9) 6.0 (3.0 - 14.4) IL-6, pg/mL 1.6 159.5 1.6 (1.6 - 6.4) 87.9 (12.9 - 879.1) CRP, mg/L 6.8 18.2 30.5 (15.1 - 63.0) 119.4 (54.6 - 173.8) Ferritin, ng/mL 606.3 824.2 502.4 (273.5 - 877.7) 1265 (597.8 - 2970.1) IL-15, ng/mL 29.1 56.1 33.2 (25.4 - 48) 38.4 (29.7 - 61.7) ^a Data were available for 66 patients.

CAR T cell pharmacokinetic and pharmacodynamic profiles were comparable in MCL patient groups with different prognostic marker status associated with lower and higher risk (defined by Ki-67 and mutant TP53), consistent with comparable clinical response rates. Levels of proinflammatory markers tended to be higher in patients with mutant TP53.

The pharmacodynamic profile of CAR T cell administration was associated with efficacy (MRD status at 1 month) and grade 4 treatment-emergent neurologic events. Patients who developed brain edema had the highest peak CAR T cell levels and serum IL-2, as well as elevated pro-inflammatory markers after treatment. Example 5

A Phase 2, single-arm clinical study of CD19-directed, genetically engineered autologous T cell immunotherapy in patients with relapsed or refractory mantle cell lymphoma (MCL) who have received one or more prior therapies, which may include anti-CD20 antibodies, chemotherapy containing anthracyclines or bendamustine, and/or bruton's tyrosine kinase inhibitors (BTKi), such as ibrutinib or acalabrutinib. Eligible patients also have disease progression after their last therapy or disease that is refractory to their most recent therapy. The study excluded patients with active or severe infection, previous allogeneic hematopoietic stem cell transplantation (HSCT), detectable cerebrospinal fluid malignancies or brain metastases, and central nervous system (CNS) lymphoma or any history of CNS disorders.

Peripheral blood mononuclear cells are obtained from the patient by a leukapheresis procedure. The monocytes are enriched for T cells by selecting for CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector containing the FMC63-28Z CAR, a chimeric antigen receptor (CAR) comprising an anti-CD19 single chain variable fragment (scFv), CD28, and CD3-ζ domains. Without being bound by any hypothesis, the selection for CD4+ and CD8+ cells reduces the potential for circulating CD19-expressing tumor cells to be selected in the patient's leukapheresis material during the ex vivo manufacturing process. The T cell product of this process is identified as KTE-X19. Anti-CD19 CAR T cells were expanded, washed, made into suspension, and cryopreserved. Prior to anti-CD19 CAR T cell therapy, patients were treated with a lymphodepleting chemotherapy regimen consisting of 500 mg/m ² cyclophosphamide intravenously and 30 mg/m ² fludarabine intravenously on the fifth, fourth, and third days before T cell infusion; patients also received acetaminophen and diphenhydramine or another H1-antihistamine approximately 30 to 60 minutes before anti-CD19 CAR T cell infusion. Prophylactic use of systemic corticosteroids was avoided because they may interfere with the activity of CAR T cells.

The target dose was 2×10 ⁶ CAR-positive live T cells or anti-CD19 CAR T cells per kg of body weight, with a maximum of 2×10 ⁸ anti-CD19 CAR T cells (for patients over 100 kg). 68 patients received a single infusion of anti-CD19 CAR T cells (by gravity or peristaltic pump, approximately 30 minutes), and 60 of these patients were followed for at least 6 months after the 4-week disease assessment to be evaluable for efficacy. 56 patients received 2×10 ⁶ anti-CD19 CAR T cells/kg; 1 patient received a dose of 1×10 ⁶ anti-CD19 CAR T cells/kg, 1 patient received a dose of 1.6×10 ⁶ anti-CD19 CAR T cells/kg, 2 patients received a dose of 1.8×10 ⁶ anti-CD19 CAR T cells/kg, and 2 patients received a dose of 1.9×10 ⁶ anti-CD19 CAR T cells/kg. Of these 60 patients, the median age was 65 years (range: 38 to 79 years), 51 were male, and 56 were white. 50 patients had stage IV disease. Based on the simplified international prognostic index for mantle cell lymphoma (s-MIPI), 25 patients were classified as low risk, 25 patients were classified as intermediate risk, 8 patients were classified as high risk, and 2 patients had unknown risk status. Twenty patients had a baseline bone marrow examination per protocol; of these patients, 10 were negative, 8 were positive, and 2 were indeterminate. The median number of prior therapies among all 60 efficacy-evaluable patients was 3 (range: two to five). Twenty-six patients relapsed after autologous HSCT or were refractory to autologous HSCT. Twenty-one patients relapsed after their last therapy for MCL, and 36 patients were refractory to their last therapy for MCL. Fourteen patients had blastoid MCL. Twenty-one patients received transitional therapy after leukapheresis and before infusion of anti-CD19 CAR T cells. Nineteen were treated with BTKi, 14 with corticosteroids, and six with both BTKi and corticosteroids. Fifty-three patients received a lymphodepleting chemotherapy regimen of 500 mg/ ^m2 cyclophosphamide intravenously and 30 mg/ ^m2 fludarabine intravenously, both given daily on the fifth, fourth, and third days before anti-CD19 CAR T therapy (day 0). The remaining seven patients received the same dose of lymphodepleting chemotherapy 4 or more days before CAR T therapy. The primary endpoint of objective response rate (ORR) among patients is shown in Table 12. The median time to response was 28 days (range: 24 to 92 days) with a median follow-up period of 12.3 months. Twenty-eight patients had a potential follow-up period of ≥24 months and twelve of these patients remained in remission. Efficacy was established based on complete response and duration of response (DOR).

Table 12 Efficacy evaluable patients N = 60 Leukocyte apheresis patients N = 74 Response rate Objective response rate (ORR) [95% CI] 52 [75, 94] 59 [69, 88] Complete remission (CR) rate [95% CI] 37 [48, 74] 41 [43, 67] Partial response (PR) rate [95% CI] 15 [15, 38] 18 [15, 36] Duration of response (DOR) ^a Median months [95% CI] Range months NR [8.6, NE] 0.0b, 29.2b NR [11.8, NE] 0.0b, 29.2b DOR, median [95% CI] months range, with CR as the best response NR [13.6, NE] 1.9b, 29.2b NR [13.6, NE] 0.0b, 29.2b DOR, median months [95% CI] range of months, with PR as the best response 2.2 [1.5, 5.1] 0.0b, 22.1b 4.2 [1.5, 5.1] 0.0b, 22.1b DOR median tracking period, month 8.6 8.1 CI, confidence interval; NE, not estimable; NR, not reached; PR, partial response. a. Among all responders. DOR was measured from the date of first objective response to the date of progression or death. b. Censored values. CRS (interleukin-1 release syndrome) was observed in 75 of 82 patients, including ≥ Grade 3 (Lee Grading System 1) CRS in 15 of 82 patients. The median time to onset of CRS was 3 days (range: 1 to 13 days) and the median duration of CRS was 10 days (range: 1 to 50 days). Among patients with CRS, key findings (i.e., findings occurring in >10% of patients) included fever (99% of patients), hypotension (60% of patients), hypoxia (37% of patients), chills (33% of patients), tachycardia (37% of patients), headache (24% of patients), fatigue (19% of patients), nausea (13% of patients), increased alanine aminotransferase (13% of patients), increased aspartate aminotransferase (12% of patients), and diarrhea (11% of patients). Serious events associated with CRS included hypotension, fever, hypoxia, acute renal injury, and tachycardia. In response to CRS, patients may receive tocilizumab and/or corticosteroids as indicated in Table 13. Table 13 CRS Level ^a Tocilizumab Corticosteroids Grade 1 : Symptoms requiring symptomatic treatment only (e.g., fever, nausea, fatigue, headache, myalgia, malaise). If no improvement after 24 hours, give 8 mg/kg ^tocilizumab intravenously over 1 hour (not to exceed 800 mg). (This may not be appropriate for other cancers) Not applicable. Grade 2 Symptoms requiring and responsive to moderate intervention. Oxygen requirement less than 40% _FiO2 or hypotension responsive to fluids or low doses of one vasopressor or Grade 2 organ ^toxicityb . Administer 8 mg/kg tosilimab (not to exceed 800 mg) intravenously over 1 hour. Repeat tosilimab every 8 hours as needed if unresponsive to intravenous fluids or increased oxygenation. Limit to a maximum of 3 doses in a 24-hour period; if signs and symptoms of CRS do not improve clinically, give a maximum of 4 doses total. If improved, discontinue tosilimab. If no improvement within 24 hours of starting tocilizumab, manage according to Grade 3. If improvement, taper corticosteroids. Grade 3 Symptoms requiring and responding to aggressive intervention. Oxygen requirement greater than or equal to 40% _FiO2 or hypotension requiring high-dose or multiple vasopressors or Grade 3 organ toxicity or Grade 4 transaminase elevations. According to Level 2 Administer 1 mg/kg methylprednisolone or equivalent dexamethasone (e.g., 10 mg IV every 6 hours) twice daily until Grade 1, then taper corticosteroids. If improvement, manage as for Grade 2. If no improvement, manage as for Grade 4. Grade 4 Life-threatening symptoms. Respiratory support or continuous venovenous hemodialysis (CVVHD) required, or grade 4 organ toxicity (excluding elevated transaminases). According to Level 2 Administer 1000 mg methylprednisolone IV daily for 3 days. If improvement, taper corticosteroids and manage as Grade 3. If no improvement, consider another immunosuppressant. a. Lee DW et al. (2014). Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 Jul 10; 124(2): 188-195. b. See Table 14 for management of neurotoxicity. c. See tocilizumab prescribing information for details

Neurological events were observed in 53 patients, of whom 20 experienced grade 3 or higher (severe or life-threatening) adverse reactions. The median time to neurological onset was 6 days (range: 1 to 32 days). Neurological events resolved in 52 of 66 patients, with a median duration of 21 days (range: 2 to 454 days). Three patients had ongoing neurological events at the time of death, including 1 patient with severe encephalopathy. The remaining unresolved neurological events were grade 1 or 2. 54 patients experienced CRS due to neurological episodes. Five patients did not experience CRS with neurological events and eight patients had neurological events after resolution of CRS. 56 patients experienced initial CRS or neurological events within 7 days after infusion of anti-CD19 CAR T cells.

The most common neurological events (occurring in >10% of patients) included encephalopathy (51% of patients), headache (35% of patients), tremors (38 of patients), aphasia (23% of patients), and delirium (16% of patients). Severe events occurred after treatment, including encephalopathy, aphasia, and seizures. Some adverse reactions observed in at least 10% of treated patients included: blood and lymphatic system disorders (coagulopathy, cardiac disorders, tachycardia, bradycardia, nonventricular arrhythmias); gastrointestinal disorders (nausea, constipation, diarrhea, abdominal pain, oral pain, vomiting, dysphagia); general disorders and administration site symptoms (fever, fatigue, chills, edema, pain); immune system disorders (interleukin-1 release syndrome , hypogammaglobulinemia); infections and infective disorders (infections-pathogen unspecified, viral infections, bacterial infections); metabolic and nutritional disorders (decreased appetite), musculoskeletal and connective tissue disorders (musculoskeletal pain, motor dysfunction); nervous system disorders (encephalopathy, tremors; headache, aphasia, vertigo, neuropathy); psychiatric disorders (insomnia, delirium, anxiety); renal and urinary disorders (renal insufficiency, decreased urine output); respiratory, thoracic and diaphragmatic disorders (hypoxia, cough, dyspnea, pleural effusion); skin and subcutaneous tissue disorders (rash); and vascular disorders (hypotension, hypertension, thrombosis). Patients experiencing Grade 2 or higher neurotoxicity may be treated according to the indications shown in Table 14 .

Table 14 Graded ^Assessmenta Concurrent CRS No concurrent CRS Level 2 Administer tocilizumab to manage Grade 2 CRS according to Table 13. If no improvement occurs within 24 hours after starting tocilizumab, administer 10 mg dexamethasone intravenously every 6 hours until the event is Grade 1 or less, followed by a gradual taper of corticosteroids. If still no improvement, manage as for Grade 3. Administer 10 mg dexamethasone intravenously every 6 hours until the event is grade 1 or less, followed by a gradual taper of corticosteroids. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 3 Administer tosilimab to manage Grade 2 CRS according to Table 13. In addition, administer 10 mg dexamethasone intravenously with the first dose of tosilimab and repeat dexamethasone doses every 6 hours. Continue dexamethasone until events are Grade 1 or less, then taper corticosteroids. If improved, discontinue tosilimab and manage as for Grade 2. If still no improvement, manage as for Grade 4 (or less). Administer 10 mg dexamethasone intravenously every 6 hours. Continue dexamethasone until events are Grade 1 or less, then taper corticosteroids. If no improvement, manage as for Grade 4. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. Level 4 Administer tocilizumab to manage Grade 2 CRS according to Table 13. Administer 1000 mg methylprednisolone IV daily with first dose of tocilizumab and continue 1000 mg methylprednisolone IV daily for 2 additional days. If improved, manage as for Grade 3. If not improved, consider another immunosuppressive agent. Administer 1000 mg methylprazosin IV daily for 3 days. If improved, manage as for grade 3. If not improved, consider another immunosuppressant. Consider non-sedative antiepileptic drugs (eg, levetiracetam) to prevent seizures. a. Severity is based on Common Adverse Event Terminology criteria.

Following infusion of anti-CD19 CAR T cells, pharmacodynamic response is assessed at four-week intervals by measuring transient increases in interleukins, chemokines, and other molecules in the blood. Levels of IL-6, IL-8, IL-10, IL-15, TNF-α, IFN-γ, and/or sIL2Rα are analyzed. Peak increases in these interleukin levels are typically observed between 4 and 8 days after infusion, and levels typically return to baseline within 28 days. Expected to be during the B-cell aplasia period. Following infusion, anti-CD19 CAR T cells initially expand, followed by a decline to near baseline levels by 3 months. Peak levels of anti-CD19 CAR T cells occur within the first 7 to 15 days after infusion. Results showed that the level of anti-CD19 CAR T cells in the blood correlated with objective response (i.e., complete remission (CR) or partial remission (PR)). The median peak anti-CD19 CAR T cell level was 102.4 cells/μL (range: 0.2 to 2589.5 cells/μL; n=51) in responders (complete remission and partial remission responders) and 12.0 cells/μL (range: 0.2 to 1364.0 cells/μL, n=8) in non-responders. The median AUC on days 0-28 (AUC _{0 - 28} ) was 1487.0 cells/μL·day (range: 3.8 to 2.77 × 10 ⁴ cells/μL·day; n=51) in patients with objective responses and 169.5 cells/μL·day in nonresponders (range: 1.8 to 1.17 10 × 10 ⁴ cells/μL·day; n=8). The median peak anti-CD19 CAR T cells (24.7 cells/μL) and AUC _{0 - 28} levels (360.4 cells/μL·day) in patients who received neither corticosteroids nor tocilizumab (n=18) were similar to those in patients who received corticosteroids alone (n=2) (peak: 24.2 cells/μL; AUC _{0 -} 28: 367.8 cells/μL·day). In patients who received tocilizumab alone (n=10), the mean peak anti-CD19 CAR T cells were 86.5 cells/μL and the AUC _{0 - 28} levels were 86.5 cells/μL. In patients who received corticosteroids with tocilizumab (n=37), the mean peak anti-CD19 CAR T cell value was 74.1 cells/μL in patients ≥65 years (n=39) and 112.5 cells/μL in patients <65 years ( _n =28). The median anti-CD19 CAR T cell AUC _{0 - 28} value was 876.5 cells/μL· _day in patients _≥65 years and 1640.2 cells/μL·day in patients <65 years. Gender-specific anti _- CD19 CAR There was no significant effect on AUC _{0 - 28} and C _max of T cells. Example 6

MCL patients who progress after BTKi therapy have a median overall survival of only 5.8 months under salvage therapy. ZUMA-2 (ClinicalTrials.gov Identifier: NCT02601313) is a Phase 2 multicenter registered study of patients with R/R MCL after 1-5 prior therapies including BTKi. Patients are administered autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy, prepared and administered as described in Example 5. This anti-CD19 CAR T cell product may be referred to as KTE-X19. In the primary analysis of ZUMA-2 (N = 60), the objective response rate (ORR) of anti-CD19 CAR T cell therapy (median follow-up period of 12.3 months) was 93% (67% complete response [CR] rate). This example describes the pharmacological profile of anti-CD19 CAR T cell therapy prepared as described in Example 5 and a comparative analysis of the efficacy by MCL morphology and prior BTKi exposure (ibrutinib [Ibr] and/or acalabrutinib [Acala]), along with basic product attribute characterization.

Eligible R/R MCL patients underwent leukapheresis and conditioning chemotherapy followed by a single infusion of 2×10 ⁶ anti-CD19 CAR T cells/kg. Product properties (e.g., IFNγ production by anti-CD19 CAR T cells when co-cultured with CD19+ cells), CAR T cell levels in blood, and interleukin levels in serum were assessed using previously described methods (see previous examples). Clinical outcomes were reported in 60 efficacy-evaluable patients; product properties and pharmacology data were reported for all 68 treated patients.

At baseline, 40 patients (59%) had classical MCL, 17 (25%) had blastoid MCL, and 4 (6%) had pleomorphic MCL, as assessed by the investigators. Prior to study entry, 52 patients (76%) had prior Ibr, 10 (15%) had prior Acala, and 6 (9%) had both; 88% had BTKi-refractory disease. In the manufactured anti-CD19 CAR T products, the median (range) CD4+/CD8+ T cell ratios for patients with classical, blastoid, and pleomorphic MCL were 0.7 (0.04-2.8), 0.6 (0.2-1.1), or 0.7 (0.5-2.0), respectively. Product T cell phenotypes (median [range]) included less differentiated CCR7+ T cells (typical 40.0% [2.6-88.8]; blastoid 35.3% [14.3-73.4]; pleomorphic 80.8% [57.3-88.8]) and effector and effector memory CCR7- T cells (typical 59.9% [11.1-97.4]; blastoid 64.8% [26.6-85.7]; pleomorphic 19.2% [11.1 - 42.7]). The median (range) interferon (IFN)-γ levels by coculture in patients with classical, blastoid, or pleomorphic MCL were 6309.5 pg/mL (424.0-20,000), 6510.0 pg/mL (2709.0-18,000), or 7687.5 pg/mL (424.0-12,000), respectively. The median (range) peak CAR T cell levels in patients with classical, blastoid, or pleomorphic MCL were 77.6 cells/μL (0.2-2241.6), 35.0 cells/μL (0.2-2589.5), or 144.9 cells/μL (39.2-431.3), respectively. The ORR/CR rates were 93%/65% in patients with classical MCL, 88%/53% in patients with blastoid MCL, and 100%/75% in patients with pleomorphic MCL. The 12-month survival rates were 86.7%, 67.9%, or 100% in patients with classical, blastoid, or pleomorphic MCL, respectively. Grade ≥3 interleukin release syndrome (CRS) and neurologic events occurred in 15% and 38% of patients with classical MCL, 6% and 8% of patients with blastoid MCL, and 25% and 50% of patients with pleomorphic MCL.

For patients who received prior Ibr, Acala, or both, the median CD4+/CD8+ T cell ratios in the produced anti-CD19 CAR T cell products were 0.7 (range, 0.04-3.7), 0.6 (range, 0.3-1.2), or 1.0 (range, 0.7-1.9), respectively. Product T cell phenotypes (median [range]) included less differentiated CCR7+ T cells (Ibr 39.3% [2.6-86.4]; Acala 42.7% [16.3-88.8]; both 49.5% [14.3 - 83.0]) and CCR7- effector and effector memory T cells (Ibr 60.6 [13.7-97.4]; Acala 57.3% [11.1-83.8]; both 50.6% [17.0 - 85.7]). The median (range) IFN-γ levels by co-culture in patients previously treated with Ibr, Acala, or both were 6496.0 pg/mL (424.0-20,000), 5972.5 pg/mL (2502.0-18,000), or 7985.5 pg/mL (2709.0-12,000), respectively. For patients previously treated with Ibr, Acala, or both, the median (range) peak CAR T cell levels were 95.9 (0.4-2589.5), 13.7 (0.2-182.4), or 115.9 (17.2-1753.6), respectively. The ORR/CR rates were 94%/65% in patients previously treated with Ibr, 80%/40% in patients previously treated with Acala, and 100%/100% in patients previously treated with two BTKi. The 12-month survival rates were 81%, 80%, or 100% in patients previously treated with Ibr, Acala, or both, respectively. Grade ≥3 CRS and neurologic events occurred in 17% and 31% of patients previously treated with Ibr, 10% and 10% of patients previously treated with Acala, and 0 and 67% of patients treated with both BTKi. Although CAR T cell levels were lower after treatment in patients with blastoid morphology or previously treated with Acala alone, all subgroups defined by MCL morphology or prior BTKi benefited from anti-CD19 CAR T cell treatment, as reflected by the similar trends in clinical outcomes. Example 7

This example provides an updated analysis of efficacy, safety, and pharmacology for all patients in ZUMA-2 with a minimum follow-up of 1 year. Eligible R/R MCL patients underwent leukapheresis and conditioning chemotherapy, followed by a single infusion of anti-CD19 CAR T cells (2×10 ⁶ CAR T cells/kg) as described in the previous example. The primary endpoint was ORR (CR+partial response) as assessed by the Lugano classification by an independent review committee. Efficacy data are reported for 60 patients treated with a follow-up of ≥ 1 year; safety data for all 68 patients treated are presented.

The median follow-up period was 17.5 months (range, 12.3-37.6). The ORR was 92% (95% CI, 81.6-97.2), including a CR rate of 67% (95% CI, 53.3-78.3). Of all efficacy-evaluable patients, 48% had a durable response as of the data cutoff. The median duration of response, progression-free survival (PFS), or overall survival was not reached; the 15-month estimates were 58.6% (95% CI, 42.5-71.7), 59.2% (95% CI, 44.6-71.2), or 76.0% (95% CI, 62.8-85.1), respectively. Among patients who achieved a CR, median PFS was not reached (15-month rate, 75.1% [95% CI, 56.8-86.5]); among patients who achieved a partial response, median PFS was 3.1 months (95% CI, 2.3-5.2). Among patients who did not respond, median PFS was 1.1 months (95% CI, 0.9-3.0). The first 28 patients treated had a median follow-up of 32.3 months (range, 30.6-37.6); 39.3% of these patients remained in remission without further therapy.

Common grade ≥3 adverse events were neutropenia (85%), thrombocytopenia (53%), anemia (53%), and infection (34%). Grade ≥3 cytopenia was reported ≥30 days after infusion in 60% of patients. Grade ≥3 interleukin release syndrome occurred in 15% of patients; 59% received tocilizumab for management of CRS. Grade ≥3 neurologic events (NE) were reported in 31% of patients, and 38% received steroids for management of NE. All CRS events and the majority of NE (37/43) resolved. There were no grade 5 CRS events or NE, and no new grade 5 events occurred during the additional follow-up period. There were 2 cases of grade 2 cytomegalovirus infection, 1 case each of grade ≥3 hypogammaglobulinemia and grade ≥3 tumor lysis syndrome, and no cases of Epstein-Barr virus-related lymphoproliferation, replication-competent retrovirus, hemophagocytic lymphohistiocytosis, or anti-CD19 CAR T cell-related secondary cancers.

The median peak CAR T-cell count and median area under the curve (days 0-28) were 98.9 cells/μL (range, 0.2-2565.8) and 1394.9 cells/μL (range, 3.8-27,700) in patients with sustained responses at 12 months, 202.6 cells/μL (range, 1.6-2589.5) and 2312.3 cells/μL (range, 19.0-27,200) in patients with relapse at 12 months, and 0.4 cells/μL (range, 0.2-95.9) and 5.5 cells/μL (range, 1.8-1089.1) in nonresponders. Among the 57 efficacy-evaluable patients with available data, 84% had detectable B cells by flow cytometry at baseline. Among patients in sustained response at 12 months, 10 of 26 patients with evaluable samples (38%) had detectable B cells at 3 months, and 10 of 18 (56%) had detectable B cells at 12 months; 5 of 28 evaluable patients had no detectable genetically marked CAR T cells at 12 months (17%). The ZUMA-2 study continues to show significant and durable clinical benefit of anti-CD19 CAR T cell therapy in patients with R/R MCL, with a manageable safety profile. In this patient population lacking curative treatment options, the majority of patients achieved durable CRs, and no new safety signals were reported. Although early CAR T cell expansion was higher in patients who achieved an objective response, patients who subsequently relapsed showed elevated CAR T cell counts, pointing to an alternative mechanism for secondary treatment failure in MCL. Example 8

Although approximately 80-85% of ALL patients achieve durable complete remission (CR) after initial treatment, the remaining 15-20% of relapsed or refractory (R/R) ALL patients have unfavorable outcomes, with a 2-year event-free survival rate of ≤40% in patients with relapsed disease. For adult patients with R/R B-cell lymphoma, anti-CD19 CAR T-cell therapy prepared as described above showed high complete response rates and a manageable safety profile (see previous examples). See, e.g., Example 5). ZUMA-4 (ClinicalTrials.gov Identifier: NCT02625480) is a Phase 1/2 study evaluating this anti-CD19 CAR T-cell therapy in pediatric and adolescent patients with R/R B-cell ALL or NHL. The end-of-phase analysis of ZUMA-4 showed the feasibility of anti-CD19 CAR T-cell therapy for the treatment of pediatric patients with R/R ALL with an optimized dosing and adverse event (AE) management strategy. The phase 2 protocol of ZUMA-4 has been amended to include broader B-cell ALL inclusion criteria, focus on patients with early relapse due to poor response, and add an NHL cohort.

Key B-cell ALL inclusion criteria included age ≤21 years, weight ≥10 kg, and B-cell ALL was primary refractory, relapsed within 18 months of initial diagnosis, R/R after ≥2 lines of systemic therapy, or R/R after allogeneic stem cell transplantation at least 100 days before inclusion. B-cell ALL was also B-progenitor ALL that was R/R after autologous stem cell transplantation at least 100 days before inclusion and had stopped immunosuppressive drugs for ≥4 weeks. Lansky (age <16 years) or Karnofsky (age ≥16 years) performance status was PS ≥80 and weight ≥6 kg. Eligible patients included those with CNS-1 disease, those with CNS-2 disease without clinically evident neurological changes, and those with >5% BM blasts or MRD-positive disease (by flow cytometry or PCR, threshold ^10-4 ). CNS-1 disease was defined by no detectable lymphoblasts in the CSF; CNS-2 disease was defined by detectable disease and a white blood cell count <5 cells/μL in the CSF. CNS-3 disease was defined by ≥5 WBCs/ ^μL in the CSF. Disease burden criteria were modified to also include patients with minimal residual disease-positive disease at inclusion. Patients with Philadelphia chromosome-positive ALL were eligible if intolerant to tyrosine kinase inhibitor therapy or if R/R after ≥2 tyrosine kinase inhibitors. Patients previously treated with blinatumomab were also included. Patients with chronic myeloid leukemia lymphoblastic crisis or clinically significant infections were not eligible. Patients with Burkitt's leukemia/lymphoma were also not eligible.

For B-cell NHL, key inclusion criteria included age <18 years, weight ≥10 kg, histologically confirmed diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS), primary septal large B-cell lymphoma, Burkitt's lymphoma (BL), Burkitt-like lymphoma, or unclassified B-cell lymphoma between DLBCL and BL, with ≥1 measurable lesion. For NHL, disease must be primary refractory, R/R after ≥2 lines of systemic therapy, or R/R after autologous or allogeneic stem cell transplantation at least 100 days prior to inclusion. Patients must have been off immunosuppressive medications for ≥4 weeks. Lansky (age <16 years) or Karnofsky (age ≥16 years) performance status of PS ≥80 and weight ≥6 kg. Patients previously treated with blinatumomab were also included. Patients must have received adequate prior therapy, at least anti-CD20 mAb and anthracycline-containing chemotherapy, and have one or more measurable lesions. Patients with acute graft-versus-host disease or chronic graft-versus-host disease requiring treatment within 4 weeks of enrollment were not eligible. Patients previously treated with CAR T-cell therapy or other genetically modified T-cell therapy were excluded, although patients receiving KTE-X19 in this study were eligible for retreatment. Patients with cardiac lymphoma involvement or requiring acute therapy due to tumor mass effects were also excluded. Additional exclusions for the ALL and NHL groups included: patients with clinically significant infections; patients with acute or chronic GVHD requiring treatment within 4 weeks of enrollment, alendronate (or other anti-CD52 antibodies) within the past 6 months, clofarabine or cladribine within the past 3 months, PEG-asparaginase within the past 3 weeks, or donor leukocyte infusion (DLI) within the past 28 days.

Patients with CNS involvement and certain abnormalities were excluded. Patients with CNS-1 disease (no detectable lymphoblasts in cerebrospinal fluid), CNS-2 disease (detectable disease but cerebrospinal fluid white blood cell count <5 cells/μL) with lymphoblasts and neurological symptoms without clinically evident neurological changes who had been previously treated with blinatumomab could be included in the ALL and NHL groups. Patients with lymphoblastoid cells and neurological symptoms, patients with CNS-3 disease (≥5 WBC/μL in CSF) and lymphoblastoid cells with or without neurological symptoms were excluded, patients with any CNS tumor mass by imaging and/or with parameningeal masses, any history of CNS disease or the presence of any CNS disease (such as cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease or any autoimmune disease with CNS involvement, reversible posterior encephalopathy syndrome or brain edema with structural defects, history of stroke or transient ischemic attack in the past 12 months, and epilepsy requiring active anticonvulsant drugs were excluded. Patients previously treated with CD19-directed therapy, except blinatumomab, were excluded.

Patients received conditioning chemotherapy with 25 mg/m ² fludarabine on days -4, -3, and -2 and 900 mg/m ² cyclophosphamide on day -2, followed by a single infusion of anti-CD19 CAR T cells at a target dose of 1×10 ⁶ anti-CD19 CAR T cells/kg on day 0. For ALL, the primary phase 2 objective was to assess anti-CD19 CAR T cell efficacy as assessed by overall CR rate (CR and CR with incomplete hematologic recovery). For NHL, the primary phase 2 objective was to assess anti-CD19 CAR T cell therapy efficacy by objective response rate (CR+partial response). Secondary phase 2 objectives for both ALL and NHL cohorts included safety and tolerability, additional efficacy endpoints, and changes in patient-reported efficacy scores.

The CAR T cell therapy used in this study (also called KTE-X19) described in previous examples, such as Example 5, is an autologous anti-CD19 CAR T cell therapy used to treat R/R mantle cell lymphoma and other R/R hematological malignancies. PBMCs from the blood cell separation product are enriched for T cells by CD4+/CD8+ positive selection to remove malignant cells. The resulting T cells are activated with anti-CD3/anti-CD28 antibodies in the presence of IL-2, retrotransduced to introduce the anti-CAR gene construct (FMC63-28Z CAR) and expanded to the desired dose. The expanded T cells can be frozen for transport and transported back to the patient for infusion. Akiramec is manufactured by different methods as described, for example, in: Park J.H. et al. N Engl J Med. 2018;378(5):449-459; and Lee D.W. et al. Lancet. 2015;385(9967):517-528. In adult patients with R/R B-ALL, KTE-X19 treatment improved CR rate, CRi rate, or safety profile in Phase 1. Shah BD et al., J Clin Oncol. 2019;37(Supplement Abs):7006.

During the DLT assessment period in Phase 1, the starting dose was 2×10 ⁶ anti-CD19 CAR T cells/kg. DLTs were defined as grade 3 non-hematologic AEs lasting >7 days and grade 4 non-hematologic AEs regardless of duration, except as specified in the protocol, or grade 4 hematologic AEs lasting >30 days. Doses of 1×10 ⁶ CAR-T cells in a 68 mL volume or 40 mL volume were also examined. Patients in the 40 mL 1×10 ⁶ group received modified AE management. Based on available data, 1×10 ⁶ cells/kg in 40 mL was used in Phase 2. Phase 1 study results showed that 94% MRD negativity and 73% CR+Cri were observed in pediatric and adolescent patients with R/R B-ALL. Results also showed a manageable AE profile consistent with known toxicities, and a low incidence and severity of NE with optimized dosing and revised safety management. Wayne AS et al., Pediatr Blood Cancer. 2019;66(Suppl):S24.

In Phase 2, patients were screened and underwent leukapheresis, followed by conditioning chemotherapy, starting on Day -4. Transition therapy may be administered after leukapheresis at the investigator's discretion and must have been completed ≥7 days or 5 half-lives prior to conditioning chemotherapy. KTE-X19 was infused on Day 0. The first disease assessment was performed on Day 28. Post-treatment assessments for safety and efficacy were performed at Weeks 2, 4, 2, and 3. Patients were followed every 3 months through Month 18 and every 6 months between Months 24 and 60. Beginning in Year 6, patients returned annually until age 15. A total of 50 R/R ALL patients and 16 R/R NHL patients were enrolled using 40 mL formulation of 1×10 ⁶ KTE-X19 cells/kg. Patients in the current Phase 2 study also include the NHL cohort and the inclusion criteria for R/R B-ALL are broadened to include patients in early first relapse due to poor efficacy and patients with MRD-positive disease. The primary objective is efficacy as assessed by overall CR rate (CR and Cri) for ALL and ORR (CR+PR) for NHL. Secondary objectives include assessment of safety, tolerability, DOR, OS, relapse-free survival (RFS)/progression-free survival (PFS) and patient-reported outcomes (PROs). For ALL, additional secondary objectives include assessment of MRD-negativity rate and allo-SCT rate. For the overall CR rate (ALL group only), the incidence rate and exact two-sided 95% CI will be determined. It will be compared with a response rate of 35% at a one-sided α-level of 0.025 using the exact binomial test. For the MRD-negativity rate (ALL group only), the incidence rate and exact two-sided 95% CI will be determined. If the statistical test for the overall CR rate is significant, the MRD-negativity rate will be compared with a response rate of 30% at a one-sided α-level of 0.025 using the exact binomial test. For DOR and OS, Kaplan-Meier estimates and two-sided 95% CI will be determined. For the allogeneic SCT rate (ALL group only), the mITT-centered incidence rate and exact two-sided 95% CI will be determined. Based on safety, the incidence of AEs will be determined, including all severe, fatal, CTCAE version 4.03 grade ≥3, and treatment-related AEs with onset on or after the infusion day. No specific assumptions will be tested for the NHL cohort. With the planned sample sizes in this cohort, assuming an observed ORR of 63% (10/16 patients), 69% (11/16), 75% (12/16), and 81% (13/16), the lower limits of the 95% exact CIs for the estimated ORR will be 35%, 41%, 48%, and 54%, respectively. Example 9

This example reports Phase 1 results of ZUMA-3 (ClinicalTrials.gov Identifier: NCT02614066) in adults with relapsed/refractory (R/R) B-cell ALL, a Phase 1/2 study evaluating autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that includes CD3ζ and CD28 co-stimulatory domains and is prepared as described in previous examples (CD4+/CD8+ enriched/depleted of malignant cells). This approach to preparing anti-CD19 CAR T cells depleted of cancer cells reduces the likelihood of activation and elimination of anti-CD19 CAR T cells during ex vivo manufacturing. The presence of leukemic blasts in the peripheral blood may limit the number of T cells available to manufacture the CAR T-cell product, which may lead to manufacturing failure. Sabatino M. et al. Blood. 2016;128(22):1227. The anti-CD19 CAR T cell product used in this study has been described in Wang M. et al. N Engl J Med. 2020;382(14):1331-1342 for use in MCL. It is different from the T cell products used in: Sabatino M. et al. Blood. 2016;128(22):1227; Park J.H. et al. N Engl J Med. 2018;378(5):449-459; and Lee D.W. et al. Lancet. 2015;385(9967):517-528. This anti-CD19 CAR T cell product has different product characteristics in terms of T cell phenotype than T cell products made by previously described methods. This anti-CD19 CAR is also referred to as KTE-X19 in this example and elsewhere in this application.

Following fludarabine/cyclophosphamide lymphodepletion, patients received 2, 1, or 0.5 × 10 ⁶ cells/kg of anti-CD19 CAR T cells. The primary endpoint was the rate of dose-limiting toxicity (DLT) within 28 days after CAR T cell infusion. Anti-CD19 CAR T cells were manufactured for 54 enrolled patients and administered to 45 (median age 46 years [range 18-77]). No DLTs occurred in the DLT-evaluable group. Grade ≥3 interleukin release syndrome (CRS) and neurologic events (NE) occurred in 31% and 38% of patients, respectively. To optimize the benefit-risk ratio, modified adverse event (AE) management of CRS and NE was evaluated at 1×10 ⁶ cells/kg anti-CD19 CAR T cells (early steroids for NE and tocilizumab for CRS only). Of the 9 patients treated according to the modified AE management, 33% had grade 3 CRS and 11% had grade 3 NE, with no grade 4/5 NE. The overall complete remission rate was associated with CAR T cell expansion and was 83% in patients treated with 1×10 ⁶ cells/kg and 69% in all patients. Minimal residual disease was undetectable in all responding patients. At a median follow-up of 22.1 months (range, 7.1-36.1), the median DOR was 17.6 months (range, 5.8-17.6) in patients treated with 1×10 ⁶ cells/kg and 14.5 months (range, 5.8-18.1) in all patients. Anti-CD19 CAR T cell therapy provides high response rates and a tolerable safety profile in adults with R/R B-ALL. Phase 2 was conducted at 1×10 ⁶ cells/kg with modified AE management.

patient

Eligible patients were ≥18 years of age with R/R B-cell ALL, defined as refractory to first-line therapy (i.e., primary refractory), relapsed ≤12 months after first remission, relapsed or refractory after ≥2 lines of prior systemic therapy, or relapsed after allogeneic stem cell transplant (SCT). Patients were required to have ≥5% bone marrow blasts, a Eastern Cooperative on Cancer performance status of 0 or 1, and adequate renal, hepatic, and cardiac function. The first six patients enrolled were required to have ≥25% blasts in the bone marrow. For patients who had previously received blinatumomab, CD19 expression of ≥90% of leukemic blasts was required. Patients with Philadelphia chromosome-positive (Ph+) disease, associated extramedullary disease, central nervous system (CNS)-2 disease (with <5 white blood cells/mm3 cerebrospinal fluid [CSF] blasts) without neurologic changes, and patients with Down syndrome were eligible. Patients with CNS-3 disease (with ≥5 white blood cells/mm3 CSF blasts) not associated with neurologic changes and a history of CNS disease were excluded.

Additional eligibility criteria included: Individuals with Philadelphia chromosome (Ph)-positive disease were eligible if they were intolerant to tyrosine kinase inhibitor (TKI) therapy or if they had relapsed/refractory disease despite treatment with ≥2 different TKIs; absolute neutrophilia was not considered necessary unless, in the investigator's opinion, the cytopenia was caused by an underlying leukemia and was potentially reversible with leukemia therapy. cytopenia ≥500/μL; platelet count ≥50,000/μL unless, in the investigator's opinion, the cytopenia is caused by underlying leukemia and may be reversible with leukemia therapy; absolute lymphocyte count ≥100/μL; adequate renal, hepatic, pulmonary, and cardiac function, defined as [creatinine clearance (as estimated by Cockcroft-Gault) ≥60 cc/min; serum alanine aminotransferase/aspartate aminotransferase ≤2.5 upper limit of normal; total bilirubin ≤1.5 mg/dL, except for individuals with Gilbert's syndrome; left ventricular ejection fraction ≥50% as determined by echocardiography, no evidence of pericardial effusion, no New York Heart Association class III or IV functional class, and no clinically significant arrhythmias; no clinically significant pleural effusion; baseline airway oxygen saturation >92%]; females of childbearing age must have had a negative serum or urine pregnancy test; females of childbearing age must have had a negative serum or urine pregnancy test.

Additional exclusion criteria include: The subjects had a history of malignancy other than melanoma, skin cancer, or carcinoma in situ (e.g., cervical, bladder, breast) unless they had been disease-free for ≥3 years; a history of severe allergic reaction to aminoglycosides or any of the agents used in this study; or central nervous system (CNS) abnormalities [presence of CNS-3 disease, defined as detectable cerebrospinal blasts in cerebrospinal fluid (CSF) samples with ≥5 white blood cells (WBC)/mm3 with or without neurologic changes and; presence of CNS-2 disease, defined as detectable cerebrospinal blasts in CSF samples with <5 WBC/mm3 with neurologic changes]. Note: Subjects with CNS-1 (no detectable leukemia in CSF) and subjects with CNS-2 without clinically evident neurologic changes were eligible for the study; history of or presence of any CNS disorder, such as epilepsy, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, any autoimmune disease with CNS involvement, posterior reversible encephalopathy syndrome, or cerebral edema]; history of severe allergic reaction to aminoglycosides or any of the agents used in this study; history of concomitant genetic syndromes related to bone marrow failure; history of clinically significant cardiac disease within 12 months of enrollment; history of symptomatic deep venous embolism or pulmonary embolism within 6 months of enrollment; primary immunodeficiency; known infection with HIV, hepatitis B, or hepatitis C virus. A history of hepatitis B or C was allowed if the viral load was undetectable by quantitative polymerase chain reaction and/or nucleic acid testing; simple urinary tract infection and uncomplicated bacterial pharyngitis were allowed if responsive to aggressive therapy and after consultation with a Kite medical monitor; acute graft-versus-host disease (GVHD) grade II-IV by Glucksberg criteria or acute or chronic GVHD requiring systemic therapy within 4 weeks prior to enrollment; prior medications [salvage systemic therapy (including chemotherapy, TKI for Ph+ disease, and blinatumomab) ≤ 1 blinatumomab; history of Common Adverse Event Terminology Criteria Grade 4 neurologic event or Grade 4 interleukin-1 release syndrome under prior CD19-directed therapy; treatment with alendronate ≤ 6 months prior to enrollment, clofarabine or cladribine ≤ 3 months prior to enrollment, or Patients were treated with PEG-asparaginase ≤ 3 months before enrollment; donor lymphocytes were transfused ≤ 4 weeks before enrollment; any drugs for GVHD and any immunosuppressive antibodies were used 4 weeks before enrollment; at least 3 half-lives had passed since any previous systemic suppressive/stimulatory immune checkpoint molecule therapy before enrollment; pharmacological doses of corticosteroid therapy (> 5 mg/day of prazosin or other corticosteroids of equivalent dose) and other immunosuppressive drugs must be avoided for 1 week before enrollment]; any indwelling lines or drains were present. Omayor reservoirs and dedicated central venous access catheters are permitted; live vaccines ≤ 4 weeks prior to enrollment; pregnant or breastfeeding women of childbearing age, because preparative chemotherapy may have hazardous effects on the fetus or infant; individuals of both sexes of childbearing age who are unwilling to implement birth control from the time of consent until 6 months after completion of anti-CD19 CAR-T cell therapy; individuals who, in the judgment of the investigator, are unlikely to complete all study visits or procedures required by the protocol, or comply with the requirements of study participation [history of autoimmune disease causing end-organ damage or requiring systemic immunosuppression or systemic disease-modifying agents within the last 2 years].

Study Design and Treatment

The Phase 1 objectives were to evaluate the safety of anti-CD19 CAR T cell therapy and determine the optimal Phase 2 dose based on the incidence of dose-limiting toxicity (DLT) and the overall safety profile. DLT was defined as anti-CD19 CAR T cell-related adverse events (AEs) occurring within the first 28 days after infusion of anti-CD19 CAR T cells, including grade 3 non-hematologic AEs lasting >7 days, grade 4 non-hematologic AEs regardless of duration (except pre-specified expected events, such as tumor lysis syndrome), and grade 4 hematologic AEs lasting >30 days, excluding lymphocytopenia ( Table 15 ).

Table 15. Dose-limiting toxicity ● DLT was defined as the following anti-CD19 CAR T cell-related events occurring within the first 28 days after infusion of anti-CD19 CAR T cells: ● Grade 4 hematologic toxicity (excluding lymphocytopenia) lasting more than 30 days and not attributable to an underlying disease ● All anti-CD19 CAR T cell-related grade 3 non-hematologic toxicities lasting >7 days and all anti-CD19 CAR T cell-related grade 4 non-hematologic toxicities regardless of duration were considered DLTs, with the following exceptions: ○ Aphasia/dysphasia or confusion/cognitive impairment that resolved to at least grade 1 or baseline within 2 weeks and to at least baseline within 4 weeks ○ Grade 3 or 4 fever ○ Immediate-onset hypersensitivity reactions (related to anti-CD19 CAR T cell infusion) occurring within 2 hours of anti-CD19 CAR T cell infusion that were reversible to grade 2 or lower within 24 hours of anti-CD19 CAR T cell infusion and standard of care ○ Renal toxicity requiring analysis ≤7 days ○ Intubation to protect the airway, ≤7 days ○ TLS, including findings attributable to TLS (e.g., electrolyte abnormalities, renal function, hyperuricemia) ○ Grade 3 elevations in transaminases, alkaline phosphatases, bilirubin, or other liver function tests, if resolved to ≤ Grade 2 within 14 days ○ Grade 4 transient serum liver enzyme abnormalities, if resolved to ≤ Grade 3 within <72 hours ○ Grade 3 or 4 hypogammaglobulinemia ○ Grade 3 nausea and/or anorexia ○ Adverse events caused by CRS correspond to the total CRS grade assessment used to determine DLT ○ All occurrences of grade 3 CRS lasting >7 days and all occurrences of grade 4 CRS are considered DLTs, except for occurrences of CRS caused by the exceptions listed above CRS, cytokine release syndrome; DLT, dose-limiting toxicity; TLS, tumor lysis syndrome

Patients were initially enrolled at a starting dose of 2×10 ⁶ CAR T cells/kg ( Figure 3 ). Based on the overall safety profile, patients subsequently received 2×10 ⁶ , 1×10 ⁶ , or 0.5×10 ⁶ CAR T cells/kg. At 0.5×10 ⁶ CAR T cells/kg. For patients receiving a lower dose of 0.5×10 ⁶ CAR T cells/kg, two formulations were studied, one with a total volume of 40 mL and the other with a volume of 68 mL. The 40 mL formulation was intended to maintain cell density and cell viability during the freeze/thaw process).

To reduce the risk of interleukin-1 release syndrome (CRS) and neurologic events (NE), the AE management guidelines were modified to limit tocilizumab to the treatment of CRS (and not isolated neurotoxicity) and to initiate corticosteroid therapy at the onset of grade 2 rather than grade 3 NE ( Table 16 ).

Table 16. Original and revised guidelines for the management of neurotoxicity NE level Original Management Guidelines Revised Management Guidelines Level 1 ● Supportive care ● Neurological examination and additional management as clinically indicated ● Supportive care ● Close monitoring of neurological status ● Consider prophylactic anti-epileptic drugs Level 2 Supportive Care and Assessment ● Neurological examination, brain MRI, and CSF evaluation; consider EEG as clinically indicated ● Consider prophylactic antiepileptic medication Supportive Care and Assessment ● Continuous cardiac telemetry and pulse oximetry as indicated ● Continuous neurological examinations, including funduscopy and Glasgow Coma Score, brain MRI, CSF evaluation, EEG; consider neurological society consultation ● Anti-epileptic drugs for epileptic patients Tocilizumab ● For patients with comorbidities (e.g., grade ≥2 CRS), consider 8 mg/kg tocilizumab IV over 1 hour (not to exceed 800 mg) Tosilimab ● For patients with concurrent CRS, give 8 mg/kg tosilimab (not to exceed 800 mg) IV over 1 hour; if unresponsive to IV fluids or increased supplemental oxygen, repeat every 4-6 hours as needed, up to 3 doses in 24 hours ● If patient improves, discontinue tosilimab Corticosteroids ● N/A Corticosteroids ● Dexamethasone 10 mg IV every 6 hours for patients without concurrent CRS ● Dexamethasone 10 mg IV every 6 hours for patients with concurrent CRS who do not improve within 24 hours of starting tocilizumab ● Taper corticosteroids if patient improves Level 3 Supportive care and assessment ● Based on Level 2 ● Monitoring with continuous cardiac telemetry and pulse oximetry Supportive care and assessment ● Management in monitored care or ICU Tosilimab ● Consider 8 mg/kg tosilimab IV over 1 hour (not to exceed 800 mg); repeat every 4-6 hours if symptoms are not stable or not improving Tosilimab ● Based on Grade 2 ● If patient improves, discontinue Tosilimab Corticosteroids ● Consider corticosteroids (eg, dexamethasone 10 mg IV q 6 h or methylprednisolone 1 mg/kg BID) for worsening symptoms despite tocilizumab Corticosteroids ● Dexamethasone 10 mg IV every 6 hours ● If patient improves, taper corticosteroids Level 4 Supportive care and assessment ● Based on Level 2 ● Monitoring with continuous cardiac telemetry and pulse oximetry Supportive care and assessment ● Based on level 3 ● May require ventilator ● Administer immunosuppressive agents if patient does not improve Tosilimab ● If not previously administered, administer tosilimab based on grade 3 Tocilizumab ● Based on level 2 Corticosteroids ● Administer corticosteroids (e.g., methylprednisolone 1 g/d x 3 days, then 250 mg BID x 2 days, then 125 mg BID x 2 days, then 60 mg BID x 2 days) Corticosteroids ● Administer high doses of corticosteroids (e.g., methylprednisolone 1 g/d x 3 days) ● If the patient improves, taper the corticosteroid

Modified AE management guidelines were implemented in a separate cohort of patients treated with 1× ¹⁰⁶ CAR T cells/kg. The Safety Review Team (SRT) continued to review safety and efficacy data and made recommendations for further Phase 1 enrollment and the recommended Phase 2 dose (RP2D) at milestones defined in the protocol and SRT mandate.

Patients underwent leukapheresis at enrollment to achieve a target of 5-10× ¹⁰⁹ mononuclear cells for anti-CD19 CAR T-cell manufacturing. Predefined transitional chemotherapy was recommended after leukapheresis ( Table 17 ), especially for patients with high disease burden at baseline (>25% leukemic blasts in the bone marrow or ≥1,000 blasts/mm3 in the peripheral circulation by local review).

Table 17. Transitional chemotherapy Predefined transitional chemotherapy regimen Weakened VAD Vincristine non-lipidic (IV 1-2 mg once a week) or liposomal (IV 2.25 mg/m ² once a week) and dexamethasone IV or PO once daily 20-40 mg × 3-4 days a week. Doxorubicin 50 mg/m ² IV × 1 (first week only) as needed 6- MP Oral 50-75 mg/m ² per day (take on an empty stomach at bedtime to improve absorption) Hydroxyurea Titrated dose between 15-50 mg/kg per day (rounded to the nearest 500 mg capsule and given continuously as a single daily oral dose) DOMP Dexamethasone 6 mg/m ² divided BID PO (or IV) on days 1-5, vincristine 1.5 mg/m ² IV (maximum dose 2 mg) on day 1, methotrexate 20 mg/m ² PO once a week, 6-MP 50-75 mg/m ² PO once a day Weakened FLAG/FLAG-IDA IV 30 mg/m ² fludarabine on days 1-2, IV 2 g/m ² cytarabine on days 1-2, SC or IV 5 μg/kg G-CSF started on day 3 and may be continued until the day before starting conditioning chemotherapy. With or without IV 6 mg/m ² idarucizumab on days 1-2 Mini -hyper CVAD ( method A and / or B ) Routine A: 150 mg/m ² cyclophosphamide every 12 hours for 3 days, 20 mg/d dexamethasone IV or PO once daily on days 1-4 and 11-14, 2 mg vincristine IV × 1 Routine B: 250 mg/m ² methotrexate IV 24 hours on day 1, 0.5 g/m ² cytarabine IV every 12 hours × 4 doses on days 2 and 3 BID, twice daily; CVAD, cyclophosphamide, vincristine, doxorubicin, and dexamethasone; DOMP, dexamethasone, 6-hydroxypurine, methotrexate, and vincristine; FLAG, fludarabine, high-dose cytarabine, and G-CSF; G-CSF, microglobulin-colony stimulating factor; IDA, idamycin; IV, intravenous; MP, 6-hydroxypurine; PO, oral; SC, subcutaneous; VAD, vincristine, doxorubicin, and dexamethasone.

After clearance from transitional chemotherapy for ≥7 days or 5 half-lives if shorter, patients received a lymphodepleting regimen of 25 mg/ ^m2 fludarabine intravenously (IV) daily on days -4, -3, and -2 and 900 mg/ ^m2 cyclophosphamide IV daily on day -2. On day 0, a single infusion of anti-CD19 CAR T cells was administered.

Effectiveness and Evaluation

The primary phase 1 endpoint was the incidence of DLT in DLT-evaluable patients. Secondary endpoints included safety, investigator-assessed overall response rate (CR+CR with incomplete hematologic recovery [CRi]), duration of response (DOR), relapse-free survival, OS, and rate of undetectable minimal residual disease (MRD) in the bone marrow. Blood levels of CAR T cells and interleukins were exploratory endpoints. AEs, including CRS and NE symptoms, were graded according to the Common AE Terminology Criteria, version 4.03. CRS was graded according to the criteria of Lee et al., Blood. 2014;124(2):188-195. For patients with extramedullary disease, responses were assessed according to the modified International Working Group Response Criteria for Malignant Lymphomas for Extramedullary and CNS Disease. Cheson BD et al J Clin Oncol. 2007;25(5):579-586. Undetectable MRD was assessed centrally using flow cytometry (NeoGenomics, Fort Myers, FL) and was defined as <1 leukemic cell per 10,000 viable cells. Borowitz MJ et al Blood. 2015;126(8):964-971; Bruggemann M. et al Blood Adv. 2017;1(25):2456-2466; and Gupta S. et al Leukemia. 2018;32(6):1370-1379.

Post-infusion hospitalization of ≥7 days is required. Patients were assessed by physical examination, vital sign measurements, and neurologic and laboratory assessments on Days 14 and 28, and at 2 and 3 months. Bone marrow assessments and response assessments were performed on Days 7-14 (as appropriate) and 28, and at 2 and 3 months. For patients undergoing SCT after anti-CD19 CAR T-cell infusion, bone marrow assessments were not required during the first 100 days post-SCT. For patients with baseline CNS-2 disease, CSF collection and analysis were required to confirm CR. Patients were followed up every 3 months through Month 18 for survival and disease status. Patients were followed up every 6 months between Months 24 and 60, and annually through age 15 years for the 3-month assessment after completing treatment. Patients who achieve CR may receive a second infusion of anti-CD19 CAR T cells if remission evolves >3 months later, provided that CD19 expression is preserved and there are no suspected neutralizing antibodies against the CAR.

Blood and serum samples were subjected to biomarker analysis to evaluate predictive pharmacokinetic and pharmacodynamic markers of anti-CD19 CAR T cells. As previously described, droplet digital polymerase chain reaction was used to measure the presence, expansion, and persistence of transduced anti-CD19 CAR+ T cells in blood. Locke FL et al. Mol Ther. 2017;25(1):285-295. Serum was evaluated for markers of interleukins, chemokines, immune effector molecules, and macrophage activation syndrome using previously reported methods. Locke FL et al. Mol Ther. 2017;25(1):285-295.

Statistical analysis

The DLT-evaluable group included the first 3 patients treated at a dose of 2×10 ^6. Safety and efficacy analyses included all patients treated with any dose of anti-CD19 CAR T cells. Kaplan-Meier estimates and two-sided 95% confidence intervals were generated for time-to-event endpoints. DOR was defined as the time from CR to relapse or death without recorded relapse. On the day of transplant, DOR was censored in patients who underwent allogeneic SCT while in remission. OS was defined as the time from anti-CD19 CAR T cell infusion to the date of death from any cause. Data were presented as of April 1, 2019. All statistical analyses were performed in SAS (version 9.4).

result

patient

Between March 9, 2016, and July 12, 2018, 54 patients were enrolled and underwent leukapheresis in phase 1 ( Figure 4 ). Anti-CD19 CAR T-cell product was successfully manufactured for all 54 patients; 1 patient required 2 leukapheresis procedures and 1 patient required 3 procedures for product manufacturing. The median time from leukapheresis to delivery of CD19 CAR-T cells to the study site was 15 days. Five patients discontinued before lymphapheresis due to AEs (n=3; Figure 4 ), withdrawal of consent (n=1), or ineligibility after leukapheresis (n=1). Four additional patients discontinued after lymphapheresis. Three patients did not receive anti-CD19 CAR T cells due to grade 4 sepsis (n=1), initiation of new therapy (n=1), and death due to grade 5 sepsis (n=1). One patient was interrupted before infusion due to deep venous embolism (exclusion criteria) but received anti-CD19 CAR T cells under compassionate use. Forty-five of the 54 patients (83%) received anti-CD19 CAR T cells at these doses: 2× ¹⁰⁶ (n=6), 1× ¹⁰⁶ (n=23), or 0.5× ¹⁰⁶ CAR T cells/kg (n=16). Nine of the 23 patients in the 1× ¹⁰⁶ CAR T cells/kg group were treated according to modified AE management guidelines, which called for early use of steroids for NE and reserved tocilizumab for treatment of CRS only. Forty-four patients received their target dose of anti-CD19 CAR T cells; 1 patient who was selected to receive 1× ¹⁰⁶ anti-CD19 CAR T cells/kg and modified AE management was treated with 0.5× ¹⁰⁶ cells/kg but was included in this analysis at the 1× ¹⁰⁶ dose.

The median age of all treated patients was 46 years (range, 18-77), and 67% received ≥3 lines of prior therapy ( Table 18 ). Prior to enrollment, 16 patients (40%) were primary refractory, 13 (29%) relapsed after SCT, and 21 (47%) received prior blinatumomab. Blinatumomab was the last line of therapy used before study entry in 8 patients (18%), and only 1 achieved a response (CR) to blinatumomab.

Table 18. Baseline characteristics of patients Baseline characteristics N = 45 Age, median (range), years 46 (18 - 77) Male, n (%) 22 (49) ECOG performance status score, n (%) 0 15 (33) 1 29 (64) Missing 1 (2) Philadelphia chromosome positive, n (%) 8 (18) Extramedullary disease, n (%) 4 (9) CNS disease at screening, n (%) CNS-1 42 (93) CNS-2 3 (7) Previous solution, n (%) 1 6 (13) 2 9 (20) ≥3 30 (67) Prior blinatumomab, n (%) 21 (47) Prior inotuzumab, n (%) 6 (13) Refractory, n (%) Primary refractory 16 (36) First relapse after ≤12 months of remission twenty four) Relapse or refractory disease after allogeneic SCT 13 (29) BM blasts during screening, median (range), % 61 (5 - 100) BM blasts at pretreatment after transition period, median (range), % 70 (0 - 97) BM, bone marrow; CNS, central nervous system; ECOG, Eastern Cooperative Oncology Group; SCT, stem cell transplantation

safety

No DLTs were observed in the DLT evaluable set (n=3). Ninety-eight percent of patients experienced grade ≥3 AEs ( Table 19 ). The most common AEs of any grade were pyrexia (89%), hypotension (69%), diarrhea (42%), and chills (42%). Common grade ≥3 AEs (≥20% of patients) were pyrexia (42%), hypotension (40%), thrombocytopenia (33%), anemia (31%), hypophosphatemia (31%), hypoxia (24%), encephalopathy (22%), febrile neutropenia (22%), and decreased neutrophil count (22%). Severe AEs of any grade occurred in 84% of patients.

Table 19. Adverse events n (%)* 2 × 10 ⁶ (n = 6) 1 × 10 ⁶ (n = 23) 0.5 × 10 ⁶ (n = 16) All patients (N = 45) Any adverse events 6 (100) 6 (100) 23 (100) 23 (100) 16 (100) 15 (94) 45 (100) 44 (98) Fever 6 (100) 3 (50) 22 (96) 11 (48) 12 (75) 5 (31) 40 (89) 19 (42) Low blood pressure 5 (83) 3 (50) 17 (74) 11 (48) 9 (56) 4 (25) 31 (69) 18 (40) Chills 3 (50) 0 13 (57) 0 3 (19) 0 19 (42) 0 (0) Diarrhea 3 (50) 0 10 (43) 1 (4) 6 (38) 0 19 (42) 1 (2) headache 1 (17) 0 10 (43) 1 (4) 7 (44) 1 (6) 18 (40) twenty four) Anemia 4 (67) 4 (67) 10 (43) 8 (35) 3 (19) 2 (13) 17 (38) 14 (31) Brain disease 4 (67) 2 (33) 11 (48) 6 (26) 2 (13) 2 (13) 17 (38) 10 (22) Hypophosphatemia 2 (33) 1 (17) 12 (52) 10 (43) 3 (19) 3 (19) 17 (38) 14 (31) Nausea 1 (17) 0 13 (57) 1 (4) 3 (19) 0 17 (38) 1 (2) Confusion 2 (33) 1 (17) 9 (39) 1 (4) 5 (31) 2 (13) 16 (36) 4 (9) Hypoxia 2 (33) 1 (17) 8 (35) 6 (26) 6 (38) 4 (25) 16 (36) 11 (24) Decreased platelet count 3 (50) 3 (50) 8 (35) 8 (35) 5 (31) 4 (25) 16 (36) 15 (33) constipate 2 (33) 0 10 (43) 0 2 (13) 0 14 (31) 0 (0) Fatigue 1 (17) 0 7 (30) 1 (4) 6 (38) 0 14 (31) 1 (2) Sinus tachycardia 2 (33) 0 10 (43) 1 (4) 2 (13) 0 14 (31) 1 (2) Hypokalemia 1 (17) 0 11 (48) 0 1 (6) 0 13 (29) 0 (0) Tachycardia 1 (17) 1 (17) 6 (26) 1 (4) 6 (38) 0 13 (29) twenty four) Tremor 1 (17) 0 8 (35) 0 4 (25) 0 13 (29) 0 (0) Decreased appetite 0 0 9 (39) 2 (9) 3 (19) 0 12 (27) twenty four) Hyperglycemia 1 (17) 0 6 (26) 0 5 (31) 1 (6) 12 (27) 1 (2) Hypomagnesemia 2 (33) 0 8 (35) 0 2 (13) 0 12 (27) 0 (0) Hyponatremia 3 (50) 2 (33) 7 (30) 3 (13) 2 (13) 0 12 (27) 5 (11) Peripheral edema 1 (17) 0 7 (30) 1 (4) 4 (25) 0 12 (27) 1 (2) * Table includes adverse events of any grade occurring in ≥25% of all patients

CRS was reported in 42 patients (93%); 14 patients (31%) experienced grade ≥3 CRS ( Table 19 ). Common grade ≥3 CRS symptoms were fever (45%), hypotension (36%), and hypoxia (17%). Vasopressors were used to treat CRS in 12 patients (27%). The median time to onset of CRS after infusion was 2 days (range 1-12); the median duration of any grade and grade ≥3 CRS was 9 days and 4.5 days, respectively. CRS-related events resolved in all patients except 2 patients who experienced grade 5 anti-CD19 CAR T cell-related AEs. One patient treated with 2× ¹⁰⁶ CAR T cells/kg had multi-organ failure secondary to CRS (day 6). One patient treated with 0.5 × 10 ⁶ cells/kg experienced a cerebrovascular accident (stroke) in the setting of CRS and NE (day 7). No other anti-CD19 CAR T cell-related grade 5 AEs were reported.

NE was reported in 35 patients (78%); Grade ≥3 events occurred in 17 patients (38%; Table 19). Grade ≥3 NE occurring in ≥5% of patients were encephalopathy (22%), aphasia (16%), and confusional state (9%). There were no cases of cerebral edema and no Grade 5 NE. The median time to onset of NE after infusion was 6 days (range 1-31); the median duration of any grade and Grade ≥3 NE was 12 days and 9 days, respectively. NE resolved in 31/35 patients (89%); 1 patient died of progressive disease and 3 patients died of AEs not considered related to anti-CD19 CAR T cells (sepsis [n = 1], cerebrovascular accident [n = 1], herpes simplex viremia [n = 1]) before resolution of neurologic events.

Fifty-three percent of all patients received tocilizumab, and 36% also received steroids for management of CRS; 31% and 44% received tocilizumab and steroids for NE, respectively. An overall safety improvement was observed in the 9 patients treated according to the revised AE management guidelines relative to the 14 patients treated at the same dose according to the original guidelines (Table 20). Four of the 14 patients treated at 1×10 ⁶ CAR T cells/kg according to the original guidelines had Grade 3 or 4 CRS. Under the revised AE management, 3/9 patients treated at 1×10 ⁶ CAR T cells/kg had Grade 3 CRS, with no Grade 4 CRS reported. The median duration of Grade ≥3 CRS in these patients was also shorter than in patients who received 1×10 ⁶ CAR T cells/kg according to the original AE criteria (4 days vs. 7 days), and the time to Grade ≥3 symptom onset was longer (6 days vs. 4.5 days, respectively). Specifically, 9/14 patients in the 1×10 ⁶ CAR T cells/kg dose group managed with the original criteria experienced Grade 3/4 NEs, compared to one Grade 3 event and no Grade 4 events in patients who received the same dose according to the revised management criteria ( Table 20 ). Based on a review of all available safety and efficacy data, the benefit/risk ratio was considered most favorable at the dose of 1×10 ⁶ CAR T cells/kg, and thus, this dose was the RP2D. All Phase 2 patients were treated according to the revised AE management criteria.

Table 20. Interleukin-1 Release Syndrome and Neurological Events Included in the Revised AE Management Criteria n (%) 2 × 10 ⁶ (n = 6) 1 × 10 ⁶ original AE administration (n = 14) 1 × 10 ⁶ Modified AE management (n = 9) 0.5 × 10 ⁶ (n = 16) Steroids For the treatment of CRS 1 (17) 5 (36) 5 (56) 5 (31) For the treatment of NE 3 (50) 7 (50) 5 (56) 5 (31) Tocilizumab For the treatment of CRS 1 (17) 9 (64) 9 (100) 5 (31) For the treatment of NE 4 (67) 5 (36) 4 (44) 1 (6) Adverse events, n (%) Any level Level ≥3 Any level Level ≥3 Any level Level ≥3 Any level Level ≥3 Interleukin-1 Release Syndrome 6 (100) 3 (50) 14 (100) 4 (29) 9 (100) 3 (33) 13 (81) 4 (25) Fever 6 (100) 3 (50) 12 (86) 5 (36) 9 (100) 6 (67) 10 (77) 5 (31) Low blood pressure 4 (67) 3 (50) 11 (79) 6 (43) 6 (67) 3 (33) 8 (62) 3 (19) Sinus tachycardia 2 (33) 0 6 (43) 0 4 (44) 1 (11) 2 (15) 0 Chills 1 (17) 0 5 (36) 0 4 (44) 0 2 (15) 0 Tachycardia 1 (17) 1 (17) 4 (29) 1 (7) 2 (22) 0 4 (31) 0 Shortness of breath 0 0 4 (29) 1 (7) 0 0 0 0 Hypoxia 2 (33) 1 (17) 2 (14) 2 (14) 3 (33) 2 (22) 3 (23) 2 (15) Nausea 0 0 2 (14) 0 0 0 0 0 Fatigue 0 0 1 (7) 0 3 (33) 0 1 (8) 0 headache 0 0 1 (7) 0 2 (22) 0 3 (23) 0 Hyponatremia 0 0 1 (7) 0 1 (11) 0 1 (8) 0 Any neurological event 5 (83) 3 (50) 13 (93) 9 (64) 7 (78) 1 (11) 10 (63) 4 (25) Confusion 2 (33) 1 (17) 3 (21) 0 6 (67) 1 (11) 5 (31) 2 (13) Tremor 1 (17) 0 4 (29) 0 4 (44) 0 4 (25) 0 Aphasia 0 0 6 (43) 4 (29) 2 (22) 1 (11) 2 (13) 2 (13) Brain disease 4 (67) 2 (33) 9 (64) 6 (43) 2 (22) 0 2 (13) 2 (13) Lethargy 0 0 1 (7) 0 2 (22) 0 2 (13) 0 Mental status changes 0 0 0 0 2 (22) 0 0 0 Restlessness 0 0 4 (29) 1 (7) 1 (11) 1 (11) 2 (13) 0 Pronunciation difficulties 0 0 1 (7) 1 (7) 1 (11) 0 0 0 Restlessness 0 0 1 (7) 1 (7) 1 (11) 1 (11) 0 0 Epilepsy 1 (17) 0 2 (14) 2 (14) 1 (11) 0 1 (6) 0 Ataxia 0 0 1 (7) 0 1 (11) 0 0 0 AE, adverse event; CRS, interleukin-1 release syndrome; NE, neurologic event

Twenty-six treated patients (58%) died from causes including disease progression in 19 patients (42%) and AEs in 7 patients (16%), including 2 treatment-related deaths as described above. The remaining 5 AE-related deaths occurred a median of 63 days (range 48-579) after infusion of anti-CD19 CAR T cells and were considered unrelated to anti-CD19 CAR T cells. They included sepsis (n = 2), cerebrovascular accident (n = 1), herpes simplex viremia (n = 1), and bacteremia (n = 1).

effect

All 45 treated patients met the efficacy analysis criteria. At a median follow-up of 22.1 months (range 7.1-36.1), the overall response rate (ORR) was 69%, with 51% of patients achieving CR and 18% achieving CRi ( Table 21 ). Among the 23 patients treated with 1× ¹⁰⁶ CAR T cells/kg, the ORR was 83%, with 14 achieving CR (61%) and 5 (22%) achieving CRi. Six of the 9 patients who received modified AE management achieved CR/CRi (4 CR, 2 CRi). The median time to achieve CR/CRi was 30 days (range 26-192) at each dose, including 1 patient with aplastic/aplastic bone marrow (BFBM) without blasts at day 28, who did not meet CR criteria until month 6. ORRs were generally consistent across key covariates, including refractory patients (56%), prior transplant (77%), prior blinatumomab (57%) or inotuzumab (50%), and patients with Ph+ disease (100%) ( Figure 5 ). Undetectable bone marrow MRD at Day 28 was achieved in 100% of responders, including 31 patients with CR/CRi, 1 patient with partial response, and 1 patient with BFBM. Residual disease assessment was not available in 1 patient with BFBM. Two of the 6 patients who underwent optional bone marrow assessment on Days 7-14 had undetectable MRD; 5 patients with available data at Day 30 had undetectable MRD.

Table 21. Response to anti-CD19 CAR T cells Reaction type, n (%) 2 × 10 ⁶ (n = 6) 1 × 10 ⁶ (n = 23) 0.5 × 10 ⁶ (n = 16) Total (N = 45) Complete remission Complete remission with incomplete hematological recovery 4 (67) 3 (50) 1 (17) 19 (83) 14 (61) 5 (22) 8 (50) 6 (38) 2 (13) 31 (69) 23 (51) 8 (18) Aplastic / aplastic bone marrow 0 1 (4) 1 (6) twenty four) Partial relief 0 1 (4)* 0 1 (2) No response 1 (17) 2 (9) 6 (3) 8 (18) Unknown or unassessable 1 (17) ^† 0 1 (6) ^‡ twenty four) * Patient had extramedullary disease at the time of response assessment. ^† Patient died on day 6 due to multiorgan failure secondary to CRS. ^‡ Patient died on day 7 due to cerebrovascular accident (stroke) in the setting of CRS and neurological events

The median DOR for the 31 patients who achieved CR/CRi was 14.5 months (95% CI, 5.8-18.1; Figure 6A ) and 17.6 months (95% CI, 5.8-17.6) in patients treated with 1×10 ⁶ CAR T cells/kg. The median DOR was similar regardless of how SCT was examined after anti-CD19 CAR T cells ( Figure 6B ). As of the data cutoff, 8 patients (26%) had a durable CR, including 2 patients who received 0.5×10 ⁶ CAR T cells/kg and 6 patients who received 1×10 ⁶ CAR T cells/kg, with a median follow-up period of 6.3 months (range, 5.9-18.2). Six patients (2 CR and 1 partial response treated with 1×10 ⁶ CAR T cells/kg; 3 CR treated with 0.5×10 ⁶ cells/kg) underwent SCT at a median of 2.7 months (range, 1.7-4.3) after infusion. As of this analysis, 3 of these patients remained in CR (2 treated with 1×10 ⁶ CAR T cells/kg and 1 treated with 0.5×10 ⁶ CAR T cells/kg). Across all doses, the median duration of relapse-free survival in patients receiving 1×10 ⁶ CAR T cells/kg was 7.3 months (95% CI, 2.7-18.7) vs. 7.7 months (95% CI, 3.2-18.7) ( Figure 6C ). The median OS was 12.1 months (95% CI, 6.1-19.1) across all doses and 16.1 months (95% CI, 10.2-not estimable) at 1×10 ⁶ CAR T cells/kg ( Figure 6D ).

As of the data cutoff, 1 patient (2%) withdrew consent, 1 (2%) was lost to follow-up, and 17 (38%) were alive, including 11/23 patients (50%) treated with 1× ¹⁰⁶ cells/kg. Four patients received a second infusion of anti-CD19 CAR T cells; 1 was in CR 15 months after reinfusion, 2 relapsed by the 3-month assessment, and 1 withdrew consent before the first response assessment.

Clinical Pharmacology

For most patients, CAR T cell levels, measured by CAR gene copies per microgram DNA in the blood, peaked 7-14 days after anti-CD19 CAR T cell infusion and remained detectable at 12 months in 2/12 evaluable patients, both of whom were in CR ( Figure 7A ; Table 22 ).

Table 22. CAR gene copies in blood over time CAR gene copies per microgram of DNA in blood 2 × 10 ⁶ 1 × 10 ⁶ Original AE Management 1 × 10 ⁶ Revised AE Management 0.5 × 10 ⁶ Baseline Median Range (n = 6) 0 0 - 0 (n = 14) 0 0 - 0 (n = 9) 0 0 - 0 (n = 16) 0 0 - 0 Day 7 Median range (n = 4) 62,411 11,097 - 162,972 (n = 12) 154,386 12,231 - 443,880 (n = 9) 91,287 0 - 353,160 (n = 15) 3702 0 - 375,030 Week 2 Median range (n = 5) 44,064 2228 - 106,110 (n = 14) 48,114 7614 - 283,500 (n = 8) 60,507 10,935 - 224,370 (n = 13) 3669 0 - 100,845 Week 4 Median range (n = 5) 1304 405 - 4860 (n = 11) 3119 1029 - 95,580 (n = 9) 16,200 235 - 56,052 (n = 13) 1588 0 - 27,540 Week 8 Median range (n = 0) - - (n = 5) 0 0 - 907 (n = 7) 527 0 - 972 (n = 7) 219 0 - 9882 Month 3 Median range (n = 4) 0 0 - 0 (n = 11) 203 0 - 1458 (n = 6) 99 0 - 478 (n = 9) 0 0 - 5508 Month 6 median range (n = 3) 0 0 - 0 (n = 8) 0 0 - 105 (n = 0) - - (n = 7) 0 0 - 518 9th month Median range (n = 1) 0 0 - 0 (n = 6) 0 0 - 138 (n = 0) - - (n = 4) 0 0 - 0 Month 12 Median range (n = 1) 65 65 - 65 (n = 4) 0 0 - 0 (n = 0) - - (n = 3) 0 0 - 57 AE, adverse event; CAR, chimeric antigen receptor

CAR T cells were undetectable in the five patients with available data at the time of relapse. Median peak CAR T cell levels were highest at 1×10 ⁶ CAR T cells/kg and were similar between patients receiving original versus revised AE management ( Figure 7B ; Figure 8 ). Median peak increases were greater in patients who achieved CR/CRi than in nonresponders, as were those with undetectable MRD versus those with detectable MRD ( Figure 7C - D ; Figure 8B - C ). Higher median peak increases were also observed in patients with grade ≥3 NE versus those with grade ≤2 NE ( Figure 7E - F ; Figure 8D - E ). Of the 13 patients who relapsed, 7 had detectable CD19-positive cells at the time of relapse, 3 did not have detectable CD19-positive cells, and 3 had no data available.

By day 7, peak levels of key interleukins, chemokines, and proinflammatory markers occurred, some of which tended to be higher in patients given 2× ¹⁰⁶ CAR T cells/kg compared with 1× ¹⁰⁶ CAR T cells/kg (IL-15, CRP, SAA, CXCL10, IFNγ) or lower in patients managed with the revised AE compared with those managed with the original AE (IL-6, ferritin, IL-1RA, IFNγ, IL-8, CXCL10, MCP-1) ( Figure 9 ; Figure 10 ). Although peak IL-15 serum levels were unexpectedly lower in patients with grade ≥3 CRS, median peak levels of several proinflammatory markers tended to be higher in patients with grade ≥3 CRS and in patients with grade ≥3 NE (IFNγ, IL-8, GM-CSF, IL-1RA, CXCL10, MCP-1, granzyme B; Figure 11 ).

Four patients tested positive for anti-CAR antibodies during the screening assay, but all were negative in the confirmatory assay at the time of leukapheresis. The characteristics of the produced CAR T cell products were as expected and previously reported ( Table 23 ).

Table 23. Product characteristics Median feature ( range ) 2 × 10 ⁶ (n = 6) 1 × 10 ⁶ original AE administration (n = 14) 1 × 10 ⁶ Modified AE management (n = 9) 0.5 × 10 ⁶ (n = 16) T cell subsets, % Native 32.9 (16.4-60.5) 41.1 (9.9-73.2) 30.2 (0.1-65.0) 33.1 (12.5-80.9) Central memory 34.5 (15.1-42.7) 21.9 (14.6-40.7) 19.3 (3.2-36.3) 18.0 (3.0-48.2) Effect 8.9 (3.7-13.4) 8.9 (4.5-41.6) 14.5 (2.4-20.9) 14.3 (2.4-38.1) Effect memory 20.7 (15.5-52.4) 18.4 (4.8-60.0) 19.9 (3.9-94.3) 22.6 (1.0-45.3) CD4 , % 44.7 (33.9-58.8) 47.6 (21.9-76.8) 56.8 (41.0-93.7) 58.8 (28.5-85.9) CD8 , % 55.4 (41.2-66.2) 49.0 (23.2-78.1) 43.3 (6.3-59.0) 41.2 (14.1-71.4) CD4/CD8 Ratio 0.8 (0.5-1.4) 1.0 (0.3-3.3) 1.4 (0.7-14.9) 1.4 (0.4-6.1) IFNγ production in co-culture (pg/mL) * 7944.0 (1679.5-11214.4) 9980.5 (3025.0-37921.9) 10317.3 (5255.0-45235.7) 9059.5 (1040.6-27859.1) * Co-culture experiments were performed using Toledo cells mixed with anti-CD19 CAR T cell products at a 1:1 ratio. IFNγ was measured in cell culture medium 24 hours after incubation using a qualified ELISA. AE, adverse event; IFNγ, interferon gamma

ZUMA-3 is the first multicenter study evaluating CAR T cell therapy in adult R/R B-ALL to complete Phase 1. In the Phase 1 portion, no protocol-defined DLTs were observed with anti-CD19 CAR T cells, and reported AEs were consistent with previous studies of anti-CD19 CAR T cell therapy. Neelapu SS. et al. N Engl J Med. 2017;377(26):2531-2544; Maude SL et al. N Engl J Med. 2018;378(5):439-448. The 1×10 ⁶ CAR T cells/kg dose combined with revised AE management guidelines had the most favorable risk/benefit ratio without compromising activity. Although patients had high disease burden and were heavily pretreated, high remission rates and undetectable bone marrow MRD were achieved, especially in patients treated at the 1×10 ⁶ dose; the ORR was 83%, including 61% CR and 22% CRi, all with undetectable MRD. Based on these results showing that anti-CD19 CAR T cells are safe and have promising efficacy, the 1×10 ⁶ CAR T cells/kg dose was selected for further evaluation in Phase 2 of ZUMA-3.

The use of anti-CD19 CAR T cells for the treatment of adult R/R B-ALL has proven difficult because the disease is highly proliferative and cannot tolerate treatment-related AEs. Previous CAR T cell trials in this population were closed early due to fatal NE, including 5 cases of cerebral edema. DeAngelo DJ, Ghobadi A, Park JH, et al., Journal for ImmunoTherapy of Cancer. 2017;5(Suppl 2):P217. In ZUMA-3, 2 patients died from grade 5 AEs based on the original AE management guidelines, which were considered related to anti-CD19 CAR T cells, secondary to CRS or outside the DLT assessment time frame in the case of CRS and NE. In addition to evaluating multiple doses to identify the dose with the most manageable toxicity, a revised AE management guideline requiring early steroid intervention for neurotoxicity and tocilizumab only for CRS was implemented in the 9 patients enrolled at the 1× ¹⁰⁶ CAR T cells/kg dose. This resulted in a shorter duration of CRS events and a lower incidence, severity, and duration of NE compared with the 14 patients treated according to the original guidelines at the same dose.

At a median follow-up of 22.1 months, responses were ongoing in 26% of patients, most of whom received 1× ¹⁰⁶ CAR T cells/kg (32% ongoing CR/CRi). Responses tended to occur early after treatment. Most occurred within the first month, although 1 patient with extramedullary disease achieved a CR at month 6. High response rates were observed in all prespecified subgroups, including a 100% CR rate in patients with Ph+ disease. Responses (CR/CRi) were associated with higher expansion of CAR T cells measured within 2 weeks of treatment. Similarly, in a single-center phase 1 study using anti-CD19 CART cell therapy that also contained CD3ζ and CD28 synergistic stimulatory domains (Park JH et al. N Engl J Med. 2018;378(5):449-459), the overall CR rate was 83%, although only half of the patients had ≥5% blasts in the bone marrow after transitional therapy, 28% had MRD, and 11% had undetectable MRD. However, the results of those trials were largely parallel to those of this study, further supporting the potential utility of anti-CD19 CAR T cell therapy using CD3ζ and CD28 synergistic stimulatory domains in adult R/R B-ALL.

Tesazinol is an anti-CD19 CART cell therapy containing a CD3ζ T cell activation domain and a 4-1BB co-stimulatory domain that is approved for the treatment of R/R B-ALL in children and adolescents (≤25 years). Maude SL et al N Engl J Med. 2018;378(5):439-448;KYMRIAH (tesazinol) [drug description]. Novartis. East Hanover, NJ; 2018. However, the dosing regimen of tesazinol in younger patients caused significant toxicity and CRS-related deaths in adults with R/R B-ALL. Frey NV et al J Clin Oncol. 2020;38(5):415-422. In a single-center study in adults with R/R B-ALL, fractionated dosing resulted in manageable CRS and a 90% CR rate in two clinical trials. Frey NV et al. J Clin Oncol. 2020;38(5):415-422. Similar to the ZUMA-3 findings, optimized dosing and toxicity management strategies could benefit patients who are susceptible to life-threatening treatment-related toxicities from CAR T-cell therapy.

Despite differences in trial design, patient populations, and OS approaches, the median OS in this study was 16.1 months at 1× ¹⁰⁶ CAR T cells/kg, compared with median OS of 6.1-7.7 months previously reported in adult R/R B-ALL with blinatumomab, which also targets CD19. Topp MS et al. Lancet Oncol. 2015;16(1):57-66; Kantarjian H. et al. N Engl J Med. 2017;376(9):836-847. Of the 10 patients evaluable for CD19 blast expression at relapse, 3 showed lack of CD19 expression, which is reminiscent of other reports attributing target loss to selection for exonic splicing variants and mutations. Sotillo E et al. Cancer Discov. 2015;5(12):1282-1295. In this study, only 1/8 patients (13%) responded to blinatumomab when it was used as the last prior therapy. This may suggest that unmanipulated T cells are immunocompromised in some patients with R/R ALL, which may limit the utility of bispecific T-cell engager therapy. Of the 21 patients who had previously used blinatumomab in any line, 12 (57%) achieved CR/CRi following anti-CD19 CAR T-cell therapy. As previously reported (Shah BD. et al. J Clin Oncol. 2018;36(Suppl):Abstract 7006), responses to anti-CD19 CAR T cells were similar regardless of prior exposure to blinatumomab in patients with persistent CD19 positivity. Additionally, 6 patients who achieved CR underwent SCT and were censored at the time of SCT; 3 remained in remission.

Adults with R/R B-ALL achieved high CR rates and undetectable bone marrow MRD with a tolerable safety profile following treatment with anti-CD19 CAR T cells. Successful manufacturing for all enrolled patients and relatively rapid turnaround time support the feasibility of offering this cell therapy approach to patients with rapidly progressive disease who require prompt treatment. The study from Phase 1 can be transitioned to an international Phase 2 study by carefully evaluating the dose range and employing a safety strategy, including the use of tocilizumab or steroids and the conditions under which they are administered to manage AEs. There were no cases of fatal cerebral edema in Phase 1, which was a limitation of previous studies in this population. Phase 2 of ZUMA-3 is ongoing at a dose of 1×10 ⁶ CAR T cells/kg under revised AE management guidelines. Example 10

This example describes the results of CD19ΔTyr260 in CD19 in B-ALL associated with resistance to CAR T cell therapy. After failure of several therapies, including the use of blinatumomab before KTE-X19, a B-ALL patient received a target dose of 1×10 ⁶ CAR T cells/kg. The patient did not respond clinically; CAR T and CD19 expressing lymphocytes were undetectable on day 28. Peripheral blood mononuclear cells (PBMCs) were collected from B-ALL patients before and at multiple time points after KTE-X19 infusion. Multicolor flow cytometry was used to examine surface expression of CD19 (clonal FMC63, HIB19, SJ25C1) on patient PBMCs and Jurkat cell lines engineered to express CD19 wild-type (WT) or CD19ΔTyr260. The presence of genetic variants was assessed using enhanced whole genome and RNA sequencing (TruSeq multi-stranded total RNA). The location of cellular protein expression was assessed using Western blotting in the presence and absence of deglycosylase.

Although local lesions inferred that B lymphoblasts were uniformly CD19 ^dark prior to infusion, additional analysis of the same samples using FMC63 (a single-chain variable fragment of KTE-X19) showed that CD19 was undetectable in B lymphoblasts prior to infusion. Results from RNA sequencing revealed an in-frame deletion at Tyr260 within the intracellular domain of CD19 in circulating leukemic blasts (CD19ΔTyr260). Additional analysis using flow cytometry showed that CD19 expression was not detected on Jurkat CD19ΔTyr260 cells but was present on Jurkat CD19-WT cells, indicating a lack of visualization of cells harboring this point mutation and their resistance to CAR T cell therapy. Longitudinal RNA and DNA sequencing analysis showed that the mutation had occurred before infusion of CAR-T therapy. Fractionated cell lysates showed WT CD19 in the cell membrane with high and low molecular weight bright bands, and CD19ΔTyr260 expressed on the surface with a single low molecular weight bright band. Under deglycosylation conditions, only one bright band was present in the WT CD19 and CD19ΔTyr260 cell lysates. Without being bound by any scientific theory or hypothesis, it is possible that the CD19ΔTyr260 mutation causes a lack of appropriate or functional CD19 glycosylation and/or inhibits detection. Mutations in B-ALL malignant cells may have potential implications for other anti-CD19 CARs or to CD19 CAR cell therapies. Example 11

MCL patients who progress after BTKi therapy generally have a poor prognosis with an overall survival of only 5.8 months on salvage therapy. Martin P et al., Blood. 2016;127:1559-1563. In the Phase 2 ZUMA-2 study, KTE-X19 was evaluated in MCL patients who were R/R to 1-5 prior therapies including BTKi. Wang M et al., N Engl J Med. 2020;382:1331-1342. In the primary efficacy analysis of ZUMA-2 (N=60), the ORR was 93% (67% complete response) at a median follow-up period of 12.3 months. Aggressive disease variants including blastoid or pleomorphic MCL are generally associated with poor clinical outcomes, but ORRs were comparable in patients with a variety of histologies in ZUMA-2. Wang M et al., N Engl J Med. 2020;382:1331-1342; Jain P, and Wang M. Am J Hematol. 2019;94:710-725. In this study, pharmacological profiles and clinical outcomes were compared in ZUMA-2 in subgroups of patients defined by MCL morphology and prior BTKi exposure, along with characterization of product attributes and other pre-treatment factors. Patients underwent leukapheresis and conditioning chemotherapy, followed by a single infusion of CD19 CAR-T cells at a target dose of 2× ¹⁰⁶ CAR T cells/kg by a single IV infusion on day 0. Some patients received transitional therapy with dexamethasone (20-40 mg PO or IV daily or equivalent for 1-4 days), ibrutinib (560 mg PO daily), or acalabrutinib (100 mg PO twice daily), administered after leukapheresis and completed ≤5 days before starting conditioning chemotherapy; PET-CT was required after the transition period. The primary endpoint was objective response rate (ORR [complete response (CR) + partial response]). Secondary endpoints were duration of response (DOR), progression-free survival (PFS), OS, frequency of adverse events (AEs), levels of CAR T cells in blood, and levels of interleukins in serum. Efficacy and safety analyses included all patients who received CD19 CAR-T cell therapy. The first tumor assessment was performed on day 28. Bone marrow biopsy was performed at screening and was not performed if positive, or was inconclusive and required to confirm CR.

Among the 60 MCL patients treated with KTE-X19 in ZUMA-2, there was a 93% ORR, 67% CR rate, and 57% of all patients and 78% of patients in CR had ongoing responses at a median follow-up period of 12.3 months. CRS and neurologic events were mostly reversible (N=68 patients treated). Approximately 15% had grade ≥3 CRS, 31% had grade ≥3 neurologic events, and 2 had grade 5 AEs (1 KTE-X19 related). Patient subgroups were defined by morphologic characteristics (classic, blastoid, or pleomorphic MCL) and by prior exposure to ibrutinib only, acalabrutinib only, or both ibrutinib and acalabrutinib. Table 24. Baseline characteristics were generally comparable across the groups. There was a trend toward higher pretreatment tumor burden in patients previously treated with ibrutinib. Product properties, CAR T cell levels in blood, and interleukin levels in serum were analyzed using previously described methods. Locke FL et al., Mol Ther. 2017;25:285-295. Product T cell properties were generally comparable across MCL morphology subgroups. Product co-culture IFN-γ and percentage of CCR7+ cells tended to be increased in products from patients with pleomorphic morphology. Table 25. Product T cell properties were also generally comparable across previously BTKi subgroups. There was a trend toward increased product co-culture IFN-γ in patients previously treated with ibrutinib. Table 26 .

Table 24. Patient baseline characteristics Ibrutinib (n = 52) Acalabrutinib (n = 10) Both (n = 6) Median age (range), years 65 (45 - 79) 57 (38 - 73) 62 (55 - 72) ≥ 65 years old, n (%) 32 (62) 4 (40) 3 (50) Male, n (%) 43 (83) 9 (90) 5 (83) Stage IV disease, n (%) 44 (85) 9 (90) 5 (83) ECOG 0/1, n (%) 52 (100) 10 (100) 6 (100) Median tumor burdena ⁽ range) mm ² 2697 (386 - 16878) 1144 (293 - 14390) 536 (260 - 1174) Ki-67 proliferation index, n/N (%) ≥ 50 25/38 (66) 3/5 (60) 6/6 (100) ＜ 50 13/38 (34) 2/5 (40) 0 MCL form typical 30 (58) 6 (60) 4 (67) Polymorphism 1 (2) 2 (20) 1 (17) Mother cell-like 12 (23) 3 (30) 2 (33) Bone marrow involvement, n (%) 28 (54) 3 (30) 6 (100) Extranodal disease, n (%) 31 (60) 3 (30) 4 (67) Median number of previous treatments (range) 3 (1 - 5) 3 (2 - 5) 3 (3 - 4) Previous bendamustine, n (%) 28 (54) 7 (70) 2 (33) ^aAs measured by the sum of the product dimensions of all target lesions at baseline. For individuals with transition therapy, measurements after transition therapy were used as baseline.

Table 25. Cellular characteristics and MCL morphology Median ( range ) MCL form Typical (n = 40) Mother cell-like (n = 17) Polymorphism (n = 4) Transduction rate, % 58.1 (35.0 - 82.4) 60.0 (46.0 - 79.4) 61.9 (50.0 - 77.1) CD4/CD8 Ratio 0.7 (0.04 - 2.8) ^a 0.6 (0.2 - 1.1) ^a 0.7 (0.5 - 2.0) CCR7+ T cells, % 40.0 (2.6 - 88.8) ^a 35.3 (14.3 - 73.4) ^a 80.8 (57.3 - 88.8) CCR7-effector + effector memory T cells, % 59.9 (11.1 - 97.4) ^a 64.8 (26.6 - 85.7) ^a 19.2 (11.1 - 42.7) (CCR7+ T cells)/(CCR7-effector + effector memory T cells) ratio 0.7 (0.03 - 8.0) ^a 0.5 (0.2 - 2.8) ^a 4.7 (1.3 - 8.0) IFN-γ by co-culture, pg/mL 6309.5 (424.0 - 2.0 × 10 ⁴ ) 6510.0 (2709.0 - 1.8 × 10 ⁴ ) 7687.5 (424.0 - 1.2 × 10 ⁴ ) ^aBased on available data: typical, n=38; mother cell-like, n=16

Table 26. Cellular characteristics and BTKi subgroups Median ( range ) BTKi exposure Ibrutinib (n = 52) Acalabrutinib (n = 10) Both (n = 6) Transduction rate, % 56.7 (32.0 - 82.4) 65.0 (35.0 - 74.0) 58.5 (46.0 - 67.0) CD4/CD8 Ratio 0.7 (0.04 - 3.7) ^a 0.6 (0.3 - 1.2) 1.0 (0.7 - 1.9) CCR7+ T cells, % 39.3 (2.6 - 86.4) ^a 42.7 (16.3 - 88.8) 49.5 (14.3 - 83.0) CCR7-effector + effector memory T cells, % 60.6 (13.7 - 97.4) ^a 57.3 (11.1 - 83.8) 50.6 (17.0 - 85.7) (CCR7+ T cells)/(CCR7-effector + effector memory T cells) ratio 0.7 (0.03 - 6.3) ^a 0.8 (0.2 - 8.0) 1.2 (0.2 - 4.9) IFN-γ by co-culture, pg/mL 6496.0 (424.0 - 2.0 × 10 ⁴ ) 5972.5 (2502.0 - 1.8 × 10 ⁴ ) 7985.5 (2709.0 - 1.2 × 10 ⁴ ) ^aBased on available data: Ibrutinib, n = 49

High response rates were achieved in the MCL morphology and prior BTKi subgroups. Table 27. Clinical benefit from KTE-X19 treatment was observed in all subgroups defined by MCL morphology or prior BTKi. A trend toward higher durable response rates at 6 months was observed in patients previously treated with ibrutinib. Table 27. CRS and neurologic events were generally comparable in the MCL morphology and prior BTKi subgroups. Table 28. A trend toward increased rates of grade ≥3 neurologic events was observed in patients with non-blastoid morphology or prior treatment with ibrutinib. Table 28 .

Table 27. Response rate MCL form BTKi exposure Typical (n = 35) Mother cell samples (n = 14) Polymorphism (n = 4) Ibrutinib (n = 45) Acalabrutinib (n = 9) Both (n = 6) ORR, n (%) 32 (91) ¹ 13 (93) ¹ 4 (100) ¹ 43 (96) 7 (78) 6 (100) CR, n (%) 22 (63) ¹ 9 (64) ¹ 3 (75) ¹ 30 (67) 4 (44) 6 (100) Sustained response at 6 months, n (%) 18 (51) 8 (57) 3 (75) 25 (56) 3 (33) 6 (100) 12-month OS, % (95% CI) 85.7 (69.0 - 93.8) ¹ 71.4 (40.6 - 88.2) ¹ 100.0 (NE - NE) ¹ 82.0 (67.2 - 90.6) 77.8 (36.5 - 93.9) 100.0 (NE - NE) ¹ Wang M et al., N Engl J Med. 2020;382:1331-1342. CR, complete response; MCL, mantle cell lymphoma; NE, not evaluable; ORR, objective response rate; OS, overall survival rate

Table 28. Adverse events n (%) MCL form BTKi exposure Typical (n = 40) Mother cell-like (n = 17) Polymorphism (n = 4) Ibrutinib (n = 52) Acalabrutinib (n = 10) Both (n = 6) CRS Any level 36 (90) 15 (88) 4 (100) 50 (96) 6 (60) 6 (100) Level ≥ 3 6 (15) 1 (6) 1 (25) 9 (17) 1 (10) 0 Neurological events Any level 25 (63) 11 (65) 3 (75) 33 (63) 4 (40) 6 (100) Level ≥ 3 15 (38) 3 (18) 2 (50) 16 (31) 1 (10) 4 (67)

Comparisons within subgroups were performed using the Kruskal-Wallis test; Dunn's post-hoc test was used for comparisons between groups. Pharmacological profiles, product properties, and safety data for all 68 patients treated with KTE-X19 (2×10 ⁶ cells/kg) are reported. The pharmacological and pharmacodynamic profiles of KTE-X19 in MCL morphology subgroups demonstrated an increase in CAR T cell expansion and select proinflammatory cytokines in patients with typical morphology compared with patients with blastoid morphology (Figures 12 and 13), or in patients previously treated with ibrutinib compared with acalabrutinib alone (Figures 14 and 15). Pre-treatment patient and product characteristics were generally comparable across MCL morphology and subsets with different prior therapies. Patients with blastoid morphology demonstrated reduced CAR T-cell expansion, circulating bone marrow-related interleukins and trend factors, and rates of grade ≥3 CRS and neurologic events, with clinical efficacy comparable to that of patients with typical morphology. The trend toward improved safety profile in patients with blastoid morphology was commensurate with lower peak CAR T-cell expansion and peak reductions in interleukins associated with bone marrow-related inflammation. Patients previously treated with ibrutinib demonstrated increased CAR T cell expansion, circulating inflammatory interleukins and trend factors, and the rate of grade ≥3 neurologic events; as well as increased durable response rate at 6 months, and ORR comparable to patients previously treated with acalabrutinib alone. Patients previously treated with acalabrutinib demonstrated reduced CAR T cell expansion and circulating T1-related interleukins and trend factors, consistent with the improved safety profile. Example 12

This example characterizes two anti-CD19 CAR T therapies, KTE-X19 and Akiramu, prepared according to Example 5. Cells were labeled with fluorescent conjugated antibodies against CD3 (pan T cell marker), CD14, CD19 (B cell marker), CD45 (pan leukocyte marker), and CD56 (activation and NK marker) and assessed by flow cytometry. Cell viability was assessed using negative staining with a viability dye (SYTOX Near IR). The lower limit of quantification (LLOQ) of the assay was 0.2% and 5% for NK cells and monocytes. The percentage of NK cells was determined (NK cells were CD45 ⁺ , CD14 ^- , CD3 ^- and CD56 ⁺ ; T cells were CD45 ⁺ , CD14 ^- and CD3 ^- ). The median percentages of NK cells from 23 batches of Alkiram and 97 batches of KTE-X19 were 1.9% (range 0.8%-3.2%) and 0.1% (range 0.0%-2.8%), respectively. The median percentages of CD3 ^- cell impurities from the same batches of Alkiram and KTE-X19 were 2.4% (range 0.9%-4.6%) and 0.5% (range 0.3%-3.9%), respectively. The results for KTE-X19 and Alkiram were ≥72% and ≥80%, respectively, for cell viability; ≥24% and ≥15%, respectively, for anti-CD19 CAR expression; ≥190 pg/mL and ≥520 pg/mL, respectively, for IFN-γ production; and ≥90% and ≥85%, respectively, for CD3 ⁺ cell percentage. Example 13

Additional results are provided for patients who received a single infusion of 2×10 ⁶ KTE-X19 cells/kg in previous examples including Example 2 and Example 7. The ORR assessed by IRRC was 92% (95% CI, 82-97) and the CR rate was 67% (95% CI, 53-78). At a median follow-up period of 17.5 months (range 12.3-37.6), 29 patients maintained sustained responses. The sustained response rate was essentially the same among patients with high-risk disease features. The first 28 patients treated had a median follow-up period of 32.3 months (range 30.6-37.6). 39% of patients maintained sustained remission without further treatment. Among all enrolled patients (N=74), the ORR was 84% (59% CR rate). Median values for DOR, PFS, and OS were not reached after a median follow-up period of 17.5 months. Table 29. Durable response rates were consistent across poor prognosis groups. Figure 16. The ZUMA-2 study continues to show significant and durable clinical benefit of KTE-X19 therapy in patients with R/R MCL at a median follow-up period of 17.5 months. No new safety signals were observed with additional follow-up. No new CRS or new grade 5 events occurred since previously reported. Table 30. AE rates decreased over time. KTE-X19 therapy shows a manageable safety profile with extended follow-up.

Table 29. Duration of response, progression-free survival, and overall survival. DOR PFS OS Median (95% CI) , month 15 -month rate ratio (95% CI) Median (95% CI) , month 15 -month rate ratio (95% CI) Median (95% CI) , month 15 -month rate ratio (95% CI) Evaluable patients (N = 60) NR (13.6 - NE) ^a 58.6 (42.5 - 71.7) ^a NR (9.6 - NE) 59.2 (44.6 - 71.2) NR (NE, NE) 76.0 (62.8, 85.1) In CR (n = 40) NR (14.4 - NE) 69.7 (49.3 - 83.2) NR (15.3 - NE) 75.1 (56.8 - 86.5) NR (NE, NE) 91.7 (76.2, 97.2) In PR (n = 15) 2.2 (1.4 - 4.3) 24.1 (5.9 - 48.9) 3.1 (2.3 - 5.2) 24.1 (5.9 - 48.9) 12.6 (3.3, NE) 46.7 (21.2, 68.7) ^aAmong 55 responding patients.

Table 30. Safety analysis All treated patients (N = 68) 3 months after infusion / after 6 months after infusion / after AE , n (%) ^a Any level Level ≥ 3 Any level Level ≥ 3 Any AE 55 (81) 33 (48) 49 (72) 25 (37) Anemia 22 (32) 9 (13) 13 (19) 4 (6) Neutropenia 20 (29) 16 (24) 14 (21) 11 (16) Thrombocytopenia 20 (29) 14 (21) 14 (21) 9 (13) Decreased white blood cell count 16 (24) 9 (13) 12 (18) 6 (9) Fatigue 10 (15) 0 10 (15) 0 pneumonia 9 (13) 5 (7) 6 (9) 4 (6) cough 8 (12) 0 7 (10) 0 Hypogammaglobulinemia 8 (12) 0 7 (10) 0 Upper respiratory tract infection 7 (10) twenty three) 5 (7) 1 (1) ^a Includes AEs of any grade occurring in ≥10% of patients.

Of the 57 efficacy-evaluable patients with available data, 48 (84%) had detectable B cells at baseline. Among patients with sustained responses at 12 months, more than 50% of evaluable patients had detectable B cells and genetically marked CAR T cells at 6, 12, 15, and 24 months. Among patients with sustained responses at 12 months, the percentage of patients with genetically marked CAR T cells generally decreased over time, with 100%, 93%, 82%, 89%, 80%, and 56% at 3, 6, 12, 15, 18, and 24 months, respectively. Peak CAR T cell expansion decreased in patients who failed to respond to KTE-X19. Peak CAR T cell expansion was increased in patients with a sustained response at 12 months or in patients who relapsed at 12 months compared to non-responders. Elevated CAR T cell levels were initially observed in patients who subsequently relapsed, which may point to an alternative mechanism for secondary treatment failure. Peak CAR T cell levels normalized by baseline tumor burden and sustained response at the 12-month data cutoff are shown in Figures 17A (INV) and 17B (CEN).

All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was individually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Figures 1A-1F: Comparable pharmacodynamic profiles in prognostic groups defined by the Ki-67 proliferation index and trends of increased cytokine levels in patients with mutant TP53.

Figures 2A-2I: Peak levels of selected interleukins in serum were increased in patients who achieved MRD-negative status.

Figure 3: ZUMA-3 study design. CAR, chimeric antigen receptor; DLT, dose-limiting toxicity.

Figure 4: ZUMA-3 CONSORT diagram. *AEs were grade 3 pulmonary mass (n=1), grade 1 subdural hematoma (n=1), and grade 3 febrile neutropenia (n=1); †AEs were grade 4 sepsis (n=1) and grade 5 sepsis (n=1); ‡One patient received KTE-X19 under compassionate use due to deep venous embolism, which was a study exclusion criterion. AE, adverse event.

Fig. 5: Subgroup analysis of complete response rate. BM, bone marrow; ORR, overall response rate; SCT, stem cell transplantation.

Figure 6: Duration of response, relapse-free survival, and overall survival according to dose.

Figure 7: Peak CAR T cell expansion and correlation with response, minimal residual disease, and toxicity.

Figure 8: Correlation of CAR T-cell area under the curve with response, minimal residual disease, and toxicity. AE, adverse event; AUC, area under the curve; CAR, chimeric antigen receptor; CRS, interleukin-1 release syndrome; MRD, minimal residual disease.

Figure 9: Peak interleukin levels over time.

Figure 10: Inflammatory markers in serum samples at baseline and at peak post-infusion. * Values represent the lower limit of quantification in the assay used. † Values represent the upper limit of quantification in the assay used. AE, adverse event; CAR, chimeric antigen receptor; CCL, C-C motif ligand; CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand; FGFBF, basal form of fibroblast growth factor; FLT-1, fms-related receptor tyrosine kinase 1; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IL, interleukin; MCP, monocyte chemokine protein-1; MDC, macrophage-derived MIP, macrophage inflammatory protein; PDL1, planned death ligand 1; PLGF, placental growth factor; Rα, receptor α; RA, receptor antagonist; SAA, serum amyloid A; SFASL, soluble Fas ligand; TARC, thymic and activation-regulated interleukin; TNF, tumor necrosis factor; VCAM, vascular cell adhesion protein; VEGF, vascular endothelial growth factor; VEGFC, vascular endothelial growth factor C; VEGFD, vascular endothelial growth factor D.

Figure 11: Correlation of serum biomarkers with interleukin-release syndrome and neurological events. * Values indicate the lower limit of quantification in the assay used. † Values indicate the upper limit of quantification in the assay used. CRP, C-reactive protein; CXCL, C-X-C motif trend factor ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNγ, interferon γ; IL, interleukin; IP, interferon γ-inducing protein; MCP, monocyte-inducing protein; Rα, receptor α; RA, receptor antagonist; SAA, serum amyloid A.

Figure 12: Pharmacodynamic profile of KTE-X19 in MCL morphological subgroups. AUC, area under the curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocytic protein-1; MIP-1β, macrophage inflammatory protein-1β; PD-L1, planned death-ligand 1; PRF, perforin; Rα, receptor α; TNF-α, tumor necrosis factor α.

Figure 13: Pharmacological profile of KTE-X19 in MCL morphological subgroups.

Figure 14: Pharmacodynamic profile of KTE-X19 in prior BTKi subgroups. AUC, area under the curve; CAR, chimeric antigen receptor; CXCL10, C-X-C motif chemokine ligand 10; IFN-g, interferon gamma; IL, interleukin; MCL, mantle cell lymphoma; MCP-1, monocytic protein-1; MIP-1β, macrophage inflammatory protein-1β; PD-L1, planned death-ligand 1; PRF, perforin; Rα, receptor α; TNF-α, tumor necrosis factor α.

Figure 15: Pharmacological profile of KTE-X19 in previous BTKi subgroups.

Figure 16: Persistent Response Rates in Subgroups

Figures 17A-17B show peak CAR T cell levels normalized by baseline tumor burden and sustained responses at the 12-month data cutoff.

Claims (28)

A use of autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR) for preparing a drug, wherein the drug is used to treat mantle cell lymphoma (MCL) or B cell ALL in an individual in need thereof, wherein the anti-CD19 CAR comprises a chimeric antigen receptor (CAR) of an anti-CD19 single chain variable fragment (scFv), a CD28 intracellular signaling region, and a CD3-ζ signaling domain, and wherein the anti-CD19 scFv comprises a heavy chain variable region and a light chain variable region of FMC63. The use as claimed in claim 1, wherein the MCL or B-cell ALL is relapsed or refractory (R/R) MCL or B-cell ALL, and depending on the circumstances, wherein the MCL is typical, blastoid or pleomorphic MCL. For use as claimed in claim 1, wherein the MCL or B-cell ALL is refractory to one or more of the following or relapses after one or more of the following: chemotherapy, radiotherapy, immunotherapy, autologous stem cell transplantation, or any combination thereof. For use as claimed in claim 1, wherein the individual has received 1-5 prior treatments, wherein at least one of the prior treatments is selected from autologous SCT, anti-CD20 antibody, chemotherapy containing anthracycline or bendamustine, and/or Bruton Tyrosine Kinase inhibitor (BTKi). For use as claimed in claim 4, wherein the BTKi is ibrutinib or acalabrutinib. The use of claim 2, wherein the R/RB cell ALL is defined as refractory to first-line therapy and after first remission

Relapse after 12 months

Relapsed or refractory after 2 lines of prior systemic therapy, or relapsed after allogeneic stem cell transplantation (SCT), where appropriate, the individual needs to have

5% bone marrow blasts, Eastern Cooperative Oncology Group performance status of 0 or 1, and/or adequate renal, liver, and heart function. The use of claim 6, wherein if the B-cell ALL individual has previously received blinatumomab, the individual needs to have CD19 expression

90% of leukemic blasts. The use as claimed in claim 1, wherein the individual receives bridging therapy after leukapheresis and before conditioning/lymphodepleting chemotherapy. The use of claim 1, wherein the MCL individual receives the following lymphodepleting chemotherapy regimen: intravenous cyclophosphamide 500 mg/m ² and intravenous fludarabine 30 mg/m ² , both of which are administered daily on the fifth day before, the fourth day before, and the third day before T cell infusion. The use of claim 1, wherein the B-cell ALL individual receives the following lymphodepleting regimen: 25 mg/m ² of fludarabine per day administered intravenously (IV) on the fourth day, the third day, and the second day before T cell infusion; and 900 mg/m ² of cyclophosphamide per day administered IV on the second day before infusion. The use of claim 8, wherein the transitional therapy is for MCL and is selected from dexamethasone, methylprednisolone, ibrutinib, acalabrutinib, immunomodulators, R-CHOP, bendamustine, alkylating agents, and/or platinum-based agents, and wherein the transitional therapy is administered after leukapheresis and, if appropriate, completed within 5 days or less prior to conditioning chemotherapy. The use of claim 8, wherein the transitional therapy is for B-cell ALL and comprises:

The use of any one of claims 1 to 12, wherein the autologous T cells comprise CD4+ and CD8+ CAR T cells, which are prepared from peripheral blood mononuclear cells (PBMCs) by positive enrichment of T cells before transduction of a polynucleotide encoding the CAR. The use of claim 13, wherein the PBMCs are enriched for T cells by positive selection for CD4+ and CD8+ cells, activated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and then transduced with a replication-defective viral vector comprising the polynucleotide. The use of claim 13, wherein the autologous T cells contain fewer cancer cells than T cells from a leukocyte separation product that have not been positively selected for CD4+ and CD8+ T cells. The use as claimed in claim 13, wherein the autologous T cells contain more naïve-like T cells than T cells from a leukocyte separation product that have not been positively selected/enriched for CD4+ and CD8+ T cells. The use of claim 16, wherein the autologous T cells further have a reduced percentage of differentiated T cells, an increased percentage of CD3+ cells, reduced IFN-γ production, and/or a reduced percentage of CD3- cells relative to T cells from a leukocyte separation product that have not been positively selected/enriched for CD4+ and CD8+ T cells. The use of claim 13, wherein one or more doses of 1.8×10 ⁶ , 1.9×10 ⁶ or 2×10 ⁶ CAR-positive live T cells per kilogram of body weight are administered to the MCL individual, wherein the maximum is 2×10 ⁸ CAR-positive live T cells (for patients weighing more than 100 kg), and 0.5×10 ⁶ , 1×10 ⁶ or 2×10 ⁶ CAR-positive live T cells per kilogram of body weight are administered to the B-cell ALL individual, wherein the maximum is 2×10 ⁸ CAR-positive live T cells (for patients weighing more than 100 kg). The use of claim 13, wherein if the individual has achieved a complete response to the first infusion, then if remission develops >3 months later, the individual may receive a second infusion of anti-CD19 CAR T cells, provided that CD19 expression has been preserved and there are no suspected neutralizing antibodies against the CAR, wherein the response is assessed using the Lugano classification. The use as claimed in claim 13, wherein the subject is monitored for signs and symptoms of cytokine release syndrome (CRS) and/or neurotoxicity after administration of T cells. For use as claimed in claim 20, wherein the subject is monitored daily for signs and symptoms of CRS and neurotoxicity for at least seven days, preferably four weeks, after infusion. For use as claimed in claim 20, the signs or symptoms associated with CRS include fever, chills, fatigue, tachycardia, nausea, hypoxia and hypotension, and the signs and symptoms associated with neurological events include encephalopathy, epilepsy, changes in mental status, speech disorders, tremors and confusion. The use of claim 20, wherein the CRS of the MCL individual is managed according to the following regimen:

The use of claim 20, wherein the neurotoxicity of the MCL subject is managed according to the following regimen:

The use of claim 13, wherein the Ki-67 tumor proliferation marker is used

50% and/or the presence of TP53 mutations, the MCL individual was considered a high-risk patient. The use of claim 20, wherein the CRS of the B-cell ALL individual is managed according to the following regimen:

The use of claim 20, wherein the neurotoxicity in the B-cell ALL individual is managed according to one of the following two regimens:

The use of claim 13, wherein the B-cell ALL individual can receive any one or more of the following transitional chemotherapy regimens: