CN113244389B

CN113244389B - Preparation method of TILs (dendritic cells) and application of TILs in tumor treatment

Info

Publication number: CN113244389B
Application number: CN202110759185.4A
Authority: CN
Inventors: 亓爱杰; 李少波; 姜广建
Original assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Current assignee: Nuosa Union Beijing Biomedical Technology Co ltd
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2021-09-17
Anticipated expiration: 2041-07-06
Also published as: CN113244389A

Abstract

The invention relates to a preparation method of TILs cell and application in tumor treatment. The invention provides the tumor infiltrating lymphocyte which is effectively separated and prepared, after the cell is transformed into TNF-alpha, the cell is highly expressed and is used together with the prepared CTLA-4 monoclonal antibody, the proliferation of lung cancer tumor can be obviously inhibited, and the effect is better.

Description

Preparation method of TILs (dendritic cells) and application of TILs in tumor treatment

Technical Field

The invention relates to the field of biology, in particular to a preparation method of TILs (dendritic cells) and application of the TILs in tumor treatment.

Background

Tumor Infiltrating Lymphocytes (TILs) are tumor antigen specific CD4+ and CD8+ cell populations separated from tumor tissues, the curative effect of the TILs is 50-100 times of that of lymphokine activated killer cells (LAK), wherein the CD8+ T cells play a role in killing tumors, and a large number of clinical studies show that TILs combined with high-dose interleukin-2 (IL-2) can achieve about 50% of positive reaction rate in patients with late-stage Multiple Myeloma (MM), and the continuous remission time is long, so that MM can be cured possibly. Under the long-term stimulation of tumor antigens, the expression levels of some inhibitory receptors (PD-1, CTLA-4, Tim-3, LAG-3 and the like) on the surface of tumor infiltrating lymphocytes are up-regulated and are in a 'failure' state, a large number of researches show that the function of TILs can be recovered by blocking the pathway of the inhibitory receptors, the tumor killing capacity of the TILs can be obviously improved, and the TILs have an extremely important function in tumor immunotherapy, thereby providing a basis for the targeted therapy of tumors. Ln-145 is a newly developed TIL treatment method, and in 2019, the FDA awards a Tumor Infiltrating Lymphocyte (TIL) treatment method Ln-145 as a breakthrough treatment designation, which is the first time that cell immunotherapy for solid tumors gets the great honor, and is expected to be marketed in 2021, which is the first cell immunotherapy for solid tumors once FDA approval, and will bring a huge survival benefit to cancer patients.

With regard to the anti-tumor mechanism of TIL, it has been demonstrated to date that: direct killing action: the killing of tumor cells by TIL is mainly dependent on the effective activation and function of CTLs therein. The transformation of T cells into effector cells requires two stimulatory signals: the first activation signal is generated by TCR interaction with an antigenic peptide: a Major Histocompatibility Complex (MHC) molecule complex; the second activation signal is provided by CD28 on T cells binding to the B7 molecule on Antigen Presenting Cells (APC). The differentiated mature effector T cells perform the killing function through four stages of recognition and combination, organelle rearrangement and particle exocytosis, CTL dissociation and target cell disintegration, and specifically crack the target cells; fas-mediated apoptosis: lymphocytes are combined with Fas on target cells through Fas ligand on the surface to cause the fragmentation of cell DNA, so that the cells are subjected to apoptosis; (iii) perforin (PFP)/granzyme B (granmeb) mediated apoptosis: CTLs can kill target cells by exocytosis of cytoplasmic granules, releasing the contents of the granules. Perforin can polymerize on the target cell membrane to form a transmembrane pore and thereby lyse the target cell. Granzyme B can induce apoptosis through a dependent/independent caspase pathway; TIL secrete some inflammatory cytokines such as TNFct, IL2 and the like, which mediate cell lysis and apoptosis and kill target cells by releasing particles or direct cell contact. Various studies have been developed that indicate that TIL treatment will be subject to constant improvement and development and will ultimately be a new weapon against cancer in humans.

Lung cancer is often associated with malignant pleural effusion, which is associated with post-pleural-metastatic effusion. In clinic, thoracocentesis is usually used for liquid extraction and intrathoracic injection of cisplatin (DDP), mitomycin C (MMC), Bleomycin (BLM) and other therapeutic drugs for treatment, but a plurality of tumor chemotherapeutic drugs can kill cancer cells in short term, and the cancer cells are easy to generate drug resistance in long term use, so that the specific killing performance to tumors is not strong. The combined or alternating use of TIL and chemotherapeutic agents may improve patient survival. There are three approaches to extracting TIL commonly used for treating lung cancer: (1) extracting from malignant pleural effusion, centrifuging heparin anticoagulation sterile pleural effusion, preparing cell with concentration of 2X10Yml, suspending in Hank solution, assisting with lymphocyte separation solution, and collecting cells on the interface. Cultured with 10% human AB serum and rIL-2, and the solution was changed as needed depending on the amplification and metabolism. (2) Extracting from tumor body or biopsy specimen removed by operation, digesting and dissociating the obtained tissue, and subjecting the digested single cell suspension toAnd (4) performing discontinuous density gradient centrifugation to obtain relatively pure TIL. (3) Metastatic lymph nodes, those obtained by surgery or biopsy, are also rich in TIL, and 1g lymph node can be isolated by about 10⁸TILs, sufficient to culture for therapeutic amounts. However, the current research is not enough, and the treatment of lung cancer is not enough.

Disclosure of Invention

The present invention overcomes the deficiencies of the prior art. A TIL cell and a corresponding monoclonal antibody for treating lung cancer are provided.

In one aspect, the present invention provides a method for preparing a TIL cell, the method comprising the steps of: soaking fresh lung cancer tumor tissue in cold Hanks' solution containing antibiotics for 30min, removing necrosis, hemorrhage and adipose tissue, weighing, cutting into lmm × lmm × lmm small pieces, adding into collagenase II type digestive enzyme solution prepared by RPMI1640, performing shake digestion for 12h, adding hyaluronidase and DNase, and stirring for 4h for digestion treatment. And (3) passing the completely digested tissue through a 120-mesh copper net to obtain a single cell suspension containing TIL and tumor cells. And lightly adding 3ml of mixed cell suspension on an lml calf serum interface, centrifuging at 500 r/min for 15s, then obtaining tumor cells at the bottom layer, and performing continuous gradient separation on the upper layer cell suspension to obtain the TIL. The isolated TIL was cultured in RPMI1640 complete medium containing 10% human AB serum at 1X 10⁶Culturing in 24-well plate at initial concentration, adding IL-210 μ g/ml and adding OKT₃Culturing 10 mu g/ml and VC 1 mu g/ml in a 5% CO2 incubator at 37 ℃, carrying out subculture once in 3-5 days, and carrying out CO-culture for 20 days to obtain the target TIL cell.

In another aspect, the invention provides a transgenic TIL cell that is highly expressed as a TNF-a transgene; specifically, the recombinant TNF-a retrovirus is used for transfecting the TIL cell prepared by separation; the TNF-a gene recombinant retrovirus is obtained by connecting a TNF-a gene to a retrovirus vector, and can be constructed in a manner commonly used in the field.

Furthermore, the invention provides a drug combination for treating lung cancer, which comprises a transgenic TIL cell and a CTLA-4 monoclonal antibody, wherein the TNF-a transgene of the TIL cell is highly expressed; the heavy chain variable region sequence of the CTLA-4 monoclonal antibody is shown as SEQ ID NO: 1, the light chain variable region sequence is shown as SEQ ID NO: 2, respectively.

In another aspect, use of a combination of TIL cells comprising a transgene and a CTLA-4 monoclonal antibody for the preparation of a pharmaceutical composition for the treatment of lung cancer; wherein, the TIL cell is high-expression of TNF-a transgene; the heavy chain variable region sequence of the CTLA-4 monoclonal antibody is shown as SEQ ID NO: 1, the light chain variable region sequence is shown as SEQ ID NO: 2, respectively.

In some embodiments, the amount of TIL sufficient for a therapeutically effective dose is about 1 × 10⁷To about 1X 10¹⁰. Any suitable dose of TIL may be administered. Preferably about 1X 10⁷To about 13X 10¹⁰Administering PIL, on average about 7-8X 10⁶Per gram.

In some embodiments, the method further comprises the step of cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation method.

In some embodiments, the harvested TIL population is used in combination with 1: 1 ratio for the cryopreservation process.

The pharmaceutical composition is a suspension of TIL in sterile buffer.

The TILs amplified using the methods of the present invention may be administered by any suitable route known in the art. Preferably, the TIL is administered as a single intra-arterial or intravenous infusion, which preferably lasts about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intramembranous.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form of administration of the compound, the sex and age of the subject to be treated, the weight of the subject to be treated and the preferences and experience of the attending physician. Clinically determined doses of TIL may also be used, if appropriate. The amount of the pharmaceutical composition, e.g., dose of PBLs, administered using the methods herein will depend on the human or mammal being treated, the severity of the condition or disorder, the rate of administration, the disposition of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some embodiments, the TIL may be administered in a single dose. Such administration may be by injection, for example, intravenously. In some embodiments, the TIL may be administered in multiple doses. The dosage may be once, twice, three times, four times, five times, six times or more per year. The dose may be monthly, biweekly, weekly or every other day. The administration of TIL may be continued as desired, and an effective amount of TIL may be administered in single or multiple doses by any acceptable mode of administration of agents having similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, transplantation or direct injection into the tumor, or by inhalation.

In some embodiments, the present invention provides a pharmaceutical composition as described in any of the preceding paragraphs that is applicable to the above improvements such that the pharmaceutical composition comprises 5 x 107 to 20 x 109TIL.

In some embodiments, the present invention provides a pharmaceutical composition as described in any of the preceding paragraphs that is applicable, the pharmaceutical composition being modified such that the pharmaceutical composition further comprises a cryopreservative.

In some embodiments, the present invention provides the applicable pharmaceutical composition described in any of the preceding paragraphs, modified such that the pharmaceutical composition further comprises at least about 5% (v/v) dimethyl sulfoxide (DMSO).

Advantageous effects

The invention provides the tumor infiltrating lymphocyte which is effectively separated and prepared, after the cell is transformed into TNF-alpha, the cell is highly expressed and is used together with the prepared CTLA-4 monoclonal antibody, the proliferation of lung cancer tumor can be obviously inhibited, and the effect is better.

Drawings

FIG. 1 is a graph of the effect of TIL cells in combination with CTLA-4 antibodies

Detailed Description

Examples will be given below and further illustrate the present invention, but the examples are merely illustrative of the embodiments of the present invention and do not limit the scope of the present invention.

Example 1 isolation and proliferation of Lung cancer tumor infiltrating lymphocytes

Soaking fresh lung cancer tumor tissue in cold Hanks' solution containing antibiotics for 30min, removing necrosis, hemorrhage and adipose tissue, weighing, cutting into lmmxlmmxlmm small pieces, adding into collagenase II type digestive enzyme solution prepared by RPMI1640, performing shake digestion for 12h, adding hyaluronidase and DNase, and stirring for 4h for digestion treatment. And (3) passing the completely digested tissue through a 120-mesh copper net to obtain a single cell suspension containing TIL and tumor cells.

And lightly adding 3ml of mixed cell suspension on an lml calf serum interface, centrifuging at 500 r/min for 15s, then obtaining tumor cells at the bottom layer, and performing continuous gradient separation on the upper layer cell suspension to obtain the TIL. The resulting TIL was assayed for viability by trypan blue exclusion and smeared with MGG staining and the percentage of lymphocytes determined under light microscopy. The results show that the yield of TIL reaches 89.3%, and the purity is 82.3%.

The isolated TIL was cultured in RPMI1640 complete medium containing 10% human AB serum at 1X 10⁶Culturing in 24-well plate at initial concentration, adding IL-210 μ g/ml and adding OKT₃10 ug/ml and VC 1 ug/ml, 37 ℃ and 5% CO₂And (5) performing incubator culture, and performing subculture once in 3-5 days for 20 days. The results show that VC can obviously help to shorten the inhibition period of TIL, the 20d amplification multiple reaches 53 times, and the TIL in vitro amplification reaches a therapeutic dose of 10⁹The desired concentration.

Lymphocyte phenotype determination cells were counted using an indirect immunofluorescent staining method with a fluorescent microscope. anti-CD 3, CD4, CD8, CDl6, CDl9, CD25 monoclonal antibody and goat anti-mouse FITC-Ig, according to the instructions. The result shows that after the induction of the three factors, the proportion of CD3+ cells is remarkably increased, the highest proportion is 99.0%, and the proportion of CD8+ cells is remarkably increased to 71.3%; no significant increase in CD4+ cells; HLA-DR cells were significantly increased to 99.4%; CD25+ cells increased to 64.3%. Indicating that the TIL cells proliferated better.

EXAMPLE 2 preparation of transgenic TIL cells

The transgenic TIL cell was prepared according to the construction method of "the construction of tumor infiltrating lymphocytes with TNF-. alpha.gene and the study of their biological properties, Royle leather et al, proceedings of the university of medical science, 2002, 19(2), p 151-154". Specifically, a cell line FIyRUFN14 containing a recombinant retrovirus for human TNF- α gene was prepared. Culturing the cell strain, taking culture solution supernatant, adding human Polybrane to the final concentration of 8 mg/L, adding the supernatant into the logarithmic growth phase TIL cell prepared in example 1, transfecting for 3 days, and adding 400ug/ml G418 for screening. After 2 weeks, the clones were subjected to limiting dilution, and the culture was continued in a culture medium containing 1000 u/ml of IL-2. Another TIL cell with the same concentration of G418 was cultured as a control.

Determination of TNF- α secretion: the TIL and the transgenic TIL are respectively prepared to have the concentration of 5 multiplied by 10 cell number⁵M1, adding into a cell culture plate, and culturing for 72h to respectively carry out TNF-alpha detection. Detection was performed according to the methods provided in the kit. The result shows that the result of the TIL cell is negative in 72h detection, and the TNF-alpha secretion amount of the transgenic TIL cell reaches 523.7pg, so that the expression effect is remarkably high. Amplifying the TIL cells positive for the transgene for standby.

EXAMPLE 3 preparation of CTLA-4 monoclonal antibody

The antigen is CTLA-4 recombinant protein, the product number is JN1568, and the antigen is Baiolaibo. Injecting 10mg of immune antigen into the neck and back of a BALB/c mouse, boosting the immunity for 1 time every 2 weeks, taking blood after 7-10 days of tail breaking after 3-immunization, and detecting the titer of a serum antibody by an indirect ELISA method, wherein the specific steps are as follows: the detection raw material mass concentration is 5 mug/mL, each hole of the 96-hole enzyme label plate is 100 mug L, and the enzyme label plate is coated overnight at 4 ℃; washing with 0.01mol/L Phosphate Buffer Solution (PBS) containing 0.1% Tween-20 by volume for 3 times (300 mL each time), and patting to dry (the same below); adding a washing solution containing 10% fetal calf serum by volume fraction into each hole, sealing, and incubating at 37 ℃ for 2 h; washing and drying; diluting the serum to be detected by 1000 times, adding 100 mu L of the serum to each hole, setting a blank control hole (PBS) and a negative control hole (negative serum), and incubating for 45min at 37 ℃; washing and drying; adding 100 μ L of 10000 times diluted HRP-labeled goat anti-mouse IgG into each well, and incubating at 37 deg.C for 30 min; washing and drying; adding 100 mu L of substrate color development liquid into each hole, and reacting for 15min at 37 ℃ in a dark place; adding 50 mu L of stop solution into each hole to stop the reaction; 450nm waveThe absorbance (A) is measured at the strong point, the absorbance of the negative control hole is A1, the absorbance of the positive control hole is A2, and the positive result is A2/A1 which is more than or equal to 2.1. And selecting the mouse with the best immune effect for cell fusion. Fusing by adopting a polyethylene glycol method, and after fusing for 8d, replacing a HT culture medium when cell colonies visible to the naked eye appear; detecting the supernatant by using an indirect ELISA method, and screening positive hybridoma cells; selecting a single clone with higher absorbance from the positive hole, and performing subcloning by a limiting dilution method until obtaining a hybridoma cell strain 3A7 stably secreting the antibody; the 3A7 hybridoma is injected into a mouse body to induce ascites, and the ascites is purified by adopting a method of caprylic acid-ammonium sulfate precipitation to obtain a purified 3A7 monoclonal antibody. The protein mass concentration of the protein is measured to reach 27.4mg/mL by an ultramicro nucleic acid protein measuring instrument, and the titer is measured to be 10 by indirect ELISA⁶The method has better effect. The subtype of the monoclonal antibody was determined to be IgG1 by indirect ELISA. Through specificity verification, the monoclonal antibody is found to be specifically combined with CTLA-4 protein only and not combined with other proteins. In addition, the dissociation constant of the antibody and the antigen is 3.7nM and has better binding property by adopting the conventional monoclonal antibody affinity determination method in the field.

The light chain variable region sequence and the heavy chain variable region sequence of the obtained antibody are respectively shown as follows by adopting the conventional light chain and heavy chain sequence identification method in the field,

SEQ ID NO: 1: heavy chain variable region sequence

EVKLVESGGGLVKPGGSLKLYCAASGFTFSHYHTIWVRQIPEKRLEWVAIFMTWYAVMCLRREYSRFTISRDNARNICYLQMNSLRSDATAMYYCARPDWSRAREGIPWGQGTTLTVSS

SEQ ID NO: 2: light chain variable region sequence

DIVLTQSPASLAVQLGQRATISCTVDTTICWIARIGDGWYQQKPGQPPKLLIYLDPNSLEGVPARFSGSGSGTDFSRNIHPVEEDDIAMYFCAPMNAWYMKFGAGTKLELK。

Example 4 preparation of Lung cancer model and therapeutic experiment

Preparation of mouse Lewis lung cancer transplantable tumor: culturing mouse Lewis lung cancer cell with 8% fetal calf serum DMEM medium, passaging for 2-3 times, collecting logarithmic growth phase cell, and culturing with the culture mediumDigesting with 0.25% trypsin, centrifuging at 1000r/min for 10min, collecting eluate, adding 0.9% physiological saline, suspending cells, and diluting to 1.0 × 10⁶0.1ml of the solution was used for inoculation of the subject mice.

Control group 1: injecting normal saline into the abdominal cavity of 10 control mice;

control group 2: non-transgenic TIL cells were grown at a cell density of 5X 10 at day 3 post tumor inoculation⁷one/mL, 0.1 mL/time from the tail vein of each tumor-bearing mouse, 1 time per day for 10 consecutive days.

Experimental group 1: on days 3 and 10 post tumor inoculation, 100 μ g of CTLA-4 antibody (3 a 7) was injected intraperitoneally into mice, respectively.

Experimental group 2: on day 3 after tumor inoculation, the transgenic positive TIL cells prepared in example 2 were used at a cell density of 5X 10⁷one/mL, 0.1 mL/time from the tail vein of each tumor-bearing mouse, 1 time per day for 10 consecutive days.

Experimental group 3: mice were injected intraperitoneally with 100 μ g CTLA-4 antibody (3 a 7) on days 3 and 10 after tumor inoculation, respectively; meanwhile, on day 3 after tumor inoculation, the cell density of the transgenic positive TIL cells prepared in example 2 was 5X 10⁷one/mL, 0.1 mL/time from the tail vein of each tumor-bearing mouse, 1 time per day for 10 consecutive days.

And (4) measuring the transplanted tumor index. The maximum diameter (a) and the transverse diameter (b) of the tumor were measured by modeling every 3d from the treatment start day, and the volume was calculated. And (4) treating 21d, taking out the spleen of the mouse after the cervical dislocation method in the 22 nd row is killed, measuring the tumor volume, and calculating the tumor growth inhibition rate. Growth inhibition rate = (control group tumor weight-experimental group tumor weight)/control group tumor weight × 100%. The results are shown in FIG. 1.

As can be seen from FIG. 1, the tumor growth inhibition ratio of the control group is 0, the tumor inhibition ratio of the non-transgenic TIL cells is about 5.4%, the tumor inhibition ratio of the transgenic TIL cells reaches 56.7%, the tumor inhibition ratio of the monoclonal antibody used alone reaches 45.2%, the tumor inhibition ratio of the monoclonal antibody used together with the transgenic TIL cells reaches 89.6%, and the control group has a good synergistic treatment effect.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Sequence listing

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Claims

1. A product, which is a drug combination for treating lung cancer, consists of transgenic TIL cells and CTLA-4 monoclonal antibodies, wherein the transgenic TIL cells are the TIL cells with high expression of transgenic TNF-a genes; the heavy chain variable region sequence of the CTLA-4 monoclonal antibody is shown as SEQ ID NO: 1, the light chain variable region sequence is shown as SEQ ID NO: 2 is shown in the specification; the TIL cell is prepared by adopting the following method: soaking fresh lung cancer tumor tissue in cold Hanks' solution containing antibiotics for 30min, removing necrosis, hemorrhage and adipose tissue, weighing, cutting into lmm × lmm × lmm small blocks, adding into collagenase II type digestive enzyme solution prepared by RPMI1640, performing shake digestion for 12h, adding hyaluronidase and DNase, and stirring for 4h for digestion treatment; passing the completely digested tissue through a 120-mesh copper net to obtain a single cell suspension containing TIL and tumor cells; lightly adding the mixed cell suspension on a calf serum interface, centrifuging for 15s at 500 r/min, taking tumor cells as a bottom layer, and performing continuous gradient separation on the upper layer cell suspension to obtain TIL; the isolated TIL was cultured in RPMI1640 complete medium containing 10% human AB serum at 1X 10⁶Culturing in 24-well plate at initial concentration, adding IL-210 μ g/ml and adding OKT₃10 ug/ml and VC 1 ug/ml, 37 ℃ and 5% CO₂And (5) culturing in an incubator, carrying out passage once for 3-5 days, and carrying out co-culture for 20 days to obtain the target TIL cell.

2. Use of a combination of transgenic TIL cells and a CTLA-4 monoclonal antibody for the preparation of a pharmaceutical composition for the treatment of lung cancer; the transgenic TIL cell is a TNF-a gene-transferred high-expression TIL cell; the heavy chain variable region sequence of the CTLA-4 monoclonal antibody is shown as SEQ ID NO: 1, the light chain variable region sequence is shown as SEQ ID NO: 2 is shown in the specification; the TIL cell is prepared by adopting the following method: soaking fresh lung cancer tumor tissue in cold Hanks' solution containing antibiotics for 30min, removing necrosis, hemorrhage and adipose tissue, weighing, cutting into lmm × lmm × lmm small blocks, adding into collagenase II type digestive enzyme solution prepared by RPMI1640, performing shake digestion for 12h, adding hyaluronidase and DNase, and stirring for 4h for digestion treatment; passing completely digested tissue through 120 mesh copper net to obtain single cell containing TIL and tumor cellSuspending the solution; lightly adding the mixed cell suspension on a calf serum interface, centrifuging for 15s at 500 r/min, taking tumor cells as a bottom layer, and performing continuous gradient separation on the upper layer cell suspension to obtain TIL; the isolated TIL was cultured in RPMI1640 complete medium containing 10% human AB serum at 1X 10⁶Culturing in 24-well plate at initial concentration, adding IL-210 μ g/ml and adding OKT₃10 ug/ml and VC 1 ug/ml, 37 ℃ and 5% CO₂And (5) culturing in an incubator, carrying out passage once for 3-5 days, and carrying out co-culture for 20 days to obtain the target TIL cell.

3. Use according to claim 2, characterized in that: the amount of TIL cells is an amount of TIL sufficient for a therapeutically effective dose, said amount being 1X 10⁷To 1X 10¹⁰。