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WO2023104099A1 - 靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用 - Google Patents

靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用 Download PDF

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WO2023104099A1
WO2023104099A1 PCT/CN2022/137265 CN2022137265W WO2023104099A1 WO 2023104099 A1 WO2023104099 A1 WO 2023104099A1 CN 2022137265 W CN2022137265 W CN 2022137265W WO 2023104099 A1 WO2023104099 A1 WO 2023104099A1
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amino acid
cells
antibody
seq
cdr
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French (fr)
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徐伟
危华锋
比尼亚 达洛夫斯基·戴安娜
许丹
姚影
普里恩斯·比安卡
博兰·奈德赛卡恩
盖根·詹姆斯
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信达细胞制药(苏州)有限公司
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Priority to CN202280081143.2A priority Critical patent/CN118317789A/zh
Priority to EP22903520.9A priority patent/EP4445912A1/en
Publication of WO2023104099A1 publication Critical patent/WO2023104099A1/zh

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Definitions

  • the present invention generally relates to the fields of antibody engineering and cellular immunology, in particular, the present invention relates to an antibody specifically binding to a B-cell maturation antigen (BCMA) comprising a P329G mutation and engineered to express a molecular switch A combination of regulatory chimeric antigen receptor immune effector cells (eg, T cells, NK cells), and relates to the use of said combination in the treatment of diseases associated with the expression of BCMA, such as cancers expressing or overexpressing BCMA.
  • BCMA B-cell maturation antigen
  • a combination of regulatory chimeric antigen receptor immune effector cells eg, T cells, NK cells
  • BCMA B cell maturation antigen
  • CD269 CD269, TNFRSF17
  • TNFRSF tumor necrosis factor receptor superfamily
  • BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of TNFR family members in the extracellular domain (ECD), which forms a ligand-binding motif.
  • Ligands of BCMA include B-cell activating factor (BAFF) and B-cell proliferation-inducing ligand (APRIL), wherein B-cell proliferation-inducing ligand (APRIL) binds to BCMA with a higher affinity to promote tumor cell proliferation.
  • BAFF B-cell activating factor
  • APRIL B-cell proliferation-inducing ligand
  • BCMA is mainly expressed on the surface of mature B cells, that is, plasma cells, and is not expressed in normal hematopoietic stem cells and non-blood-derived tissues. BCMA signaling is indispensable for the survival of long-lasting bone marrow plasma cells, but it is not necessary for overall B cell homeostasis. BCMA on the membrane surface can be cleaved by ⁇ -secretase and shed, and the resulting soluble BCMA (sBCMA) may reduce BCMA signal transduction on the membrane surface by blocking BAFF/APRIL ligand binding.
  • BCMA was found to be overexpressed in multiple myeloma (Multiple Myeloma, MM) cells, which up-regulates canonical and non-canonical NF- ⁇ B signaling, promotes the growth, survival, and adhesion of MM cells, and induces osteoclasts BCMA expression has become an important marker for the diagnosis of MM.
  • MM Multiple Myeloma
  • sBCMA in the serum of MM patients increases, which is proportional to the number of MM cells in the bone marrow, and its concentration changes are closely related to the prognosis and treatment response of MM.
  • BCMA has become a popular target for the treatment of MM.
  • targeted drugs including chimeric antigen receptor T cells.
  • CAR-T CAR-T immunotherapy, among which Abecma (idecabtagene vicleucel, ide-cel) of Bluebird (Bluebird) has been approved by the FDA in March 2021, for the treatment of relapsed and refractory MM (RRMM) with 4 lines or above
  • RRMM refractory MM
  • Ciltacabtagene autoleucel Cilta-cel
  • BLA Biologics License Application
  • Abecma as the first CAR-T cell therapy targeting BCMA, recognizes and binds to the BCMA protein on multiple myeloma cancer cells, leading to the death of cancer cells expressing BCMA protein. That is, it directly targets the surface antigen BCMA protein of tumor cells through the chimeric antigen receptor (CAR) on CAR-T cells, so as to achieve the purpose of recognizing and killing tumor cells.
  • CAR chimeric antigen receptor
  • CRS cytokine release syndrome
  • NT neurotoxicity
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • CD19-targeting CAR-T cells are clinically used to treat CD19-positive blood tumors. In addition to eliminating tumor cells, CD19 CAR-T cells also kill normal B cells.
  • CAR-T cells persist in the body, although they have long-term recurrence-free survival with patients It is closely related, but it also leads to the long-term dysplasia of B cells in the body, the loss of humoral immunity, and it is easy to cause infection. Clinical trials have shown that 27-36% of lymphoma patients developed bacterial infection within 30 days after receiving CD19CAR-T cell therapy, and 9.2-28% of patients developed viral infection one month later, requiring a median time of 6.7 months for B cells to recover, 31 -64% of patients required gamma globulin replacement therapy. In addition, the continuous activation of CAR-T cells in the body can easily lead to functional exhaustion, impair its anti-tumor effect, reduce its persistence in the body, and thus reduce the long-term therapeutic effect.
  • CAR-T cells In order to reduce the "on-target/off tumor” toxicity of CAR-T cells, the following strategies are generally adopted in the prior art.
  • One strategy is to design the antigen-binding domain contained in the CAR to target antigens that are highly expressed on the surface of tumors but not or lowly expressed in normal tissues during CAR design.
  • Another strategy is to strictly control the dose of T cells administered, because too many CAR-T cells will increase exponentially after antigen stimulation, which is more likely to cause on-target/off-tumor effects.
  • Another strategy is to introduce an inducible suicide gene, such as an inducible Caspase-9 (iCasp9) suicide gene, when constructing a CAR, and administer AP1903 (a gene that can activate iCasp9) when on-target/off-tumor toxicity is observed in the patient.
  • an inducible suicide gene such as an inducible Caspase-9 (iCasp9) suicide gene
  • AP1903 a gene that can activate iCasp9 when on-target/off-tumor toxicity is observed in the patient.
  • Dimerization chemical inducer induces CAR-T cell apoptosis and reduces toxicity
  • Zhang Huihui et al. Suicide gene as a "safety switch” to control the preclinical study of CAR-T cell toxicity, Chinese Journal of Cancer Biotherapy, 2021,28(3):225-231
  • HSV-TK herpes simplex virus thymidine kinase
  • T cells transiently express CAR by electroporation and transfection, and play a therapeutic role through transient killing function
  • CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia [J] Leukemia. 2015;29(8):1637–1647).
  • the above-mentioned regulatory methods are all carried out after toxicity occurs, and the mitigation effect of toxicity depends on the effect and efficiency of the drug to clear CAR-T cells, and CAR-T cells cannot be recovered after clearing, which affects the long-term anti-tumor efficacy.
  • the inventors designed and constructed P329G CAR-T cells targeting BCMA by binding to the Fc domain of an antibody that specifically binds to BCMA molecules containing a P329G mutation, and verified its specificity and controllability in vivo and in vitro. antitumor effect.
  • the drug combination of the present invention includes two components: BCMA-specific P329G antibody and P329G CAR-T cells.
  • the P329G CAR-T cells are redirected to the tumor cells by recognizing the Fc domain of the P329G antibody, producing tumor recognition and killing effects (see Figure 1B).
  • the P329G antibody serves as a bridge connecting P329G CAR-T cells and tumor cells, and acts as a "molecular switch" to regulate the activity of P329G CAR-T cells.
  • the present invention provides a P329G mutant antibody capable of specifically binding to BCMA molecules as a "molecular switch" for immune effector cells (for example, T cells, NK cells) expressing CAR polypeptides, including but not Limited to ADI-38497 PG Ab (also referred to herein as “ADI-38497 PG Antibody”, “38497 PG Ab”, “ADI-38497 PG IgG”, “38497 PG IgG”, “PG 38497 Antibody”), ADI-38484 PG Ab (also referred to herein as "ADI-38484 PG antibody”, “38484 PG Ab”, "ADI-38484 PG IgG”, “38484 PG IgG", “PG 38484 antibody”).
  • ADI-38497 PG Ab also referred to herein as "ADI-38497 PG Antibody”
  • 38497 PG Ab also referred to herein as "ADI-38497 PG Antibody
  • ADI-38497 PG IgG also referred to herein
  • the present invention obtains an antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering, or no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H1 CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H2; and the amino acid sequence DRGDQILDV (SEQ ID NO:27) shown in CDR H3, or the variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H3;
  • the light chain variable region comprises the amino acid sequence RASQSISRYLN (SEQ ID NO:28) shown in the CDR L1, or the CDR L1 variants with no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2, Or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR L2; and
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence NDVIS (SEQ ID NO: 31) according to Kabat numbering, or no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H1 CDR H2 shown in amino acid sequence VIIPIFGIANYAQKFQG (SEQ ID NO: 32), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of said CDR H2; and amino acid sequence GRGYYSSWLHDI (SEQ ID NO:33) shown in CDR H3, or the variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H3;
  • the light chain variable region comprises the amino acid sequence QASQDITNYLN (SEQ CDR L1 shown in ID NO:34), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of said CDR L1; CDR L2 shown in the amino acid sequence DASNLET (SEQ ID NO:35), Or a variant of no more than 2
  • amino acid changes are additions, deletions or substitutions of amino acids.
  • the invention provides an antibody or antigen-binding fragment that specifically binds a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence SSSYYWT (SEQ ID NO:25) according to Kabat numbering; CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO:26); and the amino acid sequence The CDR H3 shown in DRGDQILDV (SEQ ID NO:27);
  • the light chain variable region comprises the CDR L1 shown in the amino acid sequence RASQSISRYLN (SEQ ID NO:28) according to Kabat numbering; The amino acid sequence AASSLQS (SEQ ID NO: CDR L2 shown in 29); and CDR L3 shown in the amino acid sequence QQKYFDIT (SEQ ID NO:30);
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence NDVIS (SEQ ID NO:31) according to Kabat numbering; CDR H2 shown in the amino acid sequence VIIPIFGIANYAQKFQG (SEQ ID NO:32); and the amino acid sequence CDR H3 shown in GRGYYSSWLHDI (SEQ ID NO:33);
  • the light chain variable region comprises CDR L1 shown in the amino acid sequence QASQDITNYLN (SEQ ID NO:34) according to Kabat numbering; Amino acid sequence DASNLET (SEQ ID NO: CDR L2 shown in 35); and CDR L3 shown in the amino acid sequence QQAFDLIT (SEQ ID NO:36).
  • the present invention obtains an antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 2 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 9 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 10 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence.
  • the present invention obtains an antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the sequence of SEQ ID NO:2, and the light chain variable region comprises the sequence of SEQ ID NO:3;
  • the heavy chain variable region comprises the sequence of SEQ ID NO:9, and the light chain variable region comprises the sequence of SEQ ID NO:10.
  • an antibody of the invention that specifically binds a BCMA molecule is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody.
  • the antigen-binding fragment of an antibody of the invention that specifically binds a BCMA molecule is Fab, Fab', F(ab') 2 , Fv, single chain Fv, single chain Fab, diabody.
  • an antibody with a mutated Fc domain is obtained by mutating the amino acid at the P329 position according to EU numbering to glycine (G), wherein, with the unmutated parental antibody The Fc ⁇ receptor binding of the mutant Fc domain was reduced compared to the Fc ⁇ receptor binding of the Fc domain.
  • the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably , the mutant Fc domain is a mutant Fc domain of an IgG1 antibody;
  • the antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprises the heavy chain constant region sequence shown in SEQ ID NO: 5 or at least 90%, 91%, 92%, 93%, 94%, A sequence of 95%, 96%, 97%, 98% or 99% identity and wherein the amino acid at position P329 according to EU numbering is mutated to G;
  • the antibody or antigen-binding fragment comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the heavy chain constant region sequence shown in SEQ ID NO: 5 , a sequence of 98% or 99% identity and wherein the amino acid at position P329 according to EU numbering is mutated to G; and the light chain constant region sequence shown in SEQ ID NO: 6 or at least 90%, 91%, 92% therewith %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences.
  • the antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprises the heavy chain constant region sequence shown in SEQ ID NO:5 and the light chain constant region sequence shown in SEQ ID NO:6.
  • the present invention provides a nucleic acid encoding the antibody of the first aspect of the invention, a vector comprising the nucleic acid encoding the antibody, a cell comprising the nucleic acid molecule or vector, and a method for producing the antibody, the The method includes culturing the antibody or antigen-binding fragment encoding the antibody or antigen-binding fragment encoding the BCMA molecule described in the first aspect of the first aspect of the present invention that is introduced under conditions suitable for expressing the nucleic acid encoding the antibody or antigen-binding fragment that specifically binds to the BCMA molecule described in the first aspect of the present invention.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof, most preferably, the Host cells are HEK293 cells or CHO cells.
  • the present invention provides a pharmaceutical combination comprising
  • a first component selected from immune effector cells (for example, T cells, NK cells) expressing a molecular switch-regulated CAR polypeptide, a nucleic acid molecule encoding the CAR polypeptide, a vector comprising the nucleic acid molecule, and any combination of them; and
  • immune effector cells for example, T cells, NK cells
  • the second component which is an antibody or antigen-binding fragment (also known as a P329G mutant antibody) that specifically binds to a BCMA molecule comprising a P329G mutation, for example, the P329G mutant antibody of the first aspect of the present invention
  • the molecular switch-regulated CAR polypeptide comprises
  • scFv sequence comprises the following sequence that can specifically bind to the Fc domain of an antibody comprising a P329G mutation, but cannot specifically bind to the Fc domain of an unmutated parental antibody:
  • CDR L light chain complementarity determining region 1 shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22), or a variant of said CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • n is an integer from 1 to 10, such as an integer from 1 to 4; for example SEQ ID NO: 14 the sequence shown;
  • CD8 ⁇ hinge region or variants thereof with 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 18 or variants thereof with 1-2 amino acid modifications;
  • Transmembrane region which is selected from CD8 transmembrane domain or variants with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 15 or with 1-2 amino acids Modified variants;
  • Costimulatory signaling domain which is selected from 4-1BB costimulatory domain or variants with 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO: 16 or its variant with 1 - 2 amino acid modified variants;
  • Stimulatory signaling domain which is a CD3 ⁇ signaling domain or a variant having 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO: 17 or having 1-10, 1 - 5 amino acid modified variants;
  • amino acid modification is addition, deletion or substitution of amino acid.
  • the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the present invention comprises
  • scFv sequence comprises the following sequence that can specifically bind to the Fc domain of an antibody comprising a P329G mutation, but cannot specifically bind to the Fc domain of an unmutated parental antibody:
  • a heavy chain variable region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 12 or 99% identical sequences, and
  • transmembrane region which is selected from the CD8 transmembrane domain shown in SEQ ID NO: 15 or a variant with 1 amino acid modification;
  • co-stimulatory signaling domain which is selected from the 4-1BB co-stimulatory domain shown in SEQ ID NO: 16 or a variant with 1 amino acid modification;
  • Stimulatory signaling domain which is selected from the CD3 ⁇ signaling domain shown in SEQ ID NO: 17 or a variant with 1 amino acid modification;
  • amino acid modification is addition, deletion or substitution of amino acid.
  • the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the present invention further comprises a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 11,
  • the molecular switch-regulated CAR polypeptide in the pharmaceutical combination of the present invention has the amino acid sequence shown in SEQ ID NO: 1 or at least 90%, 91%, 92%, 93%, 94%, Sequences that are 95%, 96%, 97%, 98% or 99% identical.
  • the present invention provides a nucleic acid encoding the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the present invention, a vector comprising the nucleic acid encoding the CAR polypeptide, and a cell comprising the CAR nucleic acid molecule or vector, Or a cell expressing the CAR polypeptide, preferably, the cell is an autologous T cell or an allogeneic T cell.
  • the nucleic acid molecule encoding the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the present invention encodes the amino acid sequence shown in SEQ ID NO: 1 or has at least 90%, 91%, 92%, 93 A nucleic acid molecule having an amino acid sequence of %, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the vector comprising the nucleic acid molecule encoding the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the present invention is selected from DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors or retroviral vectors.
  • the immune effector cells in the pharmaceutical combination of the present invention are autologous T cells, NK cells or allogeneic T cells, NK cells that express the molecular switch-regulated CAR polypeptide of the present invention.
  • Cells for example, the immune effector cells are T cells isolated from human peripheral blood mononuclear cells (PBMC), T cells prepared from NK cells expressing the molecular switch-regulated CAR polypeptide of the present invention, NK cells.
  • PBMC peripheral blood mononuclear cells
  • the present invention utilizes primary P329G CAR-T cells prepared from human PBMC derived from multiple different donors, and utilizes a P329G mutated anti-BCMA humanized antibody to evaluate P329G in an in vitro co-culture system.
  • CAR-T cells target BCMA-expressing tumor cells through the effector function of antibodies. Therefore, the P329G mutated anti-BCMA antibody can be used as a "molecular switch" to regulate the recognition and killing activity of P329G CAR-T cells on BCMA-positive tumor cells.
  • the effector function in vitro is comparable to that of traditional CAR-T cells directly targeting BCMA-positive tumor cells, but the activity of traditional CAR-T cells does not depend on the P329G mutant antibody.
  • the present invention verifies the anti-tumor effect of P329G CAR-T cells combined with P329G mutant antibody in immunodeficient mice inoculated with BCMA-positive human tumor cell line-derived tumors, and the dose of the antibody, interval etc. were studied.
  • the immune effector cells expressing the CAR polypeptide of the present invention are injected at a concentration of 1 ⁇ 10 6 cells/kg body weight to 10 ⁇ 10 6 cells/kg body weight, for example, 1 ⁇ 10 6 cells/kg body weight. 10 6 cells/kg body weight, 2 ⁇ 10 6 cells/kg body weight, 3 ⁇ 10 6 cells/kg body weight, 5 ⁇ 10 6 cells/kg body weight, 7 ⁇ 10 6 cells/kg body weight, 9 ⁇ A dose of 106 cells/kg body weight, 10 x 106 cells/kg body weight is administered intravenously to the subject in single or multiple doses; and
  • the P329G mutant antibody described in the pharmaceutical combination of the present invention is dosed at 0.1-10 mg/kg, preferably 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg kg, 7 mg/kg, 9 mg/kg, 10 mg/kg dosage unit form, preferably parenterally, more preferably intravenously administered to the subject.
  • (i) and (ii) in the pharmaceutical combination of the present invention are administered separately, simultaneously or sequentially, for example, administering (ii) on the first day, administering (i) intravenously on the same day, and then administering (i) at a certain frequency multiple administrations of (ii), while determining whether to administer (i) multiple times by monitoring the in vivo PK concentration of (i) and the desired therapeutic efficacy endpoint; or
  • the present invention provides the use of the pharmaceutical combination of the present invention for treating BCMA-related diseases in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical combination defined in the aforementioned third aspect , wherein the BCMA-related disease is, for example, a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
  • the BCMA-related disease is, for example, a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
  • the present invention provides the use of the pharmaceutical combination of the present invention in the preparation of a medicament for the treatment of BCMA-related diseases, such as cancers expressing or overexpressing BCMA, said cancers
  • BCMA-related diseases such as cancers expressing or overexpressing BCMA
  • RRMM relapsed/refractory multiple myeloma
  • the present invention provides a method for treating a disease associated with BCMA, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical combination of the present invention, the disease being, for example, expressing or overexpressing BCMA Cancer, such as relapsed/refractory multiple myeloma (RRMM).
  • a disease associated with BCMA the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical combination of the present invention, the disease being, for example, expressing or overexpressing BCMA Cancer, such as relapsed/refractory multiple myeloma (RRMM).
  • RRMM relapsed/refractory multiple myeloma
  • the present invention provides a kit of parts comprising the pharmaceutical combination as defined in the aforementioned third aspect, preferably said kit is in the form of a dosage unit of the drugs.
  • the present invention provides a drug complex, which is a compound composed of
  • immune effector cells eg, T cells, NK cells
  • an antibody or antigen-binding fragment also known as a P329G mutant antibody
  • a BCMA molecule comprising a P329G mutation
  • the immune effector cells are T cells expressing the molecular switch regulatory CAR polypeptide described in the pharmaceutical combination of the present invention prepared from autologous T cells or allogeneic T cells
  • the immune effector cells are from human T cells expressing the molecular switch regulatory CAR polypeptide of the present invention prepared from T cells isolated from PBMC;
  • the P329G mutant antibody is ADI-38497 PG Ab and/or ADI-38484 PG Ab.
  • the present invention also provides the use of the drug complex for treating a BCMA-related disease in a subject, preferably, the BCMA-related disease is, for example, a cancer expressing or overexpressing BCMA, the cancer An example is relapsed/refractory multiple myeloma (RRMM).
  • the BCMA-related disease is, for example, a cancer expressing or overexpressing BCMA, the cancer
  • RRMM relapsed/refractory multiple myeloma
  • the present invention obtains a high-affinity BCMA-specific P329G antibody through in vitro binding ability, affinity and Fc effector function detection, and the antibody can simultaneously bind BCMA antigen and P329G CAR molecule to exert a bridging effect.
  • the present invention constructs an in vitro co-culture system by using the constructed P329G CAR structural molecule and combining the P329G CAR-T cells prepared by the CAR molecule with a BCMA-specific P329G antibody, and then co-culturing with BCMA-positive MM cells in vitro , in this system, it was verified that P329G antibody acts as a "molecular switch" to regulate the effect of P329G CAR-T cell activity, that is, only in the presence of P329G mutant antibody, P329G CAR-T cells can be activated, proliferate, and secrete effector cells Factors and killing effects, and these effects are P329G antibody dose-dependent, with the increase of antibody dose, P329G CAR-T cell recognition and killing effects are enhanced.
  • the WT antibody without the P329G mutation could not stimulate the effector function of P329G CAR-T cells.
  • the in vitro experiments of the present invention show that the soluble BCMA antigen does not affect the activity of P329G CAR-T cells when used in combination with BCMA-specific P329G antibodies, while the soluble BCMA antigen has a significant inhibitory effect on traditional CAR-T cells.
  • the P329G CAR-T cells of the present invention combined with P329G antibodies produced good anti-tumor effects, and the anti-tumor effects were at least as good as those of traditional CAR-T cells.
  • Figure 1A shows the expression of CAR in CD3 + cells, CD4 + , and CD8 + T cell subsets after T cells were transduced with the HuR968B and Blue21 CARs constructed in Example 1-1.
  • Figure 1B shows the mechanism of action of P329G CAR-T cells targeting BCMA-expressing target cells mediated by P329G antibody.
  • SP signal peptide
  • TMD transmembrane domain
  • ICD intracellular domain
  • CSD co-stimulatory signal domain ( costimulatory domain)
  • SSD stimulating signaling domain.
  • the extracellular domain comprises an antigen-binding part capable of specifically binding to a mutant Fc domain containing a P329G mutation, and the antigen-binding part comprises a heavy chain variable region (VH) and Light Chain Variable Region (VHL).
  • VH heavy chain variable region
  • VHL Light Chain Variable Region
  • Fig. 2A shows a schematic diagram of a method for measuring antibody affinity by surface plasmon resonance (SPR).
  • Figure 2B shows representative affinity profiles of ADI-38497 PG antibody to recombinant human, cynomolgus monkey, mouse, rat and rabbit BCMA proteins measured by SPR.
  • Figure 2C shows the binding ability of P329G BCMA antibody to CHO-GS cells stably expressing human, cynomolgus and mouse BCMA.
  • Figure 2D shows the binding activity of P329G BCMA antibody to BCMA-positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1.
  • Figure 3A shows a schematic diagram of the detection of the affinity of the specific single-chain antibody against the P329G mutation-rabbit Fc fusion protein and the ADI-38497 P329G mutation antibody by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Figure 3B shows representative affinity profiles of ADI-38497 PG antibody and wild-type antibody binding to anti-PG scFv fusion protein by SPR.
  • Fig. 3C shows a schematic diagram of the method for measuring antibody affinity (Avidity) by surface plasmon resonance (SPR).
  • Figure 3D shows representative affinity profiles of ADI-38497 PG antibody and wild-type antibody and anti-PG scFv fusion protein (interim, P329G CAR extracellular domain contains anti-PG scFv) by SPR.
  • Figure 3E shows the binding ability of ADI-38497WT antibody and ADI-38497 PG antibody to P329G CAR-T cells.
  • Figure 4A shows the ability of ADI-38497WT antibody and ADI-38497 PG antibody to mediate ADCC killing.
  • Figure 4B shows the ability of ADI-38497WT antibody and ADI-38497 PG antibody to mediate ADCP killing.
  • Figure 4C shows the ability of the ADI-38497 PG antibody to mediate lysis of target cells.
  • Figure 5A shows that for H929 cells, the ADI-38497 PG antibody containing the P329G mutation specifically mediates the activation of CAR + -T.
  • Figure 5B shows that different BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, GSK PG IgG) induced the activation effect of L363 target cells on HuR968B CAR-T cells.
  • BCMA antibodies ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, GSK PG IgG
  • Figure 5C shows the proliferation of HuR968B CAR-T cells stimulated by coated ADI-38497WT antibody or ADI-38497 PG antibody.
  • Figure 5D shows that HuR968B CAR-T cells were co-cultured with H929 cells and RPMI8226 cells. After adding different concentrations of BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG as a positive control ) after the release of effector cytokines secreted by CAR-T cells.
  • BCMA antibodies ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG as a positive control
  • FIG. 5E shows that HuR968B CAR-T cells were co-cultured with different tumor cells. After adding different concentrations of BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG), CAR-T cells The release of secreted effector cytokines results.
  • BCMA antibodies ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG
  • FIG. 5F shows that different BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG) induced HuR968B CAR-T cells to tumor cells with different BCMA expression levels (H929 ++ cells, RPMI8226 +++ cells, AMO1 + cells and L363 + cells).
  • BCMA antibodies ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or GSK PG IgG
  • Figure 5G shows the killing effect of P329G BCMA antibody-induced CAR-T production on tumor cells.
  • Figure 6 shows the effect of different concentrations of free BCMA protein on the killing effect of HuR968B CAR-T and Blue21CAR-T cells.
  • Figure 7A and Figure 7B show the results of pharmacokinetic experiments of ADI-38497 PG antibody in mice.
  • FIG 8A shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 high-expressing BCMA tumor cells.
  • cCAR-T means a conventional CAR-T, ie, Blue21CAR-T.
  • Figure 8B shows the body weight changes of immunodeficient tumor-bearing mice inoculated subcutaneously with different doses of PG antibody combined with PG CAR-T cells in human H929 high-expressing BCMA tumor cells.
  • Figure 8C shows the expansion of PG CAR-T cells in mice when different doses of PG antibody combined with PG CAR-T cells were inoculated subcutaneously in immunodeficient tumor-bearing mice with human H929 high-expressing BCMA tumor cells.
  • FIG. 9A shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human L363 low-expression BCMA tumor cells.
  • cCAR-T means conventional CAR-T, ie, Blue21CAR-T.
  • Figure 9B shows the body weight changes of immunodeficient tumor-bearing mice inoculated subcutaneously with different doses of PG antibody combined with PG CAR-T cells in human L363 low-expressing BCMA tumor cells.
  • Figure 9C shows the expansion of PG CAR-T cells in mice when different doses of PG antibody combined with PG CAR-T cells were inoculated subcutaneously in immunodeficient tumor-bearing mice with low expression of human L363 BCMA tumor cells.
  • Figure 10A shows the therapeutic effect of PG antibody combined with different doses of PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Figure 10B shows the expansion of PG CAR-T cells in mice when PG antibodies were combined with different doses of PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Fig. 11A shows the frequency of administration of the PG antibody in the experiment of Example 11-1.
  • Figure 11B shows the therapeutic effect of different administration frequencies of PG antibody when combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Figure 11C shows the expansion of PG CAR-T cells in mice in the experiment of Example 11-1.
  • Figure 12A shows the fluorescence images of the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • Figure 12B shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • Figure 12C shows the body weight changes of immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein of different doses of PG antibody combined with PG CAR-T cells.
  • Figure 13A shows the therapeutic effect of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Figure 13B shows the changes in body weight of the mice treated with PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Figure 13C and Figure 13D show the hematology and blood biochemical detection results of the mice treated with PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • BCMA and “B cell maturation antigen” are used interchangeably and include variants, isoforms, species homologs and analogs of human BCMA having at least one epitope identical to BCMA (e.g., human BCMA) .
  • BCMA proteins may also include fragments of BCMA, such as the extracellular domain and fragments of the extracellular domain, eg, fragments that retain the ability to bind to any antibody of the invention.
  • BCMA antibody As used herein, the terms “BCMA antibody”, “antibody against BCMA”, “antibody that specifically binds BCMA”, “antibody that specifically targets BCMA”, “antibody that specifically recognizes BCMA” are used interchangeably, Means an antibody capable of specifically binding to B-cell maturation antigen (BCMA).
  • antibody is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), single chain antibodies, whole antibodies and antibody fragments.
  • the antibodies of the invention are single domain antibodies or heavy chain antibodies.
  • Antibody fragment or "antigen-binding fragment” are used interchangeably herein to refer to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, diabody.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally
  • the scFvs are continuously linked by a flexible short polypeptide linker and can be expressed as a single chain polypeptide in which the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have a VL variable region and a VH variable region in any order (e.g., relative to the N-terminus and C-terminus of a polypeptide), a scFv can comprise a VL-linker-VH or can comprise VH-Joint-VL.
  • a “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or Regions containing antigen contact residues ("antigen contact points").
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • CDR H1, CDR H2, and CDR H3 The CDRs located in the variable domain of the antibody heavy chain are referred to as CDR H1, CDR H2, and CDR H3, while the CDRs located in the variable domain of the antibody light chain are referred to as CDR L1, CDR L2, and CDR L3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al., (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the present invention).
  • a reference CDR sequence eg, any of the exemplified CDRs of the present invention.
  • reference is made to numbering positions according to the Kabat numbering system.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.
  • chimeric antibody is an antibody molecule in which (a) the constant region or part thereof is altered, substituted or exchanged such that the antigen binding site is of a different or altered class and/or species of constant region or confers a chimeric (e.g., enzymes, toxins, hormones, growth factors, drugs), etc.; or (b) replace the variable region or part thereof with a variable region having different or altered antigenic specificity Alter, replace or exchange.
  • murine antibodies can be modified by exchanging their constant regions with those from human immunoglobulins. Due to the exchange of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original murine antibody.
  • a “humanized” antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • all or substantially all of the CDRs (eg, CDRs) in the humanized antibody correspond to those of a non-human antibody
  • all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human Antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions.
  • the Fc region of a human IgG heavy chain is generally defined as the segment from the amino acid residue at Cys226 or Pro230 to the carboxyl-terminus, and the lysine residue at position 447 at the C-terminus of the Fc region (according to the EU numbering system) may be present or absent.
  • whole antibody compositions can include a population of antibodies in which all K447 residues have been eliminated, a population of antibodies in which no K447 residue has been eliminated, or a population of antibodies in which antibodies with and without K447 residues are mixed.
  • the Fc region of an immunoglobulin comprises two constant domain domains, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, CH2, CH3 and CH4.
  • Binding of IgG to Fc ⁇ receptors or C1q is dependent on residues localized in the hinge region and CH2 domain. Two regions of the CH2 domain are critical for Fc ⁇ R and complement C1q binding and have unique sequences in IgG2 and IgG4. Substitution of residues 233-236 in human IgG1 and IgG2 and substitution of residues 327, 330 and 331 in human IgG4 has been shown to substantially reduce ADCC and CDC activity (Armour et al., Eur. J. Immunol. 29(8 ), 1999, 2613-2624; Shields et al., J. Biol. Chem. 276(9), 2001, 6591-6604).
  • Functional Fc region and “functional Fc region” and similar terms may be used interchangeably, referring to an Fc region having effector functions of a wild-type Fc region.
  • Variant Fc region “Fc mutant”, “Fc region carrying a mutation”, “mutant Fc region”, “Fc region variant”, “Fc variant”, “variant Fc region” and “mutated Fc region” Similar terms such as “region” can be used interchangeably, and refer to an Fc region comprising at least one amino acid modification that is distinguished from a native sequence Fc region/wild-type Fc region.
  • a variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by one or more amino acid substitutions, deletions or additions.
  • the variant Fc region has at least one amino acid substitution compared to the Fc region of wild-type IgG, the at least one amino acid substitution being a glycine (G) substitution of the amino acid at position P329 according to EU numbering.
  • Fc receptor refers to a molecule that binds the Fc region of an antibody.
  • the FcR is a native human FcR.
  • the FcR is a receptor that binds an IgG antibody, namely an Fc ⁇ R, including three receptors, Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16), and allelic variants and variants of these receptors. splice form.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA and Fc ⁇ RIIB
  • Fc ⁇ RIII receptors include Fc ⁇ RIIIA and Fc ⁇ RIIIB.
  • effector functions refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with immunoglobulin isotype.
  • immunoglobulin effector functions include: Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen Uptake of antigen by presenting cells, Clq binding and complement-dependent cytotoxicity (CDC), downregulation of cell surface receptors (eg, B cell receptors), and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK natural killer cells
  • the chimeric antigen receptors of the invention provide antibody-dependent cytotoxicity of T lymphocytes, enhance antibody-dependent cytotoxicity of NK cells.
  • the chimeric antigen receptor of the present invention induces the activation, continuous proliferation and exertion of T cells expressing the chimeric antigen receptor by binding to the antibody (or other anti-tumor molecules comprising the Fc part) that binds to the tumor cell Other anti-tumor molecules containing Fc moieties) mediated specific cytotoxicity against cancer cells of interest.
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement.
  • the complement system is part of the innate immune system made up of a series of proteins.
  • the proteins of the complement system are called “complement”, represented by abbreviations C1, C2, C3, etc., which are a group of heat-labile proteins that exist in human or vertebrate serum and interstitial fluid, and have enzymatic activity after activation.
  • CIq is the first component of the complement-dependent cytotoxicity (CDC) pathway, capable of binding six antibodies, but binding to two IgGs is sufficient to activate the complement cascade.
  • Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind the relevant antigen, activating a series of complement cascades that form holes in the target cell membrane, resulting in target cell death.
  • C1q first component of the complement system
  • a CDC assay can be performed, for example, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • variable region refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • bind or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interferometry techniques or other conventional binding assays known in the art.
  • stimulation refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its corresponding ligand, which thus mediates a signal transduction event, such as, but not limited to, via a TCR/CD3 complex body signal transduction.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, such as down-regulation of TGF- ⁇ and/or reorganization of cytoskeletal structures, etc.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates TCR complex activation in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • primary activation In one embodiment, primary signals are elicited, eg, by binding of the TCR/CD3 complex to peptide-loaded MHC molecules and result in mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like.
  • the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, eg, the primary signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as the protein given by GenBan Accession No. BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain sufficient for functional Transmits the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues for monkeys, apes, etc.).
  • the "CD3 ⁇ stimulatory signaling domain” is the sequence provided in SEQ ID NO: 17 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (eg, but not limited to, proliferation) of the cell.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "co-stimulatory molecule” is 4-1BB (ie, CD137).
  • Costimulatory signaling domain refers to the intracellular portion of a costimulatory molecule.
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2 or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) and "4-1BB co-stimulatory signaling domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.) .
  • the "4-1BB co-stimulatory domain” is the sequence provided as SEQ ID NO: 16 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • signaling pathway refers to the biochemical relationship between various signaling molecules that play a role in propagating a signal from one part of a cell to another.
  • cytokine is a general term for proteins released by one cell population to act as intercellular mediators on another cell.
  • cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7.
  • antibodies of the invention are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse phase HPLC.
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • isolated nucleic acid encoding an antibody of the invention refers to one or more nucleic acid molecules encoding a chain of an antibody of the invention or fragments thereof, including such nucleic acid molecules in a single vector or in separate vectors, and present in a host cell Such nucleic acid molecules at one or more positions in .
  • the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • amino acid change and “amino acid modification” are used interchangeably to refer to additions, deletions, substitutions and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions and other modifications can be made, provided that the final polypeptide sequence possesses the desired properties.
  • amino acid substitutions to the antibody result in decreased binding of the antibody to an Fc receptor.
  • non-conservative amino acid substitutions ie the substitution of one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred.
  • Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy lysine) substitution.
  • Amino acid changes can be made using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. A method of changing amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may be useful.
  • Various names may be used herein to refer to the same amino acid change. For example, a substitution from proline to glycine at position 329 of the Fc domain may be denoted as 329G, G329, G329 , P329G Pro329Gly, or simply "PG".
  • conservative sequence modification refers to an amino acid modification or change that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising an amino acid sequence.
  • conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • autologous refers to any substance that is derived from the same individual into whom the substance is later reintroduced.
  • allogeneic refers to any substance derived from a different animal of the same species as the individual into whom it is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently dissimilar genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • apheresis refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from a donor or patient and passed through a device that separates selected specific components and return the remainder to the donor or patient's circulation, for example, by retransfusion.
  • single sample refers to a sample obtained using apheresis.
  • immune effector cell refers to a cell that participates in an immune response, eg, participates in promoting an immune effector response.
  • immune effector cells include T cells, eg, ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • immune effector function refers to, for example, the enhancement of immune effector cells or the function or response of immune attack target cells.
  • immune effector function or response refers to T cell or NK cell properties that promote killing of target cells or inhibit growth or proliferation of target cells.
  • primary stimulation and co-stimulation are examples of immune effector functions or responses.
  • effector function refers to a specialized function of a cell.
  • the effector function of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines.
  • T cell activation refers to one or more cellular responses of T lymphocytes, especially cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and activation Expression of markers.
  • cytotoxic T lymphocytes selected from the group consisting of proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and activation Expression of markers.
  • the chimeric antigen receptors of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.
  • lentivirus refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, including inter alia self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the Lentiviral vector from Oxford BioMedica Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • BCMA-associated disease refers to any condition caused by, aggravated by, or otherwise associated with increased expression or activity of BCMA.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rodents). mouse).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rodents.
  • rodents e.g., mice and rodents.
  • an individual or subject is a human.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to the physiological disorder of unregulated cell growth in mammals.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancerous and cancerous cells and tissues.
  • Tumor immune evasion refers to the process by which tumors evade immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is “cured” when such evasions are weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • half effective concentration refers to the concentration of drug, antibody or poison that induces 50% of the response between baseline and maximum after a specified exposure time.
  • FACS fluorescence activated cell sorting
  • Such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from the Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).
  • pharmaceutically acceptable excipient refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers, etc., which are administered together with the active substance.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibody molecules of the invention are used to delay the development of a disease or to slow the progression of a disease.
  • an effective amount refers to such an amount or dose of an antibody or composition of the present invention that, after administration to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as: the species of mammal; body weight, age and general health; the particular disease involved; the extent or severity of the disease; the individual The patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount of an antibody or antibody fragment or composition thereof can vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition thereof are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • Compounds can be evaluated for their ability to inhibit a measurable parameter (eg, cancer) in animal model systems predictive of efficacy in human tumors.
  • non-fixed combination refers to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions.
  • non-fixed combination means that the active ingredients (e.g., (i) P329G CAR-T cells, and (ii) P329G mutant antibody against BCMA) are combined in separate entities at the same time, without specific time limit or in the same or different Time intervals, sequential administration to a subject, wherein such administration provides effective treatment in the subject.
  • fixed combination means that the combination of the P329G mutant antibody against BCMA and the P329G CAR-T cells of the present invention is administered to the patient at the same time in the form of a specific single dose.
  • non-fixed combination means that the combination of the 329G mutant antibody against BCMA of the present invention and the P329G CAR-T cells is administered to the patient simultaneously, concurrently or sequentially as separate entities, without specific dose and time limit, wherein such administration Therapeutically effective levels of the drug combinations of the invention in a patient are provided.
  • the drug combination is a non-fixed combination.
  • combination therapy refers to the administration of two or more components to treat a cancer as described in this disclosure.
  • Such administration includes co-administration of the components in a substantially simultaneous manner.
  • such administration includes co-administration or separate administration or sequential administration for each active ingredient in multiple or in separate containers (eg, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • administering further comprises using the P329G mutant antibody against BCMA of the invention and the P329G CAR-T cells of the invention at about the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.
  • a host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • Subject/patient sample refers to a collection of cells, tissues or body fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimen, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumors sample.
  • treating in reference to a disease means alleviating the disease (ie, slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof), preventing or delaying the onset or development or progression of the disease.
  • CAR chimeric antigen receptor
  • the present invention relates to chimeric antigen receptor polypeptides capable of specifically binding to mutant Fc domains of antibodies directed against BCMA molecules.
  • the chimeric antigen receptor of the present invention comprises a humanized anti-P329G mutation scFv sequence, and the scFv sequence can specifically bind to the Fc domain of an antibody comprising a P329G mutation, but cannot specifically bind to the Fc domain of an unmutated parental antibody. domain.
  • the Fc domain of an antibody comprising the P329G mutation has reduced binding to an Fc receptor (eg, an Fc gamma receptor) compared to the binding to an Fc receptor of an unmutated parental antibody Fc domain.
  • the recombinant CAR construct of the present invention comprises a sequence encoding a CAR, wherein the CAR comprises a humanized anti-P329G mutation scFv sequence, and the scFv sequence specifically binds to the Fc domain of an antibody with a P329G mutation.
  • the scFv sequence in the CAR construct of the present invention comprises the following sequence:
  • CDR L light chain complementarity determining region 1 shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22), or a variant of said CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid changes are additions, deletions or substitutions of amino acids.
  • the scFv can be connected with a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 11, and the scFv can be connected at the C-terminus with a sequence as provided in SEQ ID NO: 14 or SEQ ID NO: 18
  • a hinge/spacer sequence, a transmembrane region as provided in SEQ ID NO: 15, a co-stimulatory signal domain as in SEQ ID NO: 16 and an intracellular stimulatory signal comprising SEQ ID NO: 17 or a variant thereof Domains for example, where the individual domains are adjacent to each other and in the same open reading frame to form a single fusion protein.
  • the scFv domain comprises (i) a heavy chain variable region comprising or at least 90%, 91%, 92%, 93%, 94%, 95%, the sequence of SEQ ID NO: 12, A sequence of 96%, 97%, 98% or 99% identity, and (ii) a light chain variable region comprising or at least 90%, 91%, 92%, 93% of the sequence of SEQ ID NO: 13 , 94%, 95%, 96%, 97%, 98% or 99% identical sequences;
  • the scFv domain comprises (i) the heavy chain variable region set forth in SEQ ID NO: 12 and (ii) the light chain variable region set forth in SEQ ID NO: 13.
  • the scFv domain further comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4.
  • the light and heavy chain variable regions of the scFv may, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
  • an exemplary CAR construct of the invention comprises a signal peptide sequence, a humanized anti-P329G mutant scFv sequence, a hinge/spacer, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular stimulatory domain. signaling domain.
  • the present invention provides the amino acid sequence of the full-length CAR polypeptide as SEQ ID NO: 1, as shown in the sequence listing.
  • the present invention provides a recombinant nucleic acid construct comprising a nucleic acid molecule encoding the CAR of the present invention, for example, it comprises encoding the amino acid sequence shown in SEQ ID NO: 1 or having at least the same amino acid sequence as SEQ ID NO: 1
  • CAR constructs encoding the present invention can be obtained using recombinant methods well known in the art.
  • the nucleic acid of interest may be produced synthetically rather than by genetic recombination methods.
  • the present invention includes retroviral vector constructs and lentiviral vector constructs expressing a CAR that can be directly transduced into cells.
  • the nucleic acid sequence of the CAR construct of the invention is cloned into a lentiviral vector to generate a full-length CAR construct in a single coding frame, and the EF1 ⁇ promoter is used for expression.
  • CAR polypeptides of the present invention may also be modified so as to vary in amino acid sequence but not in desired activity.
  • additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues can be made to the CAR polypeptide.
  • a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family.
  • an amino acid stretch can be substituted for a structurally similar stretch that differs in the order and composition of side chain family members, for example, conservative substitutions can be made in which the amino acid residue Substitute with an amino acid residue with a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, , glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g., threonine, valine, iso leucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • the present invention contemplates the generation of functionally equivalent CAR polypeptide molecules, e.g., the VH or VL of the humanized anti-P329G mutant scFv sequence contained in the CAR can be modified to obtain at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical VH and having at least 90%, 91%, 92%, A VL that is 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the transmembrane domain contained in the CAR of the present invention is an anchored transmembrane domain, which is a component of a polypeptide chain capable of being integrated in a cell membrane. Transmembrane domains can be fused to other extracellular and/or intracellular polypeptide domains, which will also be confined to the cell membrane. In chimeric antigen receptor (CAR) polypeptides of the invention, the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention.
  • the CAR polypeptide of the present invention comprises at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising predominantly hydrophobic residues such as leucine and valine.
  • the domain may be derived from the transmembrane domain of a membrane-bound or transmembrane protein such as CD28, CD8 (eg, CD8 ⁇ , CD8 ⁇ ).
  • the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 15.
  • the transmembrane domain in the CAR of the invention is linked to the extracellular region of the CAR (ie, the humanized anti-P329G mutant scFv sequence) via a hinge/spacer.
  • the hinge can be the hinge region of CD8 ⁇ , the hinge region of CD28.
  • the hinge or spacer sequence comprises the amino acid sequence of SEQ ID NO: 18.
  • the glycine-serine doublet also provides a particularly suitable linker as hinge/spacer.
  • the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 14).
  • the cytoplasmic domain comprised in the CAR of the present invention comprises an intracellular signaling domain.
  • the intracellular signaling domain is capable of activating at least one effector function of the immune cell into which the CAR of the present invention has been introduced.
  • intracellular signaling domains useful in the CARs of the invention include those of the T cell receptor (TCR) and co-receptors that act cooperatively to initiate signal transduction upon binding of the extracellular domain to the Fc domain of an antibody with a P329G mutation.
  • TCR T cell receptor
  • the CAR of the present invention is also designed with a co-stimulatory signal domain (CSD) that can generate co-stimulatory signals.
  • CSD co-stimulatory signal domain
  • Activation of T cells is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domain) and those that act in an antigen-independent manner to provide co-stimulatory signals Those sequences of (secondary cytoplasmic domain, eg, co-stimulatory domain).
  • the CAR of the present invention comprises a primary intracellular signaling domain, e.g., the primary signaling domain of CD3 ⁇ , e.g., the CD3 ⁇ signaling domain shown in SEQ ID NO: 17.
  • the intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (ie, co-stimulatory signaling domain).
  • a co-stimulatory signaling domain refers to the portion of the CAR comprising the intracellular domain of a co-stimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for lymphocytes to respond effectively to antigens.
  • co-stimulatory molecules include, but are not limited to, CD28, 4-1BB (CD137), which cause co-stimulation that enhances the proliferation, effector function, and survival of human CART cells in vitro and enhances the anti-inflammatory effect of human T cells in vivo. tumor activity.
  • the intracellular signaling sequences of the CAR of the present invention can be connected to each other in a random order or in a specified order.
  • short oligopeptide linkers or polypeptide linkers can form linkages between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • single amino acids, eg, alanine, glycine can be used as suitable linkers.
  • the intracellular signaling domain of the CAR of the present invention is designed to include a co-stimulatory signaling domain of 4-1BB and a stimulating signaling domain of CD3 ⁇ .
  • the invention provides nucleic acid molecules encoding the CAR constructs described herein.
  • the nucleic acid molecule is provided as a DNA construct.
  • the present invention also provides a vector inserted with the CAR construct of the present invention.
  • Expression of a natural or synthetic nucleic acid encoding a CAR is achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector.
  • Vectors may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences and promoters for regulating the expression of the desired nucleic acid sequence.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject in vivo or ex vivo.
  • retroviral systems are known in the art.
  • lentiviral vectors are used.
  • Retroviruses such as lentiviruses are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in progeny cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia virus, because they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a retroviral vector may also be, for example, a gamma retroviral vector.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., encoding a CAR gene.
  • Gamma retroviral vectors may lack viral structural genes such as gag, pol, and env.
  • a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EF1a promoter has been used extensively in mammalian expression plasmids and has been shown to efficiently drive CAR expression from transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences can also be used, including but not limited to Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, and human gene promoters such as but not limited to the actin promoter , myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of
  • the invention provides methods of expressing a CAR construct of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.
  • mammalian immune effector cells eg, mammalian T cells or mammalian NK cells
  • a source of cells eg, immune effector cells, eg, T cells or NK cells
  • T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood components collected from a subject using any technique known to those of skill in the art, such as Ficoll (TM ) separation.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells can be further isolated by positive or negative selection techniques.
  • anti-CD3/anti-CD28 conjugated beads such as M-450CD3/CD28T
  • the period of time is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells wherever small numbers of T cells are present, such as for isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals.
  • TILs tumor infiltrating lymphocytes
  • Enrichment of T cell populations can be accomplished through the process of negative selection using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One method is the sorting and/or selection of cells by means of negative magnetic immunoadhesion or flow cytometry using the presence of cells on negatively selected cells Monoclonal antibody cocktail for surface markers.
  • the immune effector cells can be allogeneic immune effector cells, eg, T cells or NK cells.
  • the cell can be an allogeneic T cell, e.g., one lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) T cells.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered such that it does not express any functional TCR on its surface; engineered such that it does not express one or more subunits that make up a functional TCR (e.g. engineered so that it does not express or exhibit reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered so that it produces very few functional TCRs on its surface.
  • a T cell as described herein can, for example, be engineered such that it does not express functional HLA on its surface.
  • T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated.
  • HLA e.g., HLA class I and/or HLA class II
  • downregulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
  • the T cells may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
  • the cells transduced with the nucleic acid encoding the CAR of the present invention are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.
  • the immune effector cells expressing CAR obtained after in vitro proliferation can be tested for effector function as described in the Examples.
  • the invention provides antibodies that bind BCMA with high target specificity and high affinity, comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering, or no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H1 CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H2; and the amino acid sequence DRGDQILDV (SEQ ID NO:27) shown in CDR H3, or the variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H3;
  • the light chain variable region comprises the amino acid sequence RASQSISRYLN (SEQ ID NO:28) shown in the CDR L1, or the CDR L1 variants with no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2, Or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR L2; and
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence NDVIS (SEQ ID NO: 31) according to Kabat numbering, or no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H1 CDR H2 shown in amino acid sequence VIIPIFGIANYAQKFQG (SEQ ID NO: 32), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of said CDR H2; and amino acid sequence GRGYYSSWLHDI (SEQ ID NO:33) shown in CDR H3, or the variant of no more than 2 amino acid changes or no more than 1 amino acid change of the CDR H3;
  • the light chain variable region comprises the amino acid sequence QASQDITNYLN (SEQ CDR L1 shown in ID NO:34), or a variant of no more than 2 amino acid changes or no more than 1 amino acid change of said CDR L1; CDR L2 shown in the amino acid sequence DASNLET (SEQ ID NO:35), Or a variant of no more than 2
  • amino acid changes are additions, deletions or conservative amino acid substitutions of amino acids.
  • an antibody that binds a BCMA molecule of the invention binds mammalian BCMA, eg, human, cynomolgus monkey, mouse, rat, and rabbit BCMA.
  • an antibody that binds a BCMA molecule of the invention has one or more of the following properties:
  • ADCP antibody-dependent cellular cytotoxicity and/or antibody-dependent cellular phagocytosis
  • an antibody of the invention that binds a BCMA molecule comprises a heavy chain variable region and a light chain variable region that specifically binds BCMA, wherein:
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 2 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 9 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 10 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably amino acid substitution, more preferably amino acid conservative substitution , preferably, said amino acid changes do not occur in the CDR regions.
  • an antibody of the invention that binds a BCMA molecule is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody, e.g., a human IgG1 antibody.
  • the antibodies provided herein that bind to a BCMA molecule comprise a mutant Fc domain wherein the amino acid at position P329 is mutated to glycine (G) according to EU numbering, the same as the Fc ⁇ of the Fc domain of the parental antibody that is not mutated.
  • a mutant Fc domain has reduced Fc ⁇ receptor binding compared to receptor binding; for example, the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutant Fc domain is A mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody, for example, the mutant Fc domain is a mutant Fc domain of a human IgG1 antibody.
  • Antibodies that bind to BCMA molecules containing the P329G mutant Fc domain cannot exert antibody-dependent cellular cytotoxicity by binding to Fc ⁇ receptors, nor can they exert antibody-dependent cellular phagocytosis (ADCP).
  • the invention provides a nucleic acid encoding any of the above antibodies or fragments thereof or any chain thereof that binds a BCMA molecule.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising said nucleic acid or said vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • a nucleic acid of the invention comprises a nucleic acid encoding an antibody of the invention that binds a BCMA molecule.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • the vector is a pcDNA3.4 expression vector.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, lipid-based transfection or other conventional techniques.
  • protoplast fusion cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the particular expression vector and Mammalian host cell alteration or optimization.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.
  • a marker can, for example, confer prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance, etc. to an auxotrophic host.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.
  • a host cell comprising a polynucleotide of the invention.
  • host cells comprising an expression vector of the invention are provided.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies.
  • Suitable host cells include prokaryotic microorganisms such as E. coli.
  • the host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells and the like. Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1 ), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), Chinese hamster ovary cells (CHO cells), CHO-S cells, NSO cells, myeloma cell lines such as Y0, NSO, P3X63 and Sp2/0, etc.
  • the host cells are CHO cells or HEK293 cells.
  • the present invention provides a method for preparing an antibody (including a P329G mutant antibody) that binds to a BCMA molecule, wherein said method comprises a nucleic acid suitable for expressing said antibody (including a P329G mutant antibody) that binds to a BCMA molecule
  • the host cell comprising the nucleic acid encoding the antibody binding to BCMA molecule (including P329G mutant antibody) or the expression vector comprising said nucleic acid is cultivated under the conditions, and optionally the antibody binding to BCMA molecule (including P329G mutant antibody) is isolated ).
  • the method further comprises recovering antibodies (including P329G mutant antibodies) that bind to BCMA molecules from the host cells (or host cell culture medium).
  • Antibodies that bind to BCMA molecules of the present invention can be analyzed by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion, etc. Purification by resistance chromatography, etc. The actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of the BCMA molecule-binding antibodies of the invention, including P329G mutant antibodies can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography Chromatography, etc.
  • Antibodies provided herein that bind to BCMA molecules, including P329G mutant antibodies, can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the antigen-binding activity of the BCMA-binding antibody (including the P329G mutant antibody) of the present invention is tested, for example, by known methods such as FACS, ELISA or Western blotting. Binding to BCMA can be assayed using methods known in the art, exemplary methods are disclosed herein.
  • the binding of BCMA molecule-binding antibodies of the invention, including P329G mutant antibodies, to cell surface BCMA is determined using FACS.
  • the invention also provides assays for identifying biologically active antibodies that bind BCMA molecules, including P329G mutant antibodies.
  • Biological activity may include, for example, ADCC action, CDC action, and the like.
  • Cells for use in any of the above in vitro assays include cell lines that either naturally express BCMA or have been engineered to express BCMA.
  • the modified BCMA-expressing cell line is a cell line that does not express BCMA under normal circumstances and expresses BCMA after the DNA encoding BCMA is transfected into cells.
  • the molecular switch-regulated chimeric antigen receptor of the present invention is a regulatable CAR that can control the activity of CAR.
  • the present invention uses the Pro329Gly (the 329th proline of the Fc segment of the antibody is mutated to glycine according to the EU numbering, abbreviated as P329G) mutant antibody as the safety switch in the CAR treatment of the present invention.
  • P329G mutant antibody does not exist, the CAR activity of the present invention is turned off; when the P329G mutant antibody exists, the CAR activity of the present invention is turned on; thus, the opening and closing of the CAR molecule activity of the present invention is controlled by the P329G mutant antibody regulation.
  • the present invention provides a pharmaceutical combination comprising (i) immune effector cells (e.g., T cells, NK cells) expressing the molecular switch-regulated CAR polypeptide of the present invention; and (ii) specifically binding to BCMA Molecular P329G mutant antibody.
  • the immune effector cells are T cells expressing the molecular switch regulatory CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells, for example, the immune effector cells are prepared from T cells isolated from human PBMC T cells expressing the molecular switch regulatory CAR polypeptide of the present invention.
  • the P329G mutant antibody is ADI-38497 PG Ab and/or ADI-38484 PG Ab.
  • the present invention provides a pharmaceutical combination comprising (i) a nucleic acid molecule encoding the molecular switch-regulated CAR polypeptide of the present invention or a vector comprising the nucleic acid component; and (ii) specifically binding to a BCMA molecule P329G mutant antibody.
  • the pharmaceutical combinations of the present invention optionally further comprise pharmaceutically acceptable excipients of suitable formulations.
  • suitable formulations for example, (ii) in the pharmaceutical combination can be formulated according to conventional methods (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A).
  • the pharmaceutical combination of the present invention is used to treat BCMA-related diseases, such as cancers that express or overexpress BCMA, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
  • BCMA-related diseases such as cancers that express or overexpress BCMA, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
  • the present invention provides the aforementioned pharmaceutical combination of the present invention, which is used in the treatment of BCMA-related diseases in subjects, such as cancers expressing or overexpressing BCMA, such as relapsed/refractory multiple Relapsed/refractory multiple myeloma (RRMM).
  • BCMA-related diseases such as cancers expressing or overexpressing BCMA, such as relapsed/refractory multiple Relapsed/refractory multiple myeloma (RRMM).
  • RRMM relapsed/refractory multiple Relapsed/refractory multiple myeloma
  • the pharmaceutical combination of the present invention is used to treat a cancer that expresses or overexpresses BCMA in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting the growth of cancer cells, said
  • the cancer is, for example, relapsed/refractory multiple myeloma (RRMM).
  • the invention provides methods of treating a BCMA-associated disease (e.g., a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma) in a subject comprising administering The subject is administered a therapeutically effective amount of the pharmaceutical combination of the invention.
  • a BCMA-associated disease e.g., a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma
  • the present invention provides the use of the aforementioned pharmaceutical combination of the present invention in the preparation of medicines for treating BCMA-related diseases (for example, cancers that express or overexpress BCMA, such as relapsed/refractory multiple myeloma) use.
  • BCMA-related diseases for example, cancers that express or overexpress BCMA, such as relapsed/refractory multiple myeloma
  • the pharmaceutical combinations of the invention may also be administered to individuals whose cancer has been treated with one or more prior therapies but has subsequently relapsed or metastasized, for example, the cancer is relapsed/refractory multiple myeloma (RRMM) .
  • RRMM multiple myeloma
  • immune effector cells for example, T cells, NK cells
  • P329G specifically binding to BCMA molecules in the pharmaceutical combination of the present invention The mutated antibody is for parenteral, transdermal, intracavity, intraarterial, intravenous, intrathecal administration, or injected directly into tissue or tumor.
  • the (ii) P329G mutant antibody specifically binding to BCMA molecule in the pharmaceutical combination of the present invention is activated in (i) immune effector cells (for example, T cells, NK cells) expressing the molecular switch-regulated CAR polypeptide of the present invention. cells) before, at the same time or after administration.
  • the (i) immune effector cells expressing the molecular switch-regulated CAR polypeptide of the present invention in the pharmaceutical combination of the present invention are T cells expressing the CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells.
  • Cell; (ii) the P329G mutant antibody specifically binding to BCMA molecule in the pharmaceutical combination of the present invention is any antibody specifically binding to BCMA molecule, which contains the P329G mutation.
  • the P329G mutant antibody is ADI-38497 PG Ab and/or ADI-38484 PG Ab.
  • the present invention does not limit the component (i) in the drug combination of the present invention.
  • the order in which component (i) and component (ii) are administered to the subject does not limit the timing between the administration of component (i) and component (ii) in the pharmaceutical combination of the present invention to the subject . Therefore, (i) and (ii) in the pharmaceutical combination of the present invention may be administered separately, simultaneously or sequentially.
  • the administration of the two components can be separated by 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours, or by an interval that is readily apparent to those skilled in the art. Any suitable time difference determined.
  • administering intravenously on the first day, administering (ii) on the second day, and then administering (ii) multiple times at a certain frequency, while monitoring the in vivo PK concentration of (i) and the desired therapeutic efficacy endpoint, to determine whether Multiple administration of (i); or administration of (ii) on the first day, intravenous administration of (i) on the second day, and then multiple administrations of (ii) at a certain frequency, while monitoring the in vivo PK concentration of (i) and the expected Therapeutic efficacy endpoint, to determine whether to administer (i) multiple times; or to administer (i) and (ii) at intervals of 1 hour, 2 hours, 4 hours, 6 hours, and 12 hours on the same day, and then administer (ii) multiple times at a certain frequency ), while determining whether to administer (i) multiple times by monitoring the in vivo PK concentration of (i) and the desired therapeutic efficacy endpoint.
  • component (i) in the pharmaceutical combination of the present invention when component (i) in the pharmaceutical combination of the present invention is an immune effector cell (for example, T cell, NK cell) expressing the molecular switch-regulated CAR polypeptide of the present invention, it also includes The case where fraction (i) and component (ii) were pre-incubated together prior to administration to a subject. Thus, the two components may be preincubated for 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 1 hour, or any suitable time readily determined by a person skilled in the art, prior to administration.
  • an immune effector cell for example, T cell, NK cell
  • fraction (i) and component (ii) were pre-incubated together prior to administration to a subject.
  • the two components may be preincubated for 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 1 hour, or any suitable time readily determined by a person skilled in the art, prior to administration.
  • the pharmaceutical combination of the present invention can be administered to a subject at an appropriate dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dose for any one patient depends on many factors, including the patient's weight, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and concomitant other medications administered.
  • the component (i) in the pharmaceutical combination of the present invention is an immune effector cell (for example, T cell, NK cell) expressing the molecular switch-regulated CAR polypeptide of the present invention
  • the component (i) 1 ⁇ 10 6 cells/kg body weight-10 ⁇ 10 6 cells/kg body weight, such as 1 ⁇ 10 6 cells/kg body weight, 2 ⁇ 10 6 cells/kg body weight, 3 ⁇ 10 6 cells/kg body weight Body weight, 5 ⁇ 10 6 cells/kg body weight, 7 ⁇ 10 6 cells/kg body weight, 9 ⁇ 10 6 cells/kg body weight, 10 ⁇ 10 6 cells/kg body weight in single or multiple doses and administer component (ii) at 0.1-10 mg/kg, preferably 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg,
  • the dosage unit form of 7 mg/kg, 9 mg/kg, 10 mg/kg is preferably administered to the subject parenterally, more preferably intravenously.
  • administration of a pharmaceutical combination of the invention to an individual with cancer results in complete disappearance of the tumor. In some embodiments, administration of a pharmaceutical combination of the invention to an individual with cancer results in at least an 85% or greater reduction in tumor cells or tumor size.
  • Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetics analyze.
  • the drug combination of the present invention can reduce the "on-target/off tumor" toxicity of CAR-T cells.
  • the present invention provides a kit comprising the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit. Dosage units may thus be presented according to a dosing regimen or interval between drug administrations.
  • kit of parts of the invention comprises in the same package:
  • immune effector cells for example, T cells, NK cells
  • nucleic acid molecules encoding the molecular switch-regulated CAR polypeptide of the present invention
  • vectors comprising the nucleic acid, and any combination of them;
  • Example 1 CAR gene synthesis, construction of virus expression vector, preparation of P329G CAR-T cells and detection of CAR expression
  • the P329G CAR molecule (SEQ ID NO: 1), also known as HuR968B CAR, was constructed, consisting of a signal peptide (SP) shown in SEQ ID NO: 11, a specific single-chain antibody fragment (VH-linker-VL, VH-linker-VL, It has the VH shown in SEQ ID NO:12, the linker sequence shown in SEQ ID NO:41, the VL shown in SEQ ID NO:13), the G4S hinge region shown in SEQ ID NO:14, SEQ ID NO:15 Fusion of the CD8 transmembrane domain (CD8TM) shown, the 41BB costimulatory domain (41BB-CSD) shown in SEQ ID NO: 16, and the intracellular activation domain of the CD3 ⁇ molecule (CD3 ⁇ SSD) shown in SEQ ID NO: 17 made.
  • SP signal peptide
  • VH-linker-VL VH-linker-VL
  • VH-linker-VL VH-linker-VL
  • Blue21CAR (SEQ ID NO: 8) directly targeting BCMA was constructed and used as a control.
  • Blue21CAR contains signal peptide shown in SEQ ID NO:11, anti-BCMA single-chain antibody (from 11D53 clone), hinge region of CD8 ⁇ molecule shown in SEQ ID NO:18 and SEQ ID NO:15 from N-terminal to C-terminal.
  • the lentivirus pellet was resuspended in the medium to obtain the lentivirus concentrate,
  • Pan T Cell Isolation Kit human
  • T cell culture medium to resuspend the T cells to a certain concentration.
  • Density and add TransAct (Miltenyi, 130-111-160) for activation.
  • the lentivirus is the lentivirus encoding P329G CAR (SEQ ID NO: 1) or the control traditional CAR (SEQ ID NO: 8)) and blow the T cells evenly; the second day Remove the viral supernatant by centrifugation and resuspend the cells in fresh T cell medium.
  • Figure 1A shows the expression of CAR in CD3 + cells, CD4 + , and CD8 + T cell subsets after the two CARs constructed in Example 1-1 were used to transduce T cells, and the results showed that in these transduced T cells
  • the positive rate of CAR expression is about 18%-29%.
  • GSK company BCMA antibody clone J6M0 light and heavy chain variable region sequence Obtained from the US9273141B2 patent, GSK company BCMA antibody clone J6M0 light and heavy chain variable region sequence, as a control antibody (GSK IgG).
  • the light and heavy chain variable region sequences of GSK IgG, ADI-38497, and ADI-38484 antibodies were synthesized from the whole gene, and loaded into human IgG1 heavy chain constant region containing WT (SEQ ID NO: 4) or human IgG1 containing P329G point mutation On the pcDNA3.4 expression vector (purchased from Shanghai Boying) of heavy chain constant region (SEQ ID NO:5) and kappa light chain constant region (SEQ ID NO:6).
  • the light and heavy chain expression vectors were co-transfected into HEK293 cells through PEI at a molar ratio of 2:3, and culture supernatants were collected after 5-7 days of culture.
  • the antibody-containing supernatant medium was purified in one step through a Protein A column, and then dialyzed against PBS. The concentration was detected by reading the absorbance value at 280nm with a NanoDrop instrument, and the purity of the sample was detected by SDS-PAGE and SEC-HPLC. Obtained GSK WT antibody, GSK PG antibody; ADI-38497WT antibody, ADI-38497 PG antibody; and ADI-38484WT antibody, ADI-38484 PG antibody.
  • Antibodies with BCMA antibody clone ADI-38497 heavy chain variable region (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 3) sequences are also referred to as ADI-38497 antibodies in this application, including ADI- The 38497 PG antibody and the ADI-38497WT antibody; antibodies with the heavy chain variable region (SEQ ID NO:9) and light chain variable region (SEQ ID NO:10) sequences of BCMA antibody clone ADI-38484 are also referred to in this application as It is ADI-38484 antibody, including ADI-38484 PG antibody and ADI-38484WT antibody.
  • the specific method is as follows: After coupling anti-human Fc IgG (Ab97221, Abcam) to the surface of a CM5 chip (29149603, Cytiva), the ADI-38497 PG antibody was captured on the chip surface, and the BCMA antigen in the mobile phase was detected by detecting the antibody on the chip surface and the BCMA antigen in the mobile phase. Binding and dissociation between the obtained affinity and kinetic constants.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. Each cycle in the affinity assay consists of capture of the ADI-38497 PG antibody, binding of one concentration of antigen, and regeneration of the chip.
  • the antigen after gradient dilution (antigen concentration gradient is 1.25-40nM, 2-fold dilution) flows over the surface of the chip from low concentration to high concentration at a flow rate of 30 ⁇ l/min, the binding time is 180s, and an appropriate dissociation is set. Time (900s or 600s or 60s). Finally, the chip was regenerated using 10 mM glycine-HCl, pH 1.5 (BR-1003-54, Cytiva).
  • Figure 2B shows representative affinity profiles of the ADI-38497 PG antibody to recombinant human, cynomolgus monkey, mouse, rat, and rabbit BCMA proteins measured by SPR.
  • the results showed that the ADI-38497 PG antibody could bind to the above-mentioned BCMA proteins from different species, and the order of binding activity was human BCMA>monkey BCMA>mouse BCMA>rat BCMA>rabbit BCMA.
  • CHO GS cells expressing BCMA antigens from different species were prepared. Specifically, BCMA genes derived from human, mouse, and cynomolgus monkeys were synthesized and cloned into lentiviral vectors, and then lentiviruses containing BCMA genes from different species were packaged, and CHO GS cells were infected with the lentiviruses.
  • CHO GS cell lines expressing BCMA antigens from different species were obtained by flow cytometry sorting, namely hBCMA-CHO GS, mBCMA-CHO GS and cynoBCMA-CHO GS cells.
  • the P329G antibody bound to the cells was detected by flow cytometry, and the APC channel MFI was analyzed, with the antibody concentration as the X axis and the APC channel MFI as the Y axis for drawing and calculating the binding EC50.
  • Figure 2C shows the binding ability of different concentrations of P329G BCMA antibody to CHO-GS cells stably expressing human, cynomolgus monkey and mouse BCMA. It can be seen from Figure 2C that the ADI-38497 PG IgG antibody can bind to different species of BCMA expressed on the cell surface, while the BCMA antibody (Benchmark) derived from GSK has a high species-specificity for BCMA, and it does not recognize mouse BCMA. The results were consistent with the SPR detection results.
  • Figure 2D shows the relationship between different concentrations of P329G BCMA antibody and positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1 expressing BCMA
  • MM.1s was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60239
  • RPMI8226 was purchased From Nanjing Kebai Biotechnology Co., Ltd., CBP60244
  • U266 was purchased from Wuhan Punuosheng Life Technology Co., Ltd., CL-0510
  • H929 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60243
  • L363 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP6024
  • AMO1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60242) binding activity, ADI-38497 PG antibody, ADI-38484 PG antibody can bind to positive tumor cells expressing BCMA in a concentration
  • FIG. 3A shows a schematic diagram of the detection of the affinity of the specific single-chain antibody against the P329G mutation-rabbit Fc fusion protein and the ADI-38497 P329G mutation antibody by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the specific method is as follows: After coupling the synthetic anti-PG scFv fusion protein (SEQ ID NO: 7) to the surface of C1 chip (BR100535, Cytiva), the binding and dissociation of the antibody on the chip surface and the ADI-38497 molecule in the mobile phase was detected. Obtain affinity and kinetic constants.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. Each cycle of affinity detection includes binding of one concentration of antibody and regeneration of the chip.
  • the serially diluted ADI-38497 molecules (concentration gradient 3.125nM-100nM, 2-fold dilution) flowed over the surface of the chip at a flow rate of 30 ⁇ l/min from low concentration to high concentration, the binding time was 180s, and the dissociation time was 180s. 300s. Finally, the chip was regenerated using 10mM Glycine-HCl, pH 1.5 (BR-1003-54, Cytiva).
  • Figure 3B shows representative affinity profiles of ADI-38497 PG antibody and wild-type antibody binding to anti-PG scFv fusion protein by SPR. The results showed that only the ADI-38497 PG antibody could specifically bind to the anti-PG scFv as the extracellular domain of P329G CAR.
  • FIG. 3C shows a schematic diagram of a method for measuring antibody affinity (Avidity) by surface plasmon resonance (SPR).
  • the specific method is as follows: After coupling the anti-Fab IgG (I5260, Sigma) to the surface of the HLC chip (HLC30M, Xantec), capture the ADI-38497 molecule on the chip surface, and detect the fusion of the antibody on the chip surface with the anti-PG scFv in the mobile phase Affinities and kinetic constants are obtained for protein association and dissociation.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. Each cycle of the avidity assay consists of capture of ADI-38497 molecules, binding of a concentration of anti-PG scFv, and regeneration of the chip.
  • the anti-PG scFv after gradient dilution flows over the surface of the chip at a flow rate of 30 ⁇ l/min from low concentration to high concentration, the binding time is 180s, and the dissociation time is 300s .
  • the chip was regenerated using 10 mM glycine-HCl, pH 1.5 (BR-1003-54, Cytiva).
  • Figure 3D shows representative affinity profiles of ADI-38497 PG antibody and wild-type antibody with anti-PG scFv as P329G CAR extracellular domain measured by SPR. The results showed that only the ADI-38497 PG antibody could specifically bind to the P329G CAR.
  • the ADI-38497 PG antibody and the wild-type ADI-38497WT antibody were prepared into 5-fold gradient diluted antibody solutions of different concentrations with FACS buffer, incubated with 1E5 CAR-positive cells at 4°C for 30 minutes, washed with FACS buffer, Incubate with Fc ⁇ fragment-specific APC goat anti-human IgG (Jackson ImmunoResearch, 109-136-098) for 30 minutes at 4°C.
  • Flow cytometry was used to detect the antibody bound to the cells, and the APC channel MFI was analyzed, with the antibody concentration as the X-axis and the APC channel MFI as the Y-axis for plotting and calculating the binding EC50.
  • Figure 3E shows the binding ability of ADI-38497WT antibody and ADI-38497 PG antibody to P329G CAR-T cells. The results showed that only the P329G mutant antibody showed binding to CAR, while the WT antibody did not, which was consistent with the SPR results.
  • Table 8 summarizes the binding EC50 and EC90 values of ADI-38497 PG antibody and WT antibody to P329G CAR-T cells from different donors.
  • PBMC cells Peripheral Blood Mononuclear Cells, peripheral blood mononuclear cells
  • PBMC cells Peripheral Blood Mononuclear Cells, peripheral blood mononuclear cells
  • the killing effect on the target cells is plotted and analyzed with the antibody concentration as the X-axis and the cell lysis ratio as the Y-axis.
  • the cells were collected, washed twice with FACS buffer, and CD3, CD56, CD16 and CD107a antibodies were added, among which the CD107a antibody should be added in advance, and incubated with the cells at 37°C for 1 hour.
  • the above cell-antibody mixture was stained at 4°C for 30 minutes, washed twice, resuspended in FACS buffer, and detected by flow cytometry.
  • Figure 4A shows the ability of ADI-38497WT antibody and ADI-38497 PG antibody to mediate ADCC killing.
  • CD3, CD56, CD16 and CD107a expression all showed that only the WT antibody mediated the ADCC cytotoxic killing effect on the positive H929 tumor cells expressing BCMA, while the P329G mutant antibody lacked the ability to induce the ADCC effect.
  • ADCP reporter cell line Promega, G9871
  • H929 cells in the logarithmic growth phase
  • mix the ADCP reporter cells and H929 target cells according to the effect-to-target ratio of 2:1, 5:1, and mix with different concentrations of BCMA antibody, 37
  • luciferase detection kit Promega, E2620
  • Figure 4B shows the ability of ADI-38497WT antibody and ADI-38497 PG antibody to mediate ADCP killing.
  • the results showed that when different effect-to-target ratios (2:1 or 5:1) were tested, only the ADI-38497WT antibody mediated the ADCP killing effect on BCMA-expressing positive H929 tumor cells, while the P329G mutant antibody lacked the effect on the expression of The ability of BCMA-positive H929 tumor cells to induce ADCP killing effect.
  • H929 cells and L363 cells in the logarithmic growth phase were taken and spread in a certain number of well plates; a part of the H929 cells and L363 cells in the logarithmic growth phase were treated as target cells and treated with mitomycin C as a negative control. Then add different concentrations of ADI-38497 PG antibody to mix, continue to culture at 37°C for 48 hours, 72 hours and 120 hours respectively, use CellTiter-Glo (Promega, G9242) to detect the proportion of living cells, take the co-incubation time as the X axis, and the fluorescence The readings are plotted and analyzed on the Y-axis.
  • Figure 4C shows the ability of ADI-38497 PG antibody to mediate target cell lysis. /ml), all showed that only the ADI-38497 PG antibody lacked the ability to induce target cell lysis.
  • the CAR-T cells prepared by Donor 5 in Example 1 were revived and cultured at 37°C for overnight stability.
  • centrifuge to collect cells wash twice with FACS buffer, resuspend and add goat anti-human IgG containing LIVE/DEAD Fixable Dead Cell Stain, Biotin-F(ab') Fragment (Jackson ImmunoResearch, 109- 066-006) FACS buffer, stained at 4°C for 30 minutes, washed twice, added CD4, CD8, CD25, CD69 and APC-Streptavidin antibody combination, the above cell antibody mixture was stained at 4°C for 30 minutes, washed twice, The cells were resuspended in FACS buffer
  • Figure 5A shows that for H929 cells, only the ADI-38497 PG antibody containing the P329G mutation can specifically mediate the activation of CAR + -T, upregulate the expression levels of CD25 and CD69, and have no effect on CD4 + CAR + and CD8 + CAR + cells Differentially activated and exhibits a concentration gradient dependence of ADI-38497 PG antibody.
  • FIG. 5B shows that different BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, GSK PG IgG) induced the activation effect of L363 target cells on HuR968B CAR-T cells. It can be seen from Figure 5B that BCMA antibodies containing the P329G mutation (ADI-38497 PG antibody, ADI-38484 PG antibody, GSK PG IgG) can specifically mediate the activation of CAR+ T cells and significantly up-regulate the expression levels of CD25 and CD69. The expressions of CD25 and CD69 were also slightly up-regulated in the CAR-negative cell population, indicating a lower level of activation, but the degree of activation was not significant and could be ignored.
  • BCMA antibodies containing the P329G mutation ADI-38497 PG antibody, ADI-38484 PG antibody, GSK PG IgG
  • BCMA antibody containing the P329G mutation (ADI-38497 PG antibody, ADI- 38484 PG antibody, GSK PG IgG) can specifically mediate the activation of CAR + -T, significantly up-regulate the expression levels of CD25 and CD69, and have a gradient dependence of BCMA antibody with P329G mutation.
  • the UNT cells and CAR-T cells prepared by Donor 4 in Example 1 were revived and cultured at 37°C for overnight stability. Dilute ADI-38497WT antibody or ADI-38497 PG antibody in PBS, add to 96-well plate and incubate overnight at 4°C to coat the plate. The CAR-T cells recovered after overnight culture were added to a 96-well plate coated with antibodies.
  • CD3/CD28-coupled magnetic beads were directly added to the CAR-T cells (magnetic beads: cell ratio 3: 1); respectively incubated at 37°C for 72 hours and 120 hours, using Luminescent Cell Viability Assay (Promega, G7572) detects the luminescence value of cells.
  • FIG. 5C shows the proliferation of HuR968B CAR-T cells stimulated by coated ADI-38497WT antibody or ADI-38497 PG antibody.
  • HuR968B CAR-T cells proliferated under the stimulation of the coated ADI-38497 P329G antibody.
  • ADI-38497 PG antibody made HuR968B CAR-T cells proliferate after 3 or 5 days of stimulation, respectively
  • the stimulation with ADI-38497 PG antibody did not lead to significant proliferation of UNT cells under the stimulation of ADI-38497 PG antibody.
  • Both HuR968B CAR-T cells and UNT cells proliferated significantly under the stimulation of CD3/CD28 antibody-coupled magnetic beads.
  • the CAR-T cells prepared in Example 1 were revived and cultured at 37° C. overnight.
  • the tumor target cells and CAR-T cells were mixed according to the E:T ratio of 2:1, and 5-fold or 10-fold serial dilutions of different concentrations of BCMA antibodies (ADI-38497PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody , or as Benchmark’s GSK PG IgG) solution to a total volume of 200 ⁇ L, incubated at 37°C for about 24 hours, centrifuged, and collected the supernatant.
  • Cytokines were detected using BD TM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II ((BD, 551809)).
  • Capture Beads in the kit, and distribute the plate at 25 ⁇ L/well.
  • Figure 5D shows that the HuR968B CAR-T cells from donor 4 in Example 1 were co-cultured with H929 cells and RPMI8226 cells, after adding different concentrations of BCMA antibodies (ADI-38497 PG antibodies, ADI-38484 PG antibodies, ADI-38497WT antibodies, Or the release results of the effector cytokines secreted by CAR-T cells after GSK PG IgG as a positive control.
  • BCMA antibodies ADI-38497 PG antibodies, ADI-38484 PG antibodies, ADI-38497WT antibodies
  • FIG. 5E shows that HuR968B CAR-T cells from Donor 5 (purchased from ORiCELLS, Cat NO.: FPB004F-C, Lot NO.: PCH20210100004, Donor ID: Z0086) were co-cultured with different tumor cells, after adding different concentrations of BCMA antibody (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or Benchmark GSK PG IgG) the release results of CAR-T cells secreting effector cytokines.
  • BCMA antibody ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or Benchmark GSK PG IgG
  • the CAR-T cells prepared in Example 1 were revived and cultured at 37° C. overnight.
  • the tumor target cells and CAR-T cells were mixed according to the E:T ratio of 2:1, and different concentrations of BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or Benchmark GSK PG IgG) solution to a total volume of 200 ⁇ L, cultured at 37°C for about 24 hours, centrifuged, and transferred the cell supernatant to a 96-well white-bottomed microplate.
  • Use CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, G1780) and microplate reader (Molecular Devices, SpectraMax i3x) to measure the LDH value in the supernatant, and calculate the killing efficiency.
  • FIG. 5F shows that different BCMA antibodies (ADI-38497 PG antibody, ADI-38484 PG antibody, ADI-38497WT antibody, or Benchmark GSK PG IgG) induced HuR968B CAR-T cells to treat tumor cells with different BCMA expression levels (H929 ++ cells, Killing effect of RPMI8226 +++ cells, AMO1 + cells and L363 + cells).
  • HuR968B cells were not activated and did not produce a killing effect on tumor cells.
  • Figure 5G shows that neither the P329G BCMA antibody nor the WT BCMA antibody can mediate the killing effect of CAR-T on BCMA-negative target cells (such as BCMA-KO-H929 cells), while targeting BCMA-expressing H929 cells (H929) , WT BCMA antibody can not mediate the killing effect, only P329G BCMA antibody can induce CAR-T to produce a killing effect on tumor cells.
  • BCMA-negative target cells such as BCMA-KO-H929 cells
  • the CAR-T cells prepared by Donor 6 in Example 1 were revived and cultured at 37°C for overnight stability.
  • the tumor target cells and CAR-T cells are mixed according to the E:T ratio of 2:1, and a certain concentration of PG BCMA antibody is added.
  • BCMA antibody does not need to be added.
  • CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega, G1780
  • a microplate reader Molecular Devices, SpectraMax i3x
  • Figure 6 shows the effect of different concentrations of free BCMA protein on the killing effect of HuR968B CAR-T and Blue21CAR-T cells.
  • PG BCMA antibody combined with soluble Combined with BCMA
  • PG CAR-T can still exert normal killing effect. Therefore, the advantage of PG CAR-T cells is that their function is less affected by soluble ligands (such as soluble BCMA).
  • BALB/c mice (age 4-6 weeks, body weight 15-17g, female) were divided into 3 groups, namely ADI-38497 PG antibody, 1mg/kg antibody group; ADI-38497 PG antibody, 10mg/kg antibody group; and ADI-38497 PG antibody, 200mg/kg antibody group, 9 mice in each group; the antibody was diluted to 0.1mg/mL, 1mg/mL and 20mg/mL with 1 ⁇ PBS, and the administration volume of each mouse was 10mL/ kg, that is, the antibody doses are 1mg/kg, 10mg/mL and 200mg/mL respectively; the administration method is intravenous injection, and the administration frequency is single.
  • Coat the 96-well ELISA plate one day in advance Dilute the BCMA antigen to 1 ⁇ g/ml with coating solution (take a pack of carbonate (Thermo, 28382) powder, dissolve in 400 mL ultrapure water, dilute to 500 mL, and mix well to obtain the coating solution), 100 ⁇ L per well , seal the plate with film, and leave overnight at room temperature. Pour off the coating solution, pat dry on absorbent paper, then add 300 ⁇ L of washing solution to each well, shake and mix for 10 seconds, pat dry the washing solution, and repeat washing 3 times. Add blocking solution to each hole with a row gun, 200 ⁇ L, seal the plate with a sealing film, and incubate at room temperature for 2 hours. Then wash the plate once.
  • coating solution take a pack of carbonate (Thermo, 28382) powder, dissolve in 400 mL ultrapure water, dilute to 500 mL, and mix well to obtain the coating solution
  • 100 ⁇ L per well seal the plate with film,
  • diluted standard curve graded with known concentration of BCMA antibody to prepare the standard curve (for example, use the known concentration of ADI-38497 PG antibody to prepare the standard curve), quality control samples and samples to be tested in 100 ⁇ L per well, at room temperature Incubate for 2 hours. Pour off the pre-coating solution, pat dry on absorbent paper, then add 300 ⁇ L of washing solution to each well, shake and mix for 10 seconds, pat dry the washing solution, and repeat washing 3 times. Repeat once. Goat anti-human IgG- Dilute Fc-HRP antibody (BETHYL) 1:100,000, add 100 ⁇ L to each well, incubate at room temperature in the dark for 1 hour. Then wash the plate once.
  • BETHYL Goat anti-human IgG- Dilute Fc-HRP antibody
  • TMB substrate to the 96-well ELISA plate, 100 ⁇ L per well, and incubate at room temperature in the dark. Light color development for 5 minutes. Add 50 ⁇ L ELISA stop solution to each well, shake for 10 seconds, and read OD450nm and OD620nm values within 30 minutes.
  • FIGS 7A and 7B show the results of pharmacokinetic experiments of ADI-38497 PG antibody (hereinafter also referred to as PG Ab in vivo experiments in mice) in mice.
  • ADI-38497 PG antibody 1mg/kg AUC0-inf, Cmax, CL, T1/2 were 2480 ⁇ g ⁇ h/mL, 30ug/ml, 0.40ml/kg/h, 145h;
  • ADI -38497 PG antibody 10mg/kg: AUC0-inf, Cmax, CL, T1/2 are 24720 ⁇ g ⁇ h/mL, 187ug/ml, 0.32ml/kg/h, 219h; ADI-38497 PG antibody
  • Example 8 The effect of PG CAR-T cells combined with different doses of PG antibody against BCMA high-expressing tumors in vivo
  • mice Resuspend H929 cells with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right side of the back, subcutaneously injected with H929 cell suspension, the injection volume was 0.2mL/mouse, that is, the inoculation volume was 1 ⁇ 106 cells/mouse mouse.
  • mice with tumor volumes ranging from 50.82 to 104.36 mm3 were divided into 7 groups, namely PBS vehicle group, PG Ab group, PG only CAR-T group, traditional CAR-T group, and PG Ab group.
  • Ab+PG CAR-T 3mg/kg antibody group, PG Ab+PG CAR-T, 1mg/kg antibody group and PG Ab+PG CAR-T, 0.3mg/kg antibody group, 7 mice in each group.
  • Antibodies with a concentration of 0.3mg/mL, 0.1mg/mL and 0.03mg/mL were respectively prepared. After the grouping was completed, the antibody was administered on the 7th day. The volume of administration per mouse was 10mL/kg, and the administration frequency was Once a week, the administration method is intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended in 1 ⁇ PBS to prepare a CAR + cell suspension of 25 ⁇ 10 6 cells/mL, and 0.2 mL of the cell suspension was injected into the tail vein on the 7th day, and returned to Transfuse 5 ⁇ 10 6 CAR + cells/mouse.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week.
  • Figure 8A shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 high-expressing BCMA tumor cells.
  • the results showed that in the tumor model with high expression of BCMA, the administration of PG CAR-T cells alone did not produce obvious anti-tumor effect, and only the administration of PG antibody produced a certain anti-tumor effect. Mice produced significant anti-tumor effects, and showed an antibody dose-dependent effect.
  • TGI tumor growth inhibition
  • Figure 8B shows the body weight changes of the mice in this experiment.
  • the results showed that the body weight of mice treated with PG CAR-T cells combined with PG antibody remained stable after treatment, and the average body weight increased after treatment with PG antibody 0.3mg/kg, 1mg/kg and 3mg/kg 5.2%, 3.0%, 7.6%.
  • the results showed that PG CAR-T cells combined with PG antibody therapy had a significant anti-tumor effect without obvious toxic side effects.
  • Example 8-1 Take 30 ⁇ L of the mouse blood sample of Example 8-1, put it into a 96-well V-well plate, and mark it as the sample detection well; take 10 ⁇ L of the mouse blood sample, add it into the 96-well V-well plate, and mark it as the control well.
  • Figure 8C shows the expansion of PG CAR-T cells in mice in the experiment of Example 8-1.
  • the results showed that the in vivo expansion of PG CAR-T cells was dependent on the PG antibody, showing a certain dose-dependence of the antibody, and mice in the higher dose group had a higher level of PG CAR-T cell expansion.
  • Blue21CAR-T cells used as a positive control showed similar expansion kinetics. They also began to expand after 1 week in the reinfused mice, and reached the peak level after 2 weeks (174769 cells/100 ⁇ L peripheral blood) , and remained at a very high level (131963 cells/100 ⁇ L peripheral blood) after 4 weeks.
  • Example 9 The effect of PG CAR-T cells combined with different doses of PG antibody against BCMA low-expression tumors in vivo
  • mice with tumor volumes ranging from 74.14 to 110.29 mm3 were divided into 7 groups, namely vehicle group, PG Ab group, PG CAR-T group, traditional CAR-T group, and PG Ab+ PG CAR-T, 3mg/kg antibody group, PG Ab+PG CAR-T, 1mg/kg antibody group, and PG Ab+PG CAR-T, 0.3mg/kg antibody group, with 7 mice in each group.
  • Antibodies with a concentration of 0.3mg/mL, 0.1mg/mL and 0.03mg/mL were prepared respectively. After the grouping was completed, the antibody was administered on the 9th day.
  • the volume of administration per mouse was 10mL/kg, and the administration frequency was Once a week, the administration method is intraperitoneal injection.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week.
  • Figure 9A shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human L363 low-expressing BCMA tumor cells.
  • the results showed that in the tumor model with low expression of BCMA, only administration of PG CAR-T did not produce anti-tumor effect, while only administration of PG antibody had no obvious anti-tumor effect, with a TGI of 21%. Only the treated mice produced a significant anti-tumor effect, and showed an antibody dose-dependent effect.
  • PG CAR-T cells combined with 0.3 mg/kg dose of PG antibody, PG CAR-T cells induced a significant anti-tumor effect, with a TGI of 87%.
  • Figure 9B shows the body weight changes of the mice in this experiment.
  • the results showed that the body weight of the mice treated with PG CAR-T cells combined with PG antibody maintained a steady increase after treatment, and the average body weight increased after the combination of PG antibody 0.3mg/kg, 1mg/kg, and 3mg/kg 18.2%, 10.5%, 8.5%.
  • the results showed that PG CAR-T cells combined with PG antibody therapy had a significant anti-tumor effect and did not induce significant toxicity.
  • Figure 9C shows the expansion of PG CAR-T cells in mice in the experiment of Example 9-1.
  • the results showed that only PG CAR-T cells were administered, and they began to expand after 1 week of reinfusion into the mice, expanded to 919 cells/100 ⁇ L peripheral blood after 2 weeks, and rapidly decreased to 204 cells/100 ⁇ L peripheral blood at 3 weeks.
  • peripheral blood 100 ⁇ L peripheral blood, 8049 cells/100 ⁇ L peripheral blood and 3347 cells/100 ⁇ L peripheral blood remained at high levels after 3 weeks, respectively 2475 cells/100 ⁇ L peripheral blood, 4121 cells/100 ⁇ L peripheral blood and 1969 cells Cells/100 ⁇ L of peripheral blood was much higher than that of the group without combined administration of antibodies during the same period.
  • traditional Blue21CAR-T cells used as a positive control also expanded to a peak level (76836 cells/100 ⁇ L peripheral blood) after 2 weeks of reinfusion into mice, and maintained at a high level (36328 cells/100 ⁇ L peripheral blood) after 3 weeks. peripheral blood).
  • mice Resuspend H929 cells with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right side of the back, subcutaneously injected with H929 cell suspension, the injection volume was 0.2mL/mouse, that is, the inoculation volume was 1 ⁇ 106 cells/mouse mouse.
  • mice with tumor volumes ranging from 59.50 to 105.82 mm3 were divided into 7 groups, namely vehicle group, PG Ab group, PG CAR-T only group, and PG Ab+PG CAR-T group.
  • mice per group 10 ⁇ 10 6 group, PG Ab+PG CAR-T, 1 ⁇ 10 6 cell group, PG Ab+PG CAR-T, 0.1 ⁇ 10 6 cell group and PG Ab+PG CAR-T, 0.01 ⁇ 10 6 cell groups, 7 mice per group.
  • the CAR-T cells prepared from donor 4 were resuspended in 1 ⁇ PBS to prepare a CAR + cell suspension of 50 ⁇ 10 6 cells/mL, followed by 10-fold serial dilution, and then prepared into 5 ⁇ 10 6 , 0.5 ⁇ 10 6 and 0.05 ⁇ 10 6 cell suspensions/mL, inject 0.2 mL/mouse of the cell suspension into the tail vein on the 9th day.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week.
  • FIG. 10A shows the therapeutic effect of PG antibody combined with different doses of PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • the results showed that in the case of administering an extremely low dose of 0.01 ⁇ 10 6 CAR-T cells, CAR-T cells produced similar anti-tumor effects as those administered only with PG antibody, with TGIs of 49% and 50%, respectively.
  • Increasing the dose of CAR-T cells to 0.1 ⁇ 10 6 , 1 ⁇ 10 6 , and 10 ⁇ 10 6 CAR-T cells significantly increased the anti-tumor effect induced by PG CAR-T cells, with TGIs of 91%, 104%, and 103, respectively.
  • Administration of CAR-T cells alone did not show anti-tumor effects.
  • Figure 10B shows the expansion of PG CAR-T cells in mice in the experiment of Example 10-1.
  • the results showed that under the induction of PG antibody, CAR-T cells reinfused into the mice began to expand at 1 week, reached the peak level after 2 weeks, and remained at a high level after 3 weeks.
  • the expansion of T cells in vivo depends on the dose of CAR-T cells.
  • mice in the higher CAR-T dose group had higher levels of CAR-T cell expansion, 0.01 ⁇ 10 6 , 0.1 ⁇ 10 6 , 1 ⁇ 10 6 , 10
  • the peak expansion levels of the ⁇ 10 6 CAR-T cell dose group were 6 cells/100 ⁇ L peripheral blood, 338 cells/100 ⁇ L peripheral blood, 3640 cells/100 ⁇ L peripheral blood, and 12895 cells/100 ⁇ L peripheral blood.
  • Example 11 In vivo anti-tumor effect study of PG CAR-T cells combined with PG antibody at different administration frequencies
  • H929 cells Resuspend H929 cells with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right side of the back, subcutaneously injected with H929 cell suspension, the injection volume was 0.2mL/mouse, that is, the inoculation volume was 1 ⁇ 106 cells/mouse mouse.
  • mice with tumor volumes ranging from 51.25 to 94.97mm3 were divided into 7 groups, namely vehicle group, PG CAR-T only group, PG Ab+PG CAR-T, Q3/4 ⁇ 2 group, PG Ab+PG CAR-T, QW ⁇ 4 group, PG Ab+PG CAR-T, Q2W ⁇ 2 group, PG Ab+PG CAR-T, Q3W ⁇ 2 group and traditional CAR-T group, each Group of 7 mice.
  • the PG Ab antibody with a concentration of 0.1 mg/mL was prepared, and after the grouping was completed, the antibody was administered, with a volume of 10 mL/kg administered to each mouse, and the administration method was intraperitoneal injection.
  • the CAR-T cells prepared from donor 4 were resuspended in 1 ⁇ PBS to prepare a cell suspension of 10 ⁇ 106 CAR + cells/mL, and 0.2 mL of the cell suspension was injected into the tail vein.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week.
  • Fig. 11A shows the frequency of administration of the PG antibody in the experiment of Example 11-1.
  • Q3-4D ⁇ 2 4 weeks as a cycle, PG antibody is administered twice in the first week, with an interval of 3-4 days;
  • Q3W ⁇ 2 PG antibody is administered twice, and the dosing frequency is 3 weeks/time ;
  • Figure 11B shows the therapeutic effect of different administration frequencies of PG antibody when combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • the results showed that when PG CAR-T cells were used in combination with different administration frequencies of PG antibody, PG CAR-T cells could be induced to produce significant anti-tumor effects, and the frequency of PG antibody administration was increased, and the anti-tumor effect induced by PG CAR-T cells was significant.
  • the anti-tumor effect induced by CAR-T cells in the QW ⁇ 4 group was the most significant, showing the same anti-tumor effect as traditional Blue21 CAR-T cells.
  • Figure 11C shows the expansion of PG CAR-T cells in mice in the experiment of Example 11-1.
  • the results showed that when PG CAR-T cells were combined with PG antibody, the expansion kinetics of CAR-T cells were similar under the condition of different administration frequency of PG antibody. Expansion reached peak levels and declined to baseline levels after 3 weeks. In addition, the expansion of traditional Blue21CAR-T cells also reached the peak level 2 weeks after reinfusion into mice, and remained at a high level after 3 weeks.
  • Example 12 Study on anti-systemic tumor effect of PG CAR-T cells combined with different doses of PG antibody in vivo
  • H929-luc cells were prepared. Specifically, H929 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were used to package the lentivirus containing the GFP-luciferase gene, and the H929 cells were infected with the obtained lentivirus, and then sorted by flow cytometry to obtain H929-luc cell line expressing both GFP and luciferase.
  • H929-luc cells were resuspend with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were injected with H929-luc cell suspension through the tail vein, with an injection volume of 0.2 mL/mouse.
  • the substrate D-Luciferin (15mg/mL) was injected intraperitoneally, with an injection volume of 10mL/kg/mouse, and was analyzed by IVIS spectrum imaging 10 minutes after substrate injection.
  • mice with fluorescence signals between 1.17 ⁇ 10 7 and 1.43 ⁇ 10 8 photons/sec into 7 groups, namely vehicle group, PG Ab, 0.3mg/kg group, PG Ab, 3mg/kg group, PG CAR-T group, PG Ab+PG CAR-T, 0.3mg/kg+2 ⁇ 10 6 group, PG Ab+PG CAR-T, 3mg/kg+2 ⁇ 10 6 group and Blue21CAR-T group, 6-7 animals in each group mice.
  • Antibodies with a concentration of 0.03 mg/mL and 0.3 mg/mL were respectively prepared. After the grouping was completed, antibody administration was started on the 14th day.
  • the volume of administration was 10 mL/kg for each mouse, and the administration frequency was once a week.
  • the way of administration is intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended in 1 ⁇ PBS to prepare a CAR + cell suspension of 10 ⁇ 10 6 cells/mL, and 0.2 mL of the cell suspension was injected into the tail vein on the seventh day.
  • Figure 12A shows the fluorescence images of the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • PG antibody combined with PG CAR-T cells also produced a significant anti-tumor effect.
  • the fluorescence distribution and intensity of mice in the PG antibody 0.3 mg/kg combined with PG CAR-T cell group Compared with the control group, it was significantly reduced.
  • mice in the PG antibody 3mg/kg combined with PG CAR-T cell group had no obvious fluorescence signal, while the traditional Blue21CAR-T treatment group still had a large amount of fluorescence distribution, showing that the PG CAR-T cell group was higher than the Blue21CAR-T cell group.
  • Figure 12B shows the therapeutic effect of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • PG antibody was administered at 0.3 mg/kg
  • PG CAR-T cells produced a certain anti-tumor effect
  • increasing the dose of PG antibody to 3 mg/kg the anti-tumor effect induced by PG CAR-T cells increased significantly, and the anti-tumor effect was significantly increased.
  • the tumor effect was maintained longer, and the PG antibody was stopped, and the tumor growth began to recur in about 4 weeks, while the traditional Blue21CAR-T tumor relapsed faster when given the same cell dose, showing that PG CAR-T cells were more durable than traditional Blue21CAR-T antitumor effect. Only PG CAR-T cell group and PG antibody group did not show obvious anti-tumor effect.
  • Figure 12C shows the change in body weight of the mice in the above experiment. The results showed that the weight of the mice in each treatment group increased steadily during the treatment period, suggesting that different doses of PG antibody combined with PG CAR-T cells did not induce significant toxicity in the hematological tumor model.
  • H929 cells Resuspend H929 cells with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, and injected subcutaneously with 5 ⁇ 10 6 cells/mL of H929 cell suspension, with an injection volume of 0.2 mL/mouse.
  • mice with tumor volumes ranging from 38.49 to 104.77mm3 were divided into 8 groups, as shown in Table 11, which were the non-tumor-bearing vehicle group, PG Ab+PG CAR-T, 10mg /kg+10 ⁇ 10 6 group; and tumor-bearing vehicle group, PG CAR-T group, PG Ab group, PG Ab+PG CAR-T, 10mg/kg+10 ⁇ 10 6 group, PG Ab+PG CAR -T, 3mg/kg+10 ⁇ 10 6 group and PG Ab+PG CAR-T, 3mg/kg+1 ⁇ 10 6 group, 24 mice in each group.
  • Antibodies with a concentration of 1 mg/mL and 0.3 mg/mL were respectively prepared. After the grouping was completed, the antibody was administered, with a volume of 10 mL/kg administered to each mouse. The way of medicine is intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended with 1 ⁇ PBS, and CAR + cells were prepared at 50 ⁇ 106 /mL and 5 ⁇ 106 /mL cell suspensions, respectively. Inject 0.2 mL of cell suspension into the tail vein.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week. Peripheral blood was collected before the first antibody administration, after CAR-T reinfusion, before the third antibody administration, and at the end of the experiment. Four mice in each group were used for hematology and blood biochemical tests.
  • Figure 13A shows the therapeutic effect of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • the results showed that the PG antibody combined with PG CAR-T cell treatment group produced significant anti-tumor effects, while the anti-tumor effect of the mice in the PG antibody-only treatment group was weak, and PG CAR-T alone did not produce anti-tumor effects.
  • Figure 13B shows the change in body weight of the mice in this experiment.
  • the results showed that the weight of the mice in the tumor-bearing and non-tumor-bearing treatment groups increased steadily during the treatment period, and there was no significant change in body weight compared with the control mice, suggesting that different doses of PG antibody combined with different doses of PG CAR-T cells did not induce obvious toxic reactions.
  • Figure 13C and Figure 13D show the results of the mouse hematology and blood biochemical tests in the above experiments. The results showed that during the treatment period, the hematology and blood biochemical indicators of the mice in the tumor-bearing and non-tumor-bearing treatment groups had no significant changes compared with the control mice, indicating that the PG antibody combined with PG CAR-T cell therapy did not produce toxic reactions.
  • Embodiment 14 clinical research
  • the active components of the test drug described in this example are: P329G BCMA antibody (ADI-38497 PG antibody) and P329G CAR-T cells (the patient's own PBMC cells are transformed to obtain CAR-T cells).
  • P329G BCMA antibody is 20.0mg/ml P329G BCMA antibody, 0.76mg/ml histidine, 1.08mg/ml histidine hydrochloride, 50.00mg/ml sorbitol, 0.20mg/ml polysorbate 80, pH 6.0 ;Specification of P329G BCMA antibody: 60mg (3mL)/bottle.
  • Each bag contains 90-140 ⁇ 10 6 anti-P329G CAR-T positive cells dissolved in 7.5% DMSO cryoprotectant.
  • P329G BCMA antibody The manufacturer of P329G BCMA antibody is Innovent Biopharmaceutical (Suzhou) Co., Ltd.; the manufacturer of P329G CAR-T cells is Innovent Cell Pharmaceutical (Suzhou) Co., Ltd.
  • the proportion of monoclonal plasma cells detected by bone marrow cytology, bone marrow biopsy tissue or flow cytometry is ⁇ 5%;
  • serum immunoglobulin free light chain ⁇ 10 mg/dL and serum immunoglobulin ⁇ / ⁇ free light chain ratio is abnormal.
  • the ECOG score is 0 or 1.
  • ANC absolute neutrophil count
  • ALC absolute lymphocyte count
  • platelets ⁇ 50 ⁇ 109/L
  • Hemoglobin ⁇ 60g/L
  • Renal function serum creatinine ⁇ 2.5 ⁇ ULN; or creatinine clearance (CrCl) ⁇ 40ml/min calculated according to the Cockcroft-Gault formula;
  • Coagulation function fibrinogen ⁇ 1.0g/L; activated partial thromboplastin time, activated partial thromboplastin time ⁇ 1.5 ⁇ ULN, prothrombin time ⁇ 1.5 ⁇ ULN;
  • left ventricular ejection fraction left ventricular ejection fraction (left ventricular ejection fraction, LVEF) ⁇ 50%.
  • Subjects must sign the ICF, indicating that they understand the purpose and procedures of this study and are willing to participate in the study. Informed consent must be obtained before starting any study-related examination or procedure that is not part of the standard treatment of the subject's disease.
  • This study is a clinical study to evaluate the safety, tolerability, pharmacokinetics and preliminary efficacy of infusion of "P329G BCMA and P329G CAR-T cells" in the treatment of RRMM subjects.
  • the enrolled subjects will receive peripheral blood mononuclear cell (PBMC) apheresis (-28 ⁇ -21 days). Afterwards, the subject's own T cells were used to prepare P329G CAR-T cell preparations (hereinafter referred to as PG CAR-T cells). After successful preparation of PG CAR-T cells, the subject will first receive a preconditioning regimen of cyclophosphamide and fludarabine (-5 to -3 days), and then receive a dose of P329G 1 day after the rest assessment after pretreatment.
  • PBMC peripheral blood mononuclear cell
  • BCMA antibody (hereinafter referred to as ADI-38497 PG antibody) infusion, PG CAR-T cell infusion on the next day (0 day), and then from the PG CAR-T cell infusion day, periodic infusion of ADI every 21 days -38497 PG antibody, infused for up to 24 months, until disease progression, unacceptable toxicity, subject's withdrawal of informed consent, or other reasons for stopping study treatment (whichever occurs first).
  • ADI-38497 PG antibody dosage has 3 dosage groups: 0.3mg/kg, 1mg/kg, 3mg/kg.
  • the dose of PG CAR-T cells is 2*10 6 cells/kg.
  • the DLT observation window is 21 days after the first dose of ADI-38497 PG antibody administration, and the DLT observation period includes 1 administration of PG CAR-T cells.
  • Each dose group first enrolls 3 subjects, if no DLT is observed within the DLT observation window for the first 3 subjects, the administration of the next dose group can be started; if ⁇ 2 of the 3 subjects have DLT , the dose escalation will be terminated; if one of the 3 subjects who entered the group first at a certain dose has a DLT, 3 more subjects should be added to the same dose group (at this time, there are 6 subjects in this dose group in total). If no DLT occurred in the 3 supplementary subjects, the administration of the next dose group could be started. If ⁇ 1 of the 3 supplementary subjects had a DLT, the dose escalation was not allowed.
  • the maximum tolerated dose MTD
  • ⁇ 2 of the first 3 subjects in the initial antibody dose of 0.3 mg/kg group have DLT, they will return to a lower antibody dose of 0.1 mg/kg for dose exploration; If there is 1 case of DLT among the subjects, this dose group needs to add 3 subjects (at this time, there are 6 subjects in this dose group in total), if no DLT occurs in the 3 supplementary subjects, then the next trial can be started.
  • Dosage group administration if ⁇ 1 case of DLT occurs in the 3 supplemented subjects, then the lower antibody dose of 0.1mg/kg will be returned for dose exploration.
  • the investigator can choose the dose that may have a higher benefit/risk ratio to continue the study after discussing with the sponsor. For example, if the overall safety of the above-mentioned trials is good, it is predicted that further increasing the dose of PG CAR-T cells may increase the benefit of the subjects, and continue to explore the safety of the dose of 5*10 6 /kg PG CAR-T cells.
  • the investigator will review the safety data, combined with the PK data, to decide whether it is necessary to explore the next dose group and adjust the dose escalation scheme if necessary.
  • the first 3 subjects will adopt a staggered enrollment strategy. There must be an observation period of at least 2 weeks between the 1st and 2nd subjects, and the 2nd and 3rd subjects.
  • the current dose group will continue to receive the treatment until the disease progresses, intolerable toxicity, the subject withdraws informed consent or other conditions that require stopping Reason for study treatment (whichever occurs first), with a maximum treatment period of 24 months.
  • P329G BCMA antibody In the presence of a sufficient amount of P329G BCMA antibody, a very low dose of P329G CAR-T cells can induce a complete anti-tumor effect, and the effect is at least equivalent to that of The dose of traditional CAR-T cells was equivalent; the activity of P329G CAR-T cells was regulated by P329G BCMA antibody and showed a dose-dependent antibody dose, and different doses of P329G BCMA antibody induced different anti-tumor effects.

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Abstract

一种药物组合，所述药物组合包含(i)第一组分，其选自表达分子开关调控型 CAR 多肽的免疫效应细胞(例如，T细胞、NK 细胞)、编码所述 CAR 多肽的核酸分子、包含所述核酸分子的载体和它们的任意组合；和(ii)第二组分，其是包含 P329G 突变的特异性结合B细胞成熟抗原蛋白(BCMA)的抗体。还涉及包含所述药物组合的成套药盒、以及所述药物组合在受试者中治疗与BCMA 相关的疾病的用途。

Description

靶向BCMA的P329G抗体及其与嵌合抗原受体细胞的组合和应用 技术领域
本发明总体上涉及抗体工程和细胞免疫学领域，具体地，本发明涉及包含P329G突变的特异性结合B细胞成熟抗原蛋白(B-cell maturation antigen,BCMA)的抗体与经工程化以表达分子开关调控型嵌合抗原受体的免疫效应细胞(例如，T细胞、NK细胞)的组合，并涉及所述组合用于治疗与BCMA的表达相关的疾病，例如表达或过表达BCMA的癌症。
背景技术
B细胞成熟抗原(BCMA，即CD269，TNFRSF17)是肿瘤坏死因子受体超家族成员(TNFRSF)。BCMA是III型跨膜蛋白，在胞外结构域(ECD)中具有TNFR家族成员特征性的富半胱氨酸结构域(CRD)，该结构域形成配体结合基序。BCMA的配体包括B细胞激活因子(BAFF)和B细胞增殖诱导配体(APRIL)，其中B细胞增殖诱导配体(APRIL)与BCMA以更高的亲和力结合，促进肿瘤细胞增殖。
BCMA主要表达在成熟B细胞即浆细胞表面，在正常造血干细胞和非血源组织中不表达，BCMA信号对于长效骨髓浆细胞的生存不可或缺，但非总体B细胞稳态所必需。膜表面BCMA能够被γ分泌酶酶切而脱落，产生的可溶性BCMA(sBCMA)可能通过封闭BAFF/APRIL配体结合来降低膜表面BCMA信号传导。临床前模型以及人体肿瘤中发现BCMA在多发性骨髓瘤(Multiple Myeloma,MM)细胞中过表达，其上调经典以及非经典NF-κB信号，促进MM细胞生长、生存、粘附，诱导破骨细胞激活、血管生产、转移及免疫抑制等，BCMA表达已经成为诊断MM的重要标志物。此外，MM患者血清中sBCMA水平升高，与骨髓中MM细胞数量呈正比例相关，且其浓度变化与MM预后及治疗应答密切相关。
鉴于BCMA仅限于表达在浆细胞中，在天然和记忆性B细胞中不表达的特性，BCMA成为治疗MM的热门靶点，目前已开发了多种靶向药物，包括嵌合抗原受体T细胞(CAR-T)免疫疗法，其中蓝鸟(Bluebird)公司的Abecma(idecabtagene vicleucel,ide-cel)已于2021年3月份获得FDA批准，用于4线及以上复发难治性MM(RRMM)的治疗，强生(Johnson&Johnson)和南京传奇联合开发的Ciltacabtagene autoleucel(Cilta-cel)也已递交生物制品许可申请(BLA)。
Abecma作为首个靶向BCMA的CAR-T细胞疗法，其通过识别并结合多发性骨髓瘤癌细胞上的BCMA蛋白，导致表达BCMA蛋白的癌细胞死亡。即，其是通过CAR-T细胞上的嵌合抗原受体(CAR)直接靶向肿瘤细胞的表面抗原BCMA蛋白，从而达到识别和杀伤肿瘤细胞的目的。该传统嵌合抗原受体T细胞的不足之处在于，对于回输体内后的CAR-T细胞活性缺乏控制，这些CAR-T细胞在体内激活后会持续识别和杀伤表达其靶向抗原的细胞，在某些情况下(例如肿瘤患者负荷的肿瘤大)，大量快速激活的CAR-T细胞会释放海量炎性细胞因 子，使患者产生细胞因子释放综合征(CRS)、神经毒性(NT)等严重毒副反应。一项统计84个临床试验含有2592例患者的荟萃研究显示，接受传统CAR-T细胞治疗后3级以上CRS和NT发生率分别为29％和28％。虽然大量临床试验发现，发生毒性反应的多数患者在接受相应处理后能够快速恢复，也未显著影响抗肿瘤疗效，但毒副反应的处理占用大量临床资源，增加患者身心负担和经济负担。
由于绝大部分CAR-T细胞(例如，Abecma)靶向的抗原是肿瘤相关抗原(TAA)而非肿瘤特异性抗原(TSA)，这些TAA除了在肿瘤细胞上表达之外，在很多正常组织尤其是重要组织器官往往也存在低水平表达，CAR-T细胞对于所述正常组织的识别杀伤导致“在靶/脱肿瘤(On-target/off tumor)”毒性，机体短期内可能能够耐受，但如果长期不能恢复或者缓解，可能导致严重副作用。例如，临床使用靶向CD19的CAR-T细胞治疗CD19阳性血液肿瘤，CD19CAR-T细胞除了清除肿瘤细胞，它们也杀伤正常B细胞，CAR-T细胞在体内持续存在虽然与患者长期无复发地生存密切相关，但也同时导致机体长期B细胞发育不良，体液免疫缺失，容易引起感染。临床试验表明，接受CD19CAR-T细胞治疗后30天内27-36％淋巴瘤患者发生细菌感染，9.2-28％患者在一个月后发生病毒感染，需要中位时间6.7个月B细胞才能恢复，31-64％患者需要接受丙种球蛋白替代治疗。此外，CAR-T细胞在体内持续激活容易导致功能耗竭，损害其抗肿瘤效应，减少其在体内的存续性，从而降低长期治疗效应。
为了降低CAR-T细胞存在的“在靶/脱肿瘤(On-target/off tumor)”毒性，现有技术中通常采用以下策略。一种策略是在CAR设计时，将CAR中包含的抗原结合结构域设计为靶向肿瘤表面高表达而正常组织不表达或低表达的靶抗原。另一种策略是，严格控制施用的T细胞剂量，因为过多的CAR-T细胞受抗原刺激后会呈指数级递增，更易引起在靶/脱肿瘤效应。还有一种策略是，在构建CAR时引入诱导型自杀基因，如诱导型Caspase-9(iCasp9)自杀基因，当观察到患者发生在靶/脱肿瘤毒性时，施用AP1903(一种能够激活iCasp9的二聚化化学诱导剂)使CAR-T细胞发生凋亡，减轻毒性(张慧慧等人，自杀基因作为一种“安全性开关”控制CAR-T细胞毒性的临床前研究，中国肿瘤生物治疗杂志，2021,28(3):225-231)；也有利用单纯疱疹病毒胸苷激酶(HSV-TK)基因作为自杀基因，通过施用更昔洛韦治疗诱导CAR-T细胞发生凋亡的报导。另外，也有报导使用电穿孔转染方法使T细胞瞬时表达CAR，通过一过性杀伤功能来发挥治疗作用(Kenderian SS等人，CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia[J]Leukemia.2015；29(8):1637–1647)。然而，上述调控方法都是在发生毒性后进行，毒性缓解作用取决于药物清除CAR-T细胞的效果和效率，而且CAR-T细胞清除后就不能够恢复，影响长期抗肿瘤疗效。
发明概述
靶向BCMA的传统CAR-T细胞在治疗MM仍然面临毒性及复发问题。一项荟萃研究显示，接受靶向BCMA的传统CAR-T细胞治疗后CRS和NT发生率为74％(95％CI,56–91％) 和34％(95％CI,24–43％)，其中3级以上事件发生率分别为25％(95％CI,7–43％)和12％(95％CI,4–20％)。此外，接受治疗患者由于体内浆细胞缺失导致体液免疫受损，感染发生率增加，接受ide-cel治疗的128例复发难治性多发性骨髓瘤(RRMM)患者中69％发生感染，3级以上感染发生率22％，多数患者需要接受抗感染、升白细胞生长因子及免疫球蛋白替代治疗。同样，在接受Cilta-cel治疗的97例RRMM患者中，56％和20％发生感染及3级以上感染。这些临床结果表明，需要发展一种可调控的BCMA靶向CAR-T细胞疗法。
本发明人通过研究，设计、构建了通过与包含P329G突变的特异性结合BCMA分子的抗体的Fc结构域结合来靶向BCMA的P329G CAR-T细胞，并在体内外验证了其特异、可控的抗肿瘤效应。有别于传统BCMA靶向CAR-T细胞直接识别杀伤MM细胞，本发明的药物组合包括两个组分：BCMA特异的P329G抗体和P329G CAR-T细胞。BCMA特异的P329G抗体识别表达BCMA的肿瘤细胞后，P329G CAR-T细胞通过识别P329G抗体的Fc结构域再定向至肿瘤细胞，产生肿瘤识别及杀伤效应(参见图1B)。在使用本发明的药物组合的治疗方法中，P329G抗体作为连接P329G CAR-T细胞和肿瘤细胞的桥梁，发挥“分子开关”作用，调节P329G CAR-T细胞活性。
因此，在第一方面，本发明提供了作为表达CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)的“分子开关”的、能够特异性结合BCMA分子的P329G突变抗体，其包括但不限于ADI-38497 PG Ab(本文中也称为“ADI-38497 PG抗体”、“38497 PG Ab”、“ADI-38497 PG IgG”、“38497 PG IgG”、“PG 38497抗体”)、ADI-38484 PG Ab(本文中也称为“ADI-38484 PG抗体”、“38484 PG Ab”、“ADI-38484 PG IgG”、“38484 PG IgG”、“PG 38484抗体”)。
在一些实施方案中，本发明获得了特异性结合BCMA分子的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:25)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:26)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列DRGDQILDV(SEQ ID NO:27)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISRYLN(SEQ ID NO:28)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列AASSLQS(SEQ ID NO:29)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQKYFDIT(SEQ ID NO:30)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)所述重链可变区包含根据Kabat编号的氨基酸序列NDVIS(SEQ ID NO:31)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列VIIPIFGIANYAQKFQG(SEQ ID NO:32)所示的CDR H2、或所述CDR H2的不超过2个 氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列GRGYYSSWLHDI(SEQ ID NO:33)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列QASQDITNYLN(SEQ ID NO:34)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列DASNLET(SEQ ID NO:35)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQAFDLIT(SEQ ID NO:36)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明提供了特异性结合BCMA分子的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:25)所示的CDR H1；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:26)所示的CDR H2；和氨基酸序列DRGDQILDV(SEQ ID NO:27)所示的CDR H3；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISRYLN(SEQ ID NO:28)所示的CDR L1；氨基酸序列AASSLQS(SEQ ID NO:29)所示的CDR L2；和氨基酸序列QQKYFDIT(SEQ ID NO:30)所示的CDR L3；
(b)所述重链可变区包含根据Kabat编号的氨基酸序列NDVIS(SEQ ID NO:31)所示的CDR H1；氨基酸序列VIIPIFGIANYAQKFQG(SEQ ID NO:32)所示的CDR H2；和氨基酸序列GRGYYSSWLHDI(SEQ ID NO:33)所示的CDR H3；所述轻链可变区包含根据Kabat编号的氨基酸序列QASQDITNYLN(SEQ ID NO:34)所示的CDR L1；氨基酸序列DASNLET(SEQ ID NO:35)所示的CDR L2；和氨基酸序列QQAFDLIT(SEQ ID NO:36)所示的CDR L3。
在一些实施方案中，本发明获得了特异性结合BCMA分子的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
(a)重链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区包含SEQ ID NO:9的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:10的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明获得了特异性结合BCMA分子的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
(a)重链可变区包含SEQ ID NO:2的序列，且轻链可变区包含SEQ ID NO:3的序列；
(b)重链可变区包含SEQ ID NO:9的序列，且轻链可变区包含SEQ ID NO:10的序列。
在一些实施方案中，本发明的特异性结合BCMA分子的抗体是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是IgG1抗体。
在一些实施方案中，本发明的特异性结合BCMA分子的抗体的抗原结合片段是Fab、Fab’、 F(ab’) 2、Fv、单链Fv、单链Fab、双体抗体(diabody)。
对于所述特异性结合BCMA分子的抗体或抗原结合片段，通过将根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)获得了具有突变Fc结构域的抗体，其中，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低。
在一些实施方案中，所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域；
在一些实施方案中，特异性结合BCMA分子的抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；
例如，所述抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；和SEQ ID NO:6所示的轻链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一个实施方案中，特异性结合BCMA分子的抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列和SEQ ID NO:6所示的轻链恒定区序列。
在第二方面，本发明提供了编码本发明第一方面的抗体的核酸、包含编码所述抗体的核酸的载体、包含所述核酸分子或载体的细胞、以及制备所述抗体的方法，所述方法包括在适于表达编码本发明第一方面所述的特异性结合BCMA分子的抗体或抗原结合片段的核酸的条件下，培养导入有编码第一方面所述的特异性结合BCMA分子的抗体或抗原结合片段的核酸的表达载体的宿主细胞，分离所述特异性结合BCMA分子的抗体或抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述特异性结合BCMA分子的抗体或抗原结合片段。优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，最优选地，所述宿主细胞是HEK293细胞或CHO细胞。
在第三方面，本发明提供了药物组合，其包含
(i)第一组分，其选自表达分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)、编码所述CAR多肽的核酸分子、包含所述核酸分子的载体、和它们的任意组合；和
(ii)第二组分，其是包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段(也称为P329G突变抗体)，例如，本发明第一方面的P329G突变抗体，以及
任选地可药用辅料；
其中，所述分子开关调控型CAR多肽包含
(1)人源化抗P329G突变scFv序列，其中所述scFv序列包含能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域的如下序列：
(i)重链可变区，其包含根据Kabat编号的
(a)氨基酸序列RYWMN(SEQ ID NO:19)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:20)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:21)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(ii)轻链可变区，其包含根据Kabat编号的
(d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:22)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(e)氨基酸序列GTNKRAP(SEQ ID NO:23)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:24)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代；
(2)铰链区/间隔区，其选自
(i)(G 4S) n、(SG 4) n或G 4(SG 4) n肽接头，其中“n”是1至10的整数，例如1至4的整数；例如SEQ ID NO:14所示的序列；
(ii)CD8α铰链区或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:18所示的序列或其具有1-2个氨基酸修饰的变体；
(3)跨膜区(TM)，其选自CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:15所示的序列或其具有1-2个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其选自4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:16所示的序列或其具有1-2个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，SEQ ID NO:17所示的序列或其具有1-10个、1-5个氨基酸修饰的变体；
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明药物组合中所述的分子开关调控型CAR多肽包含
(1)人源化抗P329G突变scFv序列，其中所述scFv序列包含能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域的如下序列：
(i)重链可变区，其包含根据Kabat编号的
(a)氨基酸序列RYWMN(SEQ ID NO:19)所示的CDR H1；
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:20)所示的CDR H2；和
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:21)所示的CDR H3；和
(ii)轻链可变区，其包含根据Kabat编号的
(d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:22)所示的CDR L1；
(e)氨基酸序列GTNKRAP(SEQ ID NO:23)所示的CDR L2；和
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:24)所示的CDR L3；
例如，(i)重链可变区，其包含SEQ ID NO:12的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
(ii)轻链可变区，其包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
例如，(i)重链可变区，其包含SEQ ID NO:12的序列，和(ii)轻链可变区，其包含SEQ ID NO:13的序列；
(2)铰链区/间隔区，其选自
(i)(G 4S) n、(SG 4) n或G 4(SG 4) n肽接头，其中“n”是1至4的整数，例如SEQ ID NO:14所示的序列；
(ii)SEQ ID NO:18所示的CD8α铰链区序列或其具有1个氨基酸修饰的变体；
(3)跨膜区(TM)，其选自SEQ ID NO:15所示的CD8跨膜结构域或其具有1个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其选自SEQ ID NO:16所示的4-1BB共刺激结构域或其具有1个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，其选自SEQ ID NO:17所示的CD3ζ信号传导结构域或其具有1个氨基酸修饰的变体；
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明药物组合中所述的分子开关调控型CAR多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:11所示的信号肽序列，
在一些实施方案中，本发明药物组合中所述的分子开关调控型CAR多肽具有SEQ ID NO:1所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在第四方面，本发明提供了编码本发明药物组合中所述的分子开关调控型CAR多肽的核酸、包含编码所述CAR多肽的核酸的载体、和包含所述CAR核酸分子或载体的细胞、或表达所述CAR多肽的细胞，优选地，所述细胞是自体T细胞或同种异体T细胞。
在一些实施方案中，编码本发明药物组合中所述的分子开关调控型CAR多肽的核酸分子是编码SEQ ID NO:1所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的核酸分子。
在一些实施方案中，包含编码本发明药物组合中所述的分子开关调控型CAR多肽的核酸分子的载体选自DNA载体、RNA载体、质粒、慢病毒载体、腺病毒载体或逆转录病毒载体。
在一些实施方案中，本发明药物组合中的免疫效应细胞是自体T细胞、NK细胞或同种异体T细胞、NK细胞制备的表达本发明所述的分子开关调控型CAR多肽的T细胞、NK细胞，例如，所述免疫效应细胞是自人外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)分离的T细胞、NK细胞制备的表达本发明所述的分子开关调控型CAR多肽的T细胞、NK细胞。
在一些实施方案中，本发明利用自多个不同供者来源的人PBMC制备的原代P329G CAR-T细胞，并利用P329G突变的抗BCMA人源化抗体，在体外共培养体系中评价了P329G CAR-T细胞针对表达BCMA的肿瘤细胞通过抗体发挥的效应功能，由此，P329G突变的抗BCMA抗体能够作为“分子开关”调节P329G CAR-T细胞对BCMA阳性肿瘤细胞的识别及杀伤活性，该体外效应功能与直接靶向BCMA阳性肿瘤细胞的传统CAR-T细胞相当，但传统CAR-T细胞的活性不依赖P329G突变抗体。
又在一些实施方案中，本发明在接种BCMA表达阳性的人肿瘤细胞系来源肿瘤的免疫缺陷小鼠体内验证了P329G CAR-T细胞联合P329G突变抗体的抗肿瘤效应，并对抗体给药剂量、间隔等进行了研究。
体内外实验表明，只有在P329G抗体存在情况下，P329G CAR-T细胞活性才能“开启”，产生效应功能，识别和杀伤BCMA阳性肿瘤细胞；通过调节P329G抗体剂量及给药间隔，能够调控P329G CAR-T细胞体内扩增程度及其抗肿瘤活性强度。鉴于BCMA与其配体结合后传递促肿瘤信号，P329G抗体能够通过阻断这一信号发挥抗肿瘤效应，因此，BCMA特异的P329G抗体和P329G CAR-T细胞还能够产生协同抗肿瘤效应。
在一些实施方案中，对于本发明的药物组合，(i)将表达本发明CAR多肽的免疫效应细胞以1×10 6个细胞/kg体重－10×10 6个细胞/kg体重，例如1×10 6个细胞/kg体重、2×10 6个细胞/kg体重、3×10 6个细胞/kg体重、5×10 6个细胞/kg体重、7×10 6个细胞/kg体重、9×10 6个细胞/kg体重、10×10 6个细胞/kg体重的剂量以单次或多次静脉内施用至受试者；和
(ii)将本发明的药物组合中所述的P329G突变抗体以0.1-10mg/kg、优选地0.1mg/kg、0.3mg/kg、0.5mg/kg、1mg/kg、3mg/kg、5mg/kg、7mg/kg、9mg/kg、10mg/kg的剂量单元的形式，优选地胃肠外、更优选地静脉内施用至受试者。
在一些实施方案中，本发明的药物组合中的(i)和(ii)分开、同时或依次施用，例如，第一天施用(ii)，同一天静脉内施用(i)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者
第一天施用(i)，第二天静脉内施用(ii)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者
第一天施用(ii)，第二天静脉内施用(i)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；
例如，(i)和(ii)各施用一次，然后按照每3－4天一次、每周一次、每两周一次、每三周一次或每四周一次的给药频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)。
在第四方面，本发明提供了本发明的药物组合的用途，用于在受试者中治疗与BCMA相关的疾病，包括向受试者施用治疗有效量的前述第三方面所定义的药物组合，其中所述与 BCMA相关的疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在第五方面，本发明提供了本发明的药物组合在制备用于治疗与BCMA相关的疾病的药物中的用途，所述与BCMA相关的疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在第六方面，本发明提供了用于治疗与BCMA相关的疾病的方法，所述方法包括向受试者施用治疗有效量的本发明的药物组合，所述疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在第七方面，本发明提供了成套药盒，其包含如前述第三方面所定义的药物组合，优选地所述药盒为药物剂量单元形式。
第八方面，本发明提供了一种药物复合物，其是一种由
(i)选自表达本发明药物组合中所述的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)；和
(ii)包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段(也称为P329G突变抗体)，例如，本发明第一方面的P329G突变抗体
免疫效应细胞通过CAR多肽的胞外结构域中的人源化抗P329G突变scFv序列与P329G突变抗体的Fc结构域结合而产生的复合物；
例如，其中所述免疫效应细胞是自自体T细胞或同种异体T细胞制备的表达本发明药物组合中所述的分子开关调控型CAR多肽的T细胞，例如，所述免疫效应细胞是自人PBMC分离的T细胞制备的表达本发明所述的分子开关调控型CAR多肽的T细胞；
例如，其中P329G突变抗体是ADI-38497 PG Ab和/或ADI-38484 PG Ab。
本发明还提供了所述药物复合物的用途，用于在受试者中治疗与BCMA相关的疾病，优选地，所述与BCMA相关的疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
由此，本发明首先在第一方面，通过体外结合能力、亲和力及Fc效应功能检测，获得了高亲和力的BCMA特异的P329G抗体，该抗体能够同时结合BCMA抗原和P329G CAR分子，发挥桥接效应。其次，本发明在第三方面通过使用构建的P329G CAR结构分子并将该CAR分子制备的P329G CAR-T细胞与BCMA特异的P329G抗体组合，然后与BCMA阳性MM细胞体外共培养构建体外共培养体系，在该系统中，验证了P329G抗体作为“分子开关”调节P329G CAR-T细胞活性的效应，即，只有在P329G突变抗体存在情况下，P329G CAR-T细胞才能被激活、增殖、分泌效应细胞因子及产生杀伤效应，并且这些效应呈现P329G抗体剂量依赖性，随着抗体剂量升高，P329G CAR-T细胞识别和杀伤效应增强。而未携带P329G突变的WT抗体不能激发P329G CAR-T细胞效应功能。
此外，本发明的体外实验表明，可溶性BCMA抗原不会影响与BCMA特异的P329G抗体联用时的P329G CAR-T细胞活性，而可溶性BCMA抗原对传统CAR-T细胞产生显著抑制 效应。在高和低表达BCMA的MM细胞皮下以及全身性荷瘤免疫缺陷小鼠模型中，本发明的P329G CAR-T细胞联合P329G抗体产生良好的抗肿瘤效应，抗肿瘤效果至少与传统CAR-T细胞相当，但P329G CAR-T细胞体内扩增程度显著低于传统CAR-T细胞，提示在产生相当抗肿瘤效应同时P329G CAR-T细胞可能诱导更低的CRS、NT等急性毒副效应；进一步地，通过调节P329G抗体给药剂量和间隔能够调节P329G CAR-T细胞体内扩增程度以及抗肿瘤效应强度，在肿瘤清除后，停止给予P329G抗体，仍然能够维持持续的抗肿瘤效应，这为临床应用中“关闭”P329G CAR-T细胞活性，在维持持续抗肿瘤效应同时恢复正常浆细胞数量及体液免疫降低感染等长期毒副效应奠定了基础。
本发明的毒理实验也表明，P329G CAR-T细胞联合P329G抗体没有产生明显毒性，从而为临床转化奠定了基础。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A显示了用实施例1-1构建的HuR968B、Blue21 CAR转导T细胞后，CD3 +细胞、CD4 +、CD8 +T细胞亚群中CAR的表达。
图1B显示了P329G CAR-T细胞通过P329G抗体介导靶向表达BCMA的靶细胞的作用机制。8种CAR构建体的结构。图中，“SP”表示信号肽(signal peptide)；“TMD”表示跨膜结构域(transmembrane domain)；“ICD”表示胞内结构域(intracellular domain)；“CSD”表示共刺激信号结构域(costimulatory domain)；“SSD”表示刺激信号结构域(stimulatory signaling domain)。在图中的PG CAR所述构建体中，胞外结构域包含能够特异性结合含有P329G突变的突变Fc结构域的抗原结合部分，且所述抗原结合部分包含重链可变区(VH)和轻链可变区(VHL)。
图2A显示了采用表面等离子共振法(SPR)测定抗体亲和力的方法示意图。
图2B显示了采用SPR测定ADI-38497 PG抗体与重组人、食蟹猴、小鼠、大鼠和兔BCMA蛋白的代表性亲和力图谱。
图2C显示了P329G BCMA抗体与稳定表达人、食蟹猴及小鼠BCMA的CHO-GS细胞的结合能力。
图2D显示了P329G BCMA抗体与表达BCMA的阳性多发性骨髓瘤细胞系MM.1s、RPMI8226、U266、H929、L363及AMO1的结合活性。
图3A显示了采用表面等离子共振法(SPR)测定抗P329G突变的特异性单链抗体-兔Fc融合蛋白与ADI-38497 P329G突变抗体亲和力的检测示意图。
图3B显示了采用SPR测定ADI-38497 PG抗体及野生型抗体与抗PG scFv融合蛋白结合的代表性亲和力图谱。
图3C显示了采用表面等离子共振法(SPR)测定抗体亲合力(Avidity)的方法示意图。
图3D显示了采用SPR测定ADI-38497 PG抗体及野生型抗体与抗PG scFv融合蛋白(期中，P329G CAR胞外结构域包含抗PG scFv)的代表性亲合力图谱。
图3E显示了ADI-38497WT抗体和ADI-38497 PG抗体与P329G CAR-T细胞的结合能力。
图4A显示了ADI-38497WT抗体和ADI-38497 PG抗体介导ADCC杀伤的能力。
图4B显示了ADI-38497WT抗体和ADI-38497 PG抗体介导ADCP杀伤的能力。
图4C显示了ADI-38497 PG抗体介导靶细胞裂解的能力。
图5A显示了针对H929细胞，含P329G突变的ADI-38497 PG抗体特异性介导CAR +-T的激活。
图5B显示了不同BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、GSK PG IgG)诱导L363靶细胞对HuR968B CAR-T细胞的激活效应。
图5C显示了HuR968B CAR-T细胞受包被的ADI-38497WT抗体或ADI-38497 PG抗体刺激后的增殖情况。
图5D显示了HuR968B CAR-T细胞与H929细胞、RPMI8226细胞共培养，在加入不同浓度BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或作为阳性对照的GSK PG IgG)后的CAR-T细胞分泌效应细胞因子的释放结果。
图5E显示了HuR968B CAR-T细胞与不同肿瘤细胞共培养，在加入不同浓度BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或GSK PG IgG)后CAR-T细胞分泌效应细胞因子的释放结果。
图5F显示了不同BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或GSK PG IgG)诱导HuR968B CAR-T细胞对不同BCMA表达水平肿瘤细胞(H929 ++细胞、RPMI8226 +++细胞、AMO1 +细胞和L363 +细胞)的杀伤效应。
图5G显示了P329G BCMA抗体诱导CAR-T产生对肿瘤细胞的杀伤效应。
图6显示了不同浓度的游离BCMA蛋白对HuR968B CAR-T和Blue21CAR-T细胞杀伤效应的影响。
图7A和图7B显示了小鼠中ADI-38497 PG抗体的药代动力学实验结果。
图8A显示了在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞的治疗效应。图中，“cCAR-T”表示传统CAR-T，即，Blue21CAR-T。
图8B显示了在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗时，小鼠的体重变化。
图8C显示了在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗时，PG CAR-T细胞在小鼠体内的扩增情况。
图9A显示了在皮下接种人L363低表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂 量PG抗体联合PG CAR-T细胞的治疗效应。图中，“cCAR-T”表示传统CAR-T，即，Blue21CAR-T。
图9B显示了在皮下接种人L363低表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗时，小鼠的体重变化。
图9C显示了在皮下接种人L363低表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗时，PG CAR-T细胞在小鼠体内的扩增情况。
图10A显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合不同剂量PG CAR-T细胞的治疗效应。
图10B显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合不同剂量PG CAR-T细胞治疗时，PG CAR-T细胞在小鼠体内的扩增情况。
图11A显示了实施例11-1的实验中PG抗体的给药频率。
图11B显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中，联用PG CAR-T细胞时PG抗体不同给药时频率的治疗效应。
图11C显示了实施例11-1的实验中，PG CAR-T细胞在小鼠体内扩增情况。
图12A显示了在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗效应的荧光图像。
图12B显示了在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞的治疗效应。
图12C显示了在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗时，小鼠的体重变化。
图13A显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合PG CAR-T细胞的治疗效应。
图13B显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合PG CAR-T细胞治疗时，小鼠的体重变化。
图13C和图13D显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合PG CAR-T细胞治疗时，小鼠血液学和血生化检测结果。
发明详述
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包 括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“BCMA”和“B细胞成熟抗原”可互换地使用，其包括人BCMA的变体、同种型、物种同源物和与BCMA(例如人BCMA)具有至少一个相同表位的类似物。BCMA蛋白也可包括BCMA的片段，诸如胞外结构域以及胞外结构域的片段，例如保持与本发明任何抗体的结合能力的片段。
如本文所用的术语“BCMA抗体”、“针对BCMA的抗体”、“特异性结合BCMA的抗体”、“特异性靶向BCMA的抗体”、“特异性识别BCMA的抗体”可互换地使用，意指能够与B细胞成熟抗原(BCMA)特异性结合的抗体。
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。优选地，本发明的抗体是单结构域抗体或重链抗体。
“抗体片段”或“抗原结合片段”在本文中可互换地使用，指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab、双体抗体(diabody)。
术语“scFv”指一种融合蛋白，其包含至少一个包含轻链可变区的抗体片段和至少一个包含重链可变区的抗体片段，其中轻链可变区和重链可变区任选地借助柔性短多肽接头连续地连接，并且能够表达为单链多肽，并且其中scFv保留衍生它的完整抗体的特异性。除非另外指出，否则如本文所用，scFv可以具有按任何顺序(例如，相对于多肽的N末端和C末端)的VL可变区和VH可变区，scFv可以包含VL-接头-VH或可以包含VH-接头-VL。
“互补决定区”或“CDR区”或“CDR”或“高变区”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作CDR H1、CDR H2和CDR H3，而位于抗体轻链可变结构域内的CDR被称作CDR L1、CDR L2和CDR L3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人,(1989)Nature 342:877-883，Al-Lazikani等人, “Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
CDR也可以基于与参考CDR序列(例如本发明示例的CDR之任一)具有相同的Kabat编号位置而确定。在本发明中，当提及抗体可变区和具体CDR序列(包括重链可变区残基)时，是指根据Kabat编号系统的编号位置。
尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia或AbM定义的其余CDR残基可以被保守氨基酸残基替代。
术语“嵌合抗体”是这样的抗体分子，其中(a)将恒定区或其部分改变、替换或交换，从而抗原结合位点与不同的或改变的类别和/或物种的恒定区或赋予嵌合抗体新性能的完全不同的分子(例如，酶、毒素、激素、生长因子、药物)等连接；或(b)将可变区或其部分用具有不同或改变的抗原特异性的可变区改变、替换或交换。例如，鼠抗体可以通过将其恒定区更换为来自人免疫球蛋白的恒定区进行修饰。由于更换为人类恒定区，该嵌合抗体可以保留其在识别抗原方面的特异性，同时如与原始鼠抗体相比，具有在人类中降低的抗原性。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体中的所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
“人抗体”指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
术语“Fc区”指免疫球蛋白重链的C端区域，包括天然序列Fc区和变异Fc区。人IgG重链Fc区通常定义为自其Cys226或Pro230位置的氨基酸残基至羧基末端的区段，Fc区的C末端447位的赖氨酸残基(依照EU编号系统)可以存在或者缺失。因而，完整抗体组合物可以 包括所有K447残基都被消除的抗体群、无K447残基被消除的抗体群、或者混合了有K447残基的抗体和没有K447残基的抗体的抗体群。
在某些实施方案中，免疫球蛋白的Fc区包含两个恒定结构域域，即CH2和CH3，在另一些实施方案中，免疫球蛋白的Fc区包含三个恒定结构域，即CH2、CH3和CH4。
IgG与Fcγ受体或C1q的结合依赖于定位在铰链区和CH2结构域中的残基。CH2结构域的两个区域对FcγR和补体C1q结合至关重要，并且在IgG2和IgG4中具有唯一的序列。已显示取代人IgG1和IgG2中233-236位的残基和取代人IgG4中327、330和331位的残基可大幅降低ADCC和CDC活性(Armour等人,Eur.J.Immunol.29(8)，1999，2613-2624；Shields等人,J.Biol.Chem.276(9)，2001，6591-6604)。
“功能性Fc区”与“有功能Fc区”等类似术语可以互换使用，指具有野生型Fc区的效应功能的Fc区。
“变异Fc区”、“Fc突变体”、“携带突变的Fc区”、“突变Fc区”、“Fc区变体”、“Fc变体”、“变体Fc区”和“突变的Fc区”等类似术语可以互换使用，指包含至少一处氨基酸修饰而区别于天然序列Fc区/野生型Fc区的Fc区。
在一些实施方案中，变异Fc区包含与天然序列Fc区的氨基酸序列相差一处或多处氨基酸取代、缺失或添加的氨基酸序列。在一些实施方案中，变异Fc区与野生型IgG的Fc区相比具有至少一处氨基酸取代，所述至少一处氨基酸取代是将根据EU编号的P329位置处的氨基酸取代为甘氨酸(G)。
“Fc受体”或“FcR”指结合抗体Fc区的分子。在一些实施方案中，FcR是天然人FcR。在一些实施方案中，FcR是结合IgG抗体的受体，即FcγR，包括FcγRI(CD64)、FcγRII(CD32)和FcγRIII(CD16)三种受体，以及这些受体的等位变体和可变剪接形式。FcγRII受体包括FcγRIIA和FcγRIIB，FcγRIII受体包括FcγRIIIA和FcγRIIIB。
术语“效应功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括：Fc受体结合作用、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞摄取抗原、C1q结合和补体依赖的细胞毒性(CDC)、下调细胞表面受体(例如B细胞受体)和B细胞活化。
术语“抗体依赖的细胞介导的细胞毒性(ADCC)”是某些细胞毒性效应细胞(例如天然杀伤(NK)细胞)介导对靶细胞和外来宿主细胞杀伤的主要机制之一。在一些实施方案中，本发明的嵌合抗原受体提供T淋巴细胞的抗体依赖性细胞毒作用、增强NK细胞的抗体依赖性细胞毒作用。本发明的嵌合抗原受体通过与结合肿瘤细胞的抗体(或包含Fc部分的其他抗肿瘤分子)结合，诱发表达该嵌合抗原受体的T细胞活化、持续增殖和发挥经该抗体(或包含Fc部分的其他抗肿瘤分子)介导的对目的癌细胞的特异性细胞毒性。
术语“抗体依赖的细胞吞噬作用(ADCP)”指一种细胞反应，其中通过结合靶细胞的抗体与巨噬细胞表面的FcγRIIIa结合，诱导激活巨噬细胞，从而使靶细胞内化和被吞噬体酸化降 解。ADCP也可由FcγRIIa和FcγRI介导，但是占比较小。
术语“补体依赖的细胞毒性作用(CDC)”是指在补体存在下靶细胞的裂解。补体系统是由一系列蛋白质组成的先天免疫系统的一部分。补体系统的蛋白质称为“补体”，以缩写符号C1、C2、C3等表示，其是存在于人或脊椎动物血清、组织液中的一组不耐热的，经活化后具有酶活性的蛋白质。C1q是依赖补体的细胞毒性(CDC)途径的第一成分，其能够结合六个抗体，但与两个IgG结合就足以活化补体级联。经典补体途径的激活由补体系统的第一组分(C1q)与结合相关抗原的抗体(适当的亚类)结合而启动，活化一系列补体级联反应，在靶细胞膜中形成孔洞，从而导致靶细胞死亡。为了评估补体活化，可以执行CDC测定法，通过例如Gazzano-Santoro等人，J.Immunol.Methods 202:163(1996)中所述的方法。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
术语“刺激”指由刺激分子(例如，TCR/CD3复合体)与其相应配体的结合所诱导的初次应答，所述初次应答因而介导信号转导事件，例如但不限于借助TCR/CD3复合体的信号转导。刺激可以介导某些分子改变的表达，如下调TGF-β和/或细胞骨架结构的再组织等。
术语“刺激分子”指由提供初级胞质信号传导序列的T细胞表达的分子，所述的初级胞质信号传导序列在T细胞信号传导途径的至少某个方面以刺激性方式调节TCR复合体的初级活化。在一个实施方案中，初级信号例如通过TCR/CD3复合体与载有肽的MHC分子的结合引发并且导致介导T细胞反应，包括但不限于增殖、活化、分化等。在本发明的具体CAR中，本发明的任一种或多种CAR中的胞内信号结构域包含胞内信号传导序列，例如，CD3ζ的初级信号传导序列。
术语“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质或其等同物，并且“CD3ζ刺激信号结构域”定义为来自CD3ζ链胞质结构域的氨基酸残基，所述氨基酸残基足以在功能上传播T细胞活化必需的初始信号。在一个实施方案中，CD3ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至残基164或作为其功能直向同源物的来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“CD3ζ刺激信号结构域”是在SEQ ID NO:17中提供的序列或其变体。
术语“共刺激分子”是指细胞上的与共刺激配体特异性结合从而介导细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活 NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是4-1BB(即CD137)。共刺激信号结构域是指共刺激分子的胞内部分。
术语“4-1BB”指TNFR超家族成员，所述成员具有作为GenBank登录号AAA62478.2提供的氨基酸序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基；并且“4-1BB共刺激信号结构域”定义为GenBank登录号AAA62478.2的氨基酸残基214-255或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“4-1BB共刺激结构域”是作为SEQ ID NO:16提供的序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。
术语“信号传导途径”指在从细胞一个部分传播信号至细胞的另一个部分中发挥作用的多种信号传导分子之间的生物化学关系。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括γ-干扰素。
“分离的”抗体是指已经与其天然环境的组分分离。在一些实施方案中，将本发明的抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。“分离的编码本发明抗体的核酸”是指一个或多个核酸分子，其编码本发明抗体的链或其片段，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之 间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“氨基酸变化”和“氨基酸修饰”可互换地使用，是指氨基酸的添加、缺失、取代和其他修饰。可以进行氨基酸的添加、缺失、取代和其他修饰的任意组合，条件是最终的多肽序列具有所需的特性。在一些实施方案中，对抗体的氨基酸取代导致抗体与Fc受体的结合降低。为了改变例如Fc区结合特征的目的，特别优选非保守氨基酸取代，即用具有不同结构和/或化学性质的另一种氨基酸取代一种氨基酸。氨基酸取代包括用非天然存在的氨基酸或二十种标准氨基酸的天然存在的氨基酸衍生物(例如、4-羟基脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)的取代。可以使用本领域公知的遗传或化学方法产生氨基酸变化。遗传方法可包括定点诱变、PCR、基因合成等。通过除基因工程化之外的方法(如化学修饰)改变氨基酸侧链基团的方法可能是有用的。本文可使用多种名称来表示相同的氨基酸变化。例如，从Fc结构域的329位的脯氨酸到甘氨酸的取代可以表示为329G、G329、G 329、P329G Pro329Gly、或简称为“PG”。
术语“保守序列修饰”、“保守序列变化”指未显著影响或改变含有氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰或变化。这类种保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的抗体或抗体片段引入修饰。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因而，可以将本发明CAR内部的一个或多个氨基酸残基替换为来自相同侧链家族的其他氨基酸残基，并且可以使用本文所述的功能测定法测试改变的CAR。
术语“自体的”指这样的任何物质，所述物质从稍后将向个体再次引入所述物质的相同个体衍生。
术语“同种异体的”指这样的任何物质，所述物质从与引入所述物质的个体相同的物种的不同动物衍生。当一个或多个基因座处的基因不相同时，两位或更多位个体据称彼此是同种异体的。在一些方面，来自相同物种的个体的同种异体物质可以在遗传上足够地不相似以发生抗原性相互作用。
术语“异种的”指从不同物种的动物衍生的移植物。
如本文所用的术语“单采血液成分术”指本领域认可的体外方法，借助所述方法，供体或患者的血液从供体或患者取出并且穿过这样的装置，所述装置分离选择的特定组分并将剩余部分返回供体或患者的循环，例如，通过再输血。因此，在“单采样品”的语境中，指使用单采血液成分术获得的样品。
术语“免疫效应细胞”指参与免疫应答，例如参与促进免疫效应反应的细胞。免疫效应细胞的例子包括T细胞，例如，α/βT细胞和γ/δT细胞、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞、和髓细胞衍生的吞噬细胞。
“免疫效应功能”、“免疫效应应答”或“免疫效应反应”指例如免疫效应细胞的增强或促进免疫攻击靶细胞的功能或应答。例如，免疫效应功能或应答指促进杀伤靶细胞或抑制靶细胞生长或增殖的T细胞或NK细胞特性。在T细胞的情况下，初级刺激和共刺激是免疫效应功能或应答的例子。
术语“效应功能”指细胞的特化功能。T细胞的效应功能例如可以是溶细胞活性或辅助活性，包括分泌细胞因子。
术语“T细胞激活”是指T淋巴细胞，特别是细胞毒性T淋巴细胞的一种或多种细胞应答，选自：增殖、分化、细胞因子分泌、细胞毒性效应分子释放、细胞毒活性和活化标志物的表达。本发明的嵌合抗原受体能够诱导T细胞激活。用于测量T细胞激活的合适测定法在实施例中描述，并是本领域中已知的。
术语“慢病毒”指逆转录病毒科(Retroviridae)的一个属。慢病毒在逆转录病毒当中的独特之处在于能够感染非分裂性细胞；它们可以递送显著量的遗传信息至宿主细胞，从而它们是基因递送载体的最高效方法之一。HIV、SIV和FIV均是慢病毒的例子。
术语“慢病毒载体”指从慢病毒基因组的至少一部分衍生的载体，尤其包括如Milone等人,Mol.Ther.17(8):1453–1464(2009)中提供的自我失活慢病毒载体。可以在临床使用的慢病毒载体的其他例子例如包括但不限于来自Oxford BioMedica的
Figure PCTCN2022137265-appb-000001
基因递送技术、来自Lentigen的LENTIMAX TM载体系统等。非临床类型的慢病毒载体也是可获得的并且是本领域技术人员已知的。
术语“与BCMA相关的疾病”是指由BCMA增加的表达或活性引起、加重或以其它方式与其相关的任何病症。
术语“个体”或“受试者”可互换地使用，包括哺乳动物。哺乳动物包括但不限于驯化动物(例如，牛、羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体或受试者是人。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
术语“癌症”和“癌性”是指哺乳动物中细胞生长不受调节的生理疾患。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
“肿瘤免疫逃逸”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。
术语“半数有效浓度(EC 50)”是指在特定的暴露时间后诱导在基线和最大值之间的50％的应答的药物、抗体或毒剂的浓度。
术语“荧光激活细胞分选”或“FACS”是指专门类型的流式细胞术。它提供了根据每个细胞的特定光散射和荧光特征，将生物细胞的异质混合物以每次一个细胞分拣到两个或更多个容器中的方法(FlowMetric.“Sorting Out Fluorescence Activated Cell Sorting”.2017-11-09)。用于进行FACS的仪器是本领域技术人员已知的并且对于公众是可商购获得的。这种仪器的实例包括Becton Dickinson(Foster City，CA)的FACS Star Plus、FACScan和FACSort仪器、来自Coulter Epics Division(Hialeah，FL)的Epics C和来自Cytomation(Colorado Springs，Colorado)的MoFlo。
术语“可药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、缓冲剂或稳定剂等。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
术语“有效量”指本发明的抗体或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；体重、年龄和一般健康状况；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。 可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)P329G CAR-T细胞、以及(ii)针对BCMA的P329G突变抗体)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于受试者，其中这类施用在受试者体内提供有效治疗。术语“固定组合”是指本发明的针对BCMA的P329G突变抗体和P329G CAR-T细胞的组合各自以特定的单一剂量的形式同时施用于患者。术语“非固定组合”意指本发明的针对BCMA的329G突变抗体和P329G CAR-T细胞的组合作为分开的实体同时、并行或依次施用于患者，没有特定的剂量和时间限制，其中这样的施用提供了患者体内本发明药物组合的治疗有效水平。在一个优选的实施方案中，药物组合是非固定组合。
术语“组合疗法”或“联合疗法”是指施用两种或更多种组分以治疗如本公开所述的癌症。这种施用包括以基本上同时的方式共同施用这些组分。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如，胶囊、粉末和液体)中的共同施用或分开施用或依次施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。在一些实施方案中，施用还包括以大致相同的时间，或在不同的时间以顺序的方式，使用本发明的针对BCMA的P329G突变抗体和P329G CAR-T细胞。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
在本文中当谈及核酸时使用的术语“载体(vector)”是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
“受试者/患者样品”指从患者或受试者得到的细胞、组织或体液的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿 液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
在谈及疾病时，术语“治疗”是指减轻所述疾病(即，减缓或阻止或减少所述疾病或其至少一个临床症状的发展)、防止或延迟所述疾病的发作或发展或进展。
II.本发明的分子开关调控型嵌合抗原受体(CAR)
本发明涉及能够特异性结合针对BCMA分子的抗体的突变Fc结构域的嵌合抗原受体多肽。具体地，本发明的嵌合抗原受体包含人源化抗P329G突变scFv序列，且所述scFv序列能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域。与未突变的亲本抗体Fc结构域对Fc受体的结合相比较，包含P329G突变的抗体Fc结构域对Fc受体(例如，Fcγ受体)的结合降低。
本发明的重组CAR构建体包含编码CAR的序列，其中CAR包含人源化抗P329G突变scFv序列，所述scFv序列特异性结合P329G突变的抗体Fc结构域。
在一个实施方案中，本发明的CAR构建体中的scFv序列包含如下序列：
(i)重链可变区，其包含根据Kabat编号的
(a)氨基酸序列RYWMN(SEQ ID NO:19)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:20)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:21)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(ii)轻链可变区，其包含根据Kabat编号的
(d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:22)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(e)氨基酸序列GTNKRAP(SEQ ID NO:23)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:24)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代。
进一步地，scFv可以在N端连接有信号肽序列，例如，SEQ ID NO:11所示的信号肽序列，并且scFv可以在C端连接有如SEQ ID NO:14或SEQ ID NO:18中提供的任选铰链区/间隔区序列、如SEQ ID NO:15中提供的跨膜区、如SEQ ID NO:16的共刺激信号结构域和包含SEQ ID NO:17或其变体的胞内刺激信号结构域，例如，其中各个结构域彼此邻接并处于相同的可读框以形成单个融合蛋白。
在一些实施方案中，scFv结构域包含(i)重链可变区，其包含SEQ ID NO:12的序列或与 其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和(ii)轻链可变区，其包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
在一些实施方案中，scFv结构域包含(i)SEQ ID NO:12所示的重链可变区和(ii)SEQ ID NO:13所示的轻链可变区。在一个实施方案中，scFv结构域还包含(Gly4-Ser)n接头，其中n是1、2、3、4、5或6、优选地3或4。scFv的轻链可变区和重链可变区可以例如处于以下任何取向：轻链可变区-接头-重链可变区或重链可变区-接头-轻链可变区。
在一些实施方案中，本发明的示例性CAR构建体包含信号肽序列、人源化抗P329G突变scFv序列、铰链区/间隔区、跨膜结构域、胞内共刺激信号结构域和胞内刺激信号结构域。
在一个实施方案中，本发明将全长CAR多肽的氨基酸序列作为SEQ ID NO:1提供，如序列表中所示。
在一些实施方案中，本发明提供了重组核酸构建体，其包含编码本发明的CAR的核酸分子，例如，其包含编码SEQ ID NO:1所示的氨基酸序列或与SEQ ID NO:1具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的核酸分子。可以使用本领域公知的重组方法获得编码本发明的CAR构建体。备选地，可以合成地产生目的核酸，而非通过基因重组方法产生目的核酸。
本发明包括表达可以直接转导入细胞中的CAR的逆转录病毒载体构建体和慢病毒载体构建体。
在一些实施方案中，将本发明CAR构建体的核酸序列克隆至慢病毒载体中以在单个编码框中产生全长CAR构建体，并用EF1α启动子用于表达。
本领域普通技术人员将理解，本发明的CAR多肽还可以进行修饰，从而在氨基酸序列上变动，但是在所需的活性方面不变动。例如，可以对CAR多肽进行导致“非必需”氨基酸残基处氨基酸置换的额外核苷酸置换。例如，可以将分子中的非必需氨基酸残基替换为来自相同侧链家族的另一个氨基酸残基。在另一个实施方案中，可以将一个氨基酸片段替换为结构上相似的片段，所述的片段在侧链家族成员的顺序和组成方面中不同，例如，可以进行保守性置换，其中将氨基酸残基替换为具有相似侧链的氨基酸残基。
本领域中已经定义了具有相似侧链的氨基酸残基家族，所述侧链包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)、β分枝侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
在一些实施方案中，本发明构思了产生功能上等同的CAR多肽分子，例如，可以修饰CAR中包含的人源化抗P329G突变scFv序列的VH或VL，获得与SEQ ID NO:12具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的VH和与SEQ ID NO:13具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的VL。
本发明的CAR中包含的跨膜结构域是锚定的跨膜结构域，其是能够整合在细胞膜中的多肽链的组成部分。跨膜结构域可以与其他胞外和/或胞内多肽结构域融合，其中这些胞外和/或胞内多肽结构域也将被限制在细胞膜上。在本发明的嵌合抗原受体(CAR)多肽中，跨膜结构域赋予本发明的CAR多肽的膜附着。本发明的CAR多肽包含至少一个跨膜结构域，其可以衍生自天然来源或重组来源，包含优势疏水的残基如亮氨酸和缬氨酸。在来源是天然的情况下，结构域可以衍生自膜结合蛋白或跨膜蛋白例如CD28、CD8(例如，CD8α，CD8β)的跨膜结构域。在一个实施方案中，跨膜结构域包含SEQ ID NO:15的氨基酸序列。
在一些实施方案中，本发明的CAR中的跨膜结构域借助铰链区/间隔区与CAR的胞外区(即，人源化抗P329G突变scFv序列)连接。例如，在一个实施方案中，铰链可以是CD8α铰链区、CD28的铰链区。在一些实施方案中，铰链区或间隔区序列包含SEQ ID NO:18的氨基酸序列。
另外，甘氨酸-丝氨酸双联体也提供特别合适的接头作为铰链区/间隔区。例如，在一个实施方案中，接头包含GGGGS的氨基酸序列(SEQ ID NO:14)。
本发明的CAR中包含的胞质结构域包含胞内信号结构域。胞内信号结构域能够活化引入了本发明CAR的免疫细胞的至少一个效应功能。
用于本发明CAR中的胞内信号结构域的例子包括协同发挥作用以在胞外结构域结合P329G突变的抗体Fc结构域后启动信号转导的T细胞受体(TCR)和共受体的胞质序列，以及这些序列的任何衍生物或变体和具有相同功能性能力的任何重组序列。
考虑到仅通过TCR生成的信号尚不足以完全活化T细胞，因此本发明的CAR还设计了能够产生共刺激信号的共刺激信号结构域(CSD)。T细胞的活化由两类不同的胞质信号传导序列介导：通过TCR启动抗原依赖性初级活化的那些序列(初级胞内信号结构域)和以抗原非依赖性方式发挥作用以提供共刺激信号的那些序列(次级胞质结构域，例如，共刺激结构域)。
在一个实施方案中，本发明的CAR包含初级胞内信号结构域，例如，CD3ζ的初级信号结构域，例如，SEQ ID NO:17所示的CD3ζ信号传导结构域。
本发明CAR中的胞内信号结构域还包次级信号结构域(即，共刺激信号结构域)。共刺激信号结构域指包含共刺激分子的胞内结构域的CAR部分。共刺激分子是除抗原受体或其配体之外淋巴细胞对抗原作出有效反应所需要的细胞表面分子。在一些实施方案中，共刺激分子包括但不限于CD28、4-1BB(CD137)，其引起的共刺激作用在体外增强人CART细胞的增殖、效应功能和存活并且在体内增进人T细胞的抗肿瘤活性。
在本发明CAR的胞内信号传导序列可以彼此按随机顺序或按指定的顺序连接。任选地，短寡肽接头或多肽接头可以在胞内信号传导序列之间形成键接。在一个实施方案中，甘氨酸-丝氨酸双联体可以用作合适的接头。在一个实施方案中，单个氨基酸，例如，丙氨酸、甘氨酸，可以用作合适的接头。
在一个实施方案中，本发明CAR的胞内信号结构域设计成包含4-1BB的共刺激信号结构域和CD3ζ的刺激信号结构域。
III.编码本发明的CAR的核酸分子、载体和表达本发明CAR的细胞
本发明提供了编码本文所述的CAR构建体的核酸分子。在一个实施方案中，核酸分子作为DNA构建体提供。
本发明还提供了插入有本发明CAR构建体的载体。通过将编码CAR多肽的核酸有效连接至启动子并将构建体并入表达载体中，实现编码CAR的天然或合成的核酸的表达。载体可以适合在真核生物中复制和整合。常见的克隆载体含有用于调节所需核酸序列的表达的转录和翻译终止子、起始序列和启动子。
已经开发了众多基于病毒的系统用于转移基因至哺乳动物细胞中。例如，逆转录病毒提供了用于基因递送系统的便利平台。可以使用本领域已知的技术，将选择的基因插入载体并且包装在逆转录病毒粒子中。随后可以分离重组病毒并将其在体内或离体递送至受试者的细胞。众多逆转录病毒系统是本领域已知的。在一些实施方案中，使用慢病毒载体。
衍生自逆转录病毒(如慢病毒)的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期、稳定整合和其在子代细胞中增殖。慢病毒载体具有胜过衍生自癌-逆转录病毒(如鼠白血病病毒)的载体的额外优点，因为它们可以转导非增殖性细胞，如肝细胞。它们还具有额外的低免疫原性优点。逆转录病毒载体也可以例如是γ逆转录病毒载体。γ逆转录病毒载体可以例如包含启动子、包装信号(ψ)、引物结合位点(PBS)、一个或多个(例如，两个)长末端重复序列(LTR)和目的转基因，例如，编码CAR的基因。γ逆转录病毒载体可以缺少病毒结构性基因如gag、pol和env。
能够在哺乳动物T细胞中表达CAR转基因的启动子的例子是EF1a启动子。天然EF1a启动子驱动延伸因子-1复合体的α亚基表达，所述α亚基负责酶促递送氨酰基tRNA至核糖体。已经在哺乳动物表达质粒中广泛使用了EF1a启动子并且已经显示有效驱动从克隆至慢病毒载体中的转基因表达CAR。参见，例如，Milone等人,Mol.Ther.17(8):1453–1464(2009)。
启动子的另一个例子是立即早期巨细胞病毒(CMV)启动子序列。这个启动子序列是能够驱动与之有效连接的任何多核苷酸序列高水平表达的组成型强启动子序列。但是，也可以使用其他组成型启动子序列，所述其他组成型启动子序列包括但不限于猴病毒40(SV40)早期启动子、小鼠乳腺瘤病毒(MMTV)、人类免疫缺陷病毒(HIV)长末端重复序列(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、埃巴病毒立即早期启动子、劳斯肉瘤病毒启动子以及人基因启动子，如但不限于肌动蛋白启动子、肌球蛋白启动子、延伸因子-1α启动子、血红蛋白启动子和肌酸激酶启动子。另外，本发明不应当限于使用组成型启动子。还构思了诱导型启动子作为本发明的部分。
在一些实施方案中，本发明提供了在哺乳动物免疫效应细胞(例如哺乳动物T细胞或哺乳动物NK细胞)中表达本发明的CAR构建体的方法和由此产生的免疫效应细胞。
从受试者获得细胞来源(例如，免疫效应细胞，例如，T细胞或NK细胞)。术语“受试者”意在包括可以激发免疫应答的活生物(例如，哺乳动物)。可以从众多来源获得T细胞，包括外周血单个核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、 胸腔积液、脾组织和肿瘤。
可以使用本领域技术人员已知的任何技术(如Ficoll TM分离法)，从采集自受试者的血液成分中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体循环血液的细胞。单采产物一般含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核的白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采血液成分术采集的细胞，以除去血浆级分并且以在用于后续加工步骤的适宜缓冲液或培养基中放置细胞。在本发明的一个方面，用磷酸盐缓冲盐水(PBS)洗涤细胞。
可以通过正向或负向选择技术进一步分离特定的T细胞亚群，如CD3+、CD28+、CD4+、CD8+、CD45RA+和CD45RO+T细胞。例如，在一个实施方案中，通过与抗CD3/抗CD28缀合的珠(如
Figure PCTCN2022137265-appb-000002
M-450CD3/CD28T)温育一段足够正向选择所需T细胞的时间，分离T细胞。在一些实施方案中，该时间段是约30分钟至36小时之间或更长时间。较长的温育时间可以用来在存在少量T细胞的任何情况下分离T细胞，如用于从肿瘤组织或从免疫受损个体分离肿瘤浸润型淋巴细胞(TIL)。另外，使用较长的温育时间可以增加捕获CD8+T细胞的效率。因此，通过简单地缩短或延长该时间，允许T细胞与CD3/CD28珠结合和/或通过增加或减少珠对T细胞的比率，可以在培养伊始或在培养过程期间的其他时间点偏好地选择T细胞亚群。
可以用抗体的组合，通过负选择过程完成T细胞群体的富集，其中所述抗体针对负向选择的细胞独有的表面标志物。一种方法是借助负向磁力免疫粘附法或流式细胞术分选和/或选择细胞，所述负向磁力免疫粘附法或流式细胞术使用针对负向选择的细胞上存在的细胞表面标志物的单克隆抗体混合物。
在一些实施方案中，免疫效应细胞可以是同种异体免疫效应细胞，例如，T细胞或NK细胞。例如，细胞可以是同种异体T细胞，例如，缺少功能性T细胞受体(TCR)和/或人白细胞抗原(HLA)(例如，HLA I类和/或HLA II类)表达的同种异体T细胞。
缺少功能性TCR的T细胞可以例如经工程化，从而它在其表面上不表达任何功能性TCR；经工程化，从而它不表达构成功能性TCR的一个或多个亚基(例如，经工程化，从而它不表达或显示出减少表达的TCRα、TCRβ、TCRγ、TCRδ、TCRε和/或TCRζ)；或经工程化，从而它在其表面上产生非常少的功能性TCR。
本文所述的T细胞例如可以如此工程化，从而它不在其表面上表达功能性HLA。例如，本文所述的T细胞可以如此工程化，从而HLA(例如，HLA I类和/或HLA II类)的细胞表面表达下调。在一些方面，可以通过减少或消除β-2微球蛋白(B2M)表达实现HLA的下调。
在一些实施方案中，T细胞可以缺少功能性TCR和功能性HLA，例如，HLA I类和/或HLA II类。
在一个实施方案中，对编码本发明所述的CAR的核酸转导的细胞进行增殖，例如，将细胞在培养下增殖2小时至约14天。
对经体外增殖后获得的表达CAR的免疫效应细胞可以如实施例中所述进行效应功能的检测。
IV.特异性结合BCMA分子的抗体和其包含突变Fc结构域的抗体
本发明提供了以高靶特异性和高亲和性结合BCMA的抗体，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:25)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:26)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列DRGDQILDV(SEQ ID NO:27)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISRYLN(SEQ ID NO:28)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列AASSLQS(SEQ ID NO:29)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQKYFDIT(SEQ ID NO:30)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)所述重链可变区包含根据Kabat编号的氨基酸序列NDVIS(SEQ ID NO:31)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列VIIPIFGIANYAQKFQG(SEQ ID NO:32)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列GRGYYSSWLHDI(SEQ ID NO:33)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列QASQDITNYLN(SEQ ID NO:34)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列DASNLET(SEQ ID NO:35)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQAFDLIT(SEQ ID NO:36)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或保守氨基酸取代。
在一些实施方案中，本发明的结合BCMA分子的抗体结合哺乳动物BCMA，例如人、食蟹猴、小鼠、大鼠和兔BCMA。
在一些实施方案中，本发明的结合BCMA分子的抗体具有以下一个或多个特性：
(1)特异性结合BCMA；
(2)结合人BCMA并且与食蟹猴、小鼠、大鼠和兔BCMA交叉反应；
(3)在不包含突变Fc结构域，例如，在具有未突变的亲本抗体Fc结构域的情形，能够通过抗体依赖性细胞毒性和/或抗体依赖的细胞吞噬作用(ADCP)杀死BCMA-阳性癌症细胞。
在一些实施方案中，本发明的结合BCMA分子的抗体包含特异性结合BCMA的重链可变区和轻链可变区，其中：
(a)重链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:3的序列或与 其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区包含SEQ ID NO:9的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:10的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
其中所述至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列中的氨基酸变化优选氨基酸取代，更优选氨基酸保守取代，优选地，所述氨基酸变化不发生在CDR区中。
在一些实施方案中，本发明的结合BCMA分子的抗体是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是IgG1抗体，例如，人IgG1抗体。
在一些实施方案中，本文中所提供的结合BCMA分子的抗体包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；例如，所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域，例如，所述突变Fc结构域是人IgG1抗体的突变Fc结构域。
包含P329G突变Fc结构域的结合BCMA分子的抗体不能通过与Fcγ受体结合来发挥抗体依赖性细胞毒性，也不能发挥抗体依赖的细胞吞噬作用(ADCP)。
在一些实施方案中，本发明提供了编码以上任何结合BCMA分子的抗体或其片段或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
例如，本发明的核酸包含编码本发明的结合BCMA分子的抗体的核酸。在一些实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是pcDNA3.4表达载体。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性 (例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
在一个实施方案中，提供了包含本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体的其它细胞。合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293或293F细胞)、293细胞、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(HepG2)、中国仓鼠卵巢细胞(CHO细胞)、CHO-S细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO细胞或HEK293细胞。
在一个实施方案中，本发明提供了制备结合BCMA分子的抗体(包括P329G突变抗体)的方法，其中所述方法包括在适于表达编码所述结合BCMA分子的抗体(包括P329G突变抗体)的核酸的条件下培养包含编码所述结合BCMA分子的抗体(包括P329G突变抗体)的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述结合BCMA分子的抗体(包括P329G突变抗体)。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收结合BCMA分子的抗体(包括P329G突变抗体)。
如本文所述制备的本发明的结合BCMA分子的抗体(包括P329G突变抗体)可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的结合BCMA分子的抗体(包括P329G突变抗体)的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。
可以通过本领域中已知的多种测定法对本文中提供的结合BCMA分子的抗体(包括P329G突变抗体)鉴定、筛选或表征其物理/化学特性和/或生物学活性。一方面，对本发明的结合BCMA分子的抗体(包括P329G突变抗体)测试其抗原结合活性，例如通过已知的方法诸如FACS、ELISA或Western印迹等来进行。可使用本领域已知方法来测定对BCMA的结合，本文中公开了例示性方法。在一些实施方案中，使用FACS测定本发明的结合BCMA分子的抗体(包括P329G突变抗体)对细胞表面BCMA(例如人BCMA)的结合。
本发明还提供了用于鉴定具有生物学活性的结合BCMA分子的抗体(包括P329G突变 抗体)的测定法。生物学活性可以包括例如ADCC作用、CDC作用等。
供任何上述体外测定法使用的细胞包括天然表达BCMA或经改造而表达BCMA细胞系。所述经改造而表达BCMA细胞系是正常情况下不表达BCMA的、将编码BCMA的DNA转染入细胞之后表达BCMA的细胞系。
V.本发明的药物组合
对于优化CAR疗法的安全性和疗效而言，本发明的分子开关调控型嵌合抗原受体是可以控制CAR活性的可调控型CAR。本发明使用将Pro329Gly(抗体Fc段根据EU编号的第329位脯氨酸突变为甘氨酸，简写为P329G)突变抗体作为本发明CAR治疗中的安全开关。在所述P329G突变抗体不存在时，本发明的CAR活性关闭；在所述P329G突变抗体存在时，本发明的CAR活性开启；由此，本发明的CAR分子活性的开启和关闭受到P329G突变抗体的调控。
在一些实施方案中，本发明提供了药物组合，其包含(i)表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)；和(ii)特异性结合BCMA分子的P329G突变抗体。例如，所述免疫效应细胞是自自体T细胞或同种异体T细胞制备的表达本发明的分子开关调控型CAR多肽的T细胞，例如，所述免疫效应细胞是自人PBMC分离的T细胞制备的表达本发明的分子开关调控型CAR多肽的T细胞。在一些实施方案中，所述P329G突变抗体是ADI-38497 PG Ab和/或ADI-38484 PG Ab。
在一些实施方案中，本发明提供了药物组合，其包含(i)编码本发明的分子开关调控型CAR多肽的核酸分子或包含所述核酸组分的载体；和(ii)特异性结合BCMA分子的P329G突变抗体。
在一些实施方案中，本发明的药物组合任选地进一步包含合适制剂的可药用辅料。例如，所述药物组合中的(ii)可根据常规方法制剂化(例如Remington’s Pharmaceutical Science,最新版,Mark Publishing Company,Easton,U.S.A)。
在一些实施方案中，本发明的药物组合用于治疗与BCMA相关的疾病，例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
VI.本发明的药物组合的用途和使用本发明的药物组合的治疗方法
本发明提供了前述本发明的药物组合，其用于在受试者中治疗与BCMA相关的疾病，所述疾病例如是表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在一个实施方案中，本发明的药物组合用于在受试者中治疗表达或过表达BCMA的癌症，并且能够减轻癌症的至少一种症状或指征的严重性或抑制癌细胞生长，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
本发明提供了在受试者中治疗与BCMA相关的疾病(例如，表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤)的方法，其包括向有需要的个体施用治疗有效量的本发明的药物组合。
本发明提供了前述本发明的药物组合在制备用于治疗与BCMA相关的疾病(例如，表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤)的药物中的用途。
本发明的药物组合也可以施用于已经用一种或多种先前疗法治疗癌症但随后复发或转移的个体，例如，癌症是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在一些实施方案中，本发明的药物组合中的(i)表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)和(ii)特异性结合BCMA分子的P329G突变抗体用于胃肠外、经皮、腔内、动脉内、静脉内、鞘内施用，或直接注入组织或肿瘤中。在一些实施方案中，本发明的药物组合中的(ii)特异性结合BCMA分子的P329G突变抗体在(i)表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)之前、同时或之后施用。
在一些实施方案中，本发明的药物组合中的(i)表达本发明的分子开关调控型CAR多肽的免疫效应细胞是自自体T细胞或同种异体T细胞制备的表达本发明CAR多肽的T细胞；本发明的药物组合中的(ii)特异性结合BCMA分子的P329G突变抗体是任何特异性结合BCMA分子的抗体，其包含P329G突变。优选地，所述P329G突变抗体是ADI-38497 PG Ab和/或ADI-38484 PG Ab。
当本发明的药物组合中的组分(i)是表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)时，本发明不限制本发明的药物组合中的组分(i)和组分(ii)向受试者施用的顺序，也不限制向受试者施用本发明的药物组合中的组分(i)和组分(ii)之间的时间安排。因此，本发明的药物组合中的(i)和(ii)可以分开、同时或依次施用。当两种组分没有同时施用时，这两种组分的施用可以间隔1小时、2小时、4小时、6小时、12小时、24小时、48小时或72小时，或间隔本领域技术人员容易确定的任何合适的时间差。例如，第一天静脉内施用(i)，第二天施用(ii)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者第一天施用(ii)，第二天静脉内施用(i)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者同一天间隔1小时、2小时、4小时、6小时、12小时分别施用(i)和(ii)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)。
在一些实施方案中，本发明的药物组合中的组分(i)是表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)时，还包括将所述组分(i)和组分(ii)在施用于受试者之前预先一起孵育的情况。因此，可以在施用前预先孵育这两种组分1分钟、5分钟、10分钟、15分钟、30分钟、45分钟或1小时，或本领域技术人员容易确定的任何合适时间。
本发明的药物组合可以以合适的剂量施用于受试者。剂量方案将由主治医生和临床因素决定。如医学领域中公知的，用于任何一名患者的剂量取决于许多因素，包括患者的体重、身体表面积、年龄、待施用的特定化合物、性别、施用时间和途径、一般健康状况、和待并行施用的其他药物。
在一些实施方案中，本发明的药物组合中的组分(i)是表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)时，将组分(i)以1×10 6个细胞/kg体重－10×10 6个细胞/kg体重，例如1×10 6个细胞/kg体重、2×10 6个细胞/kg体重、3×10 6个细胞/kg体重、5×10 6个细胞/kg体重、7×10 6个细胞/kg体重、9×10 6个细胞/kg体重、10×10 6个细胞/kg体重的剂量以单次或多次静脉内施用至受试者；并将组分(ii)以0.1-10mg/kg、优选地0.1mg/kg、0.3mg/kg、0.5mg/kg、1mg/kg、3mg/kg、5mg/kg、7mg/kg、9mg/kg、10mg/kg的剂量单元的形式，优选地胃肠外、更优选地静脉内施用至受试者。
在一些实施方案中，向患有癌症的个体施用本发明的药物组合导致肿瘤的完全消失。在一些实施方案中，向患有癌症的个体施用本发明的药物组合导致肿瘤细胞或肿瘤大小减少至少85％或更多。可以通过本领域已知的任何方法测量肿瘤的减少，例如X-线、正电子发射断层扫描(PET)、计算机断层扫描(CT)、磁共振成像(MRI)、细胞学、组织学或分子遗传分析。
在一些实施方案中，本发明的药物组合可以降低CAR-T细胞存在的“在靶/脱肿瘤(On-target/off tumor)”毒性。
VII.本发明的药盒
本发明提供了一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式。由此可以依据给药方案或药物施用间隔提供剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
(i)选自表达本发明的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)、编码本发明的分子开关调控型CAR多肽的核酸分子、包含所述核酸的载体、和它们的任意组合；
(ii)特异性结合BCMA分子的P329G突变抗体。
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
实施例1、CAR基因合成、病毒表达载体的构建、P329G CAR-T细胞制备及CAR表达检测
(1-1)CAR基因合成和病毒表达载体的构建
构建了P329G CAR分子(SEQ ID NO:1)，也称为HuR968B CAR，由SEQ ID NO:11所示的信号肽(SP)、识别P329G抗体的特异单链抗体片段(VH-接头-VL，具有SEQ ID NO:12所示的VH、SEQ ID NO:41所示的接头序列、SEQ ID NO:13所示的VL)、SEQ ID NO:14所示的G4S铰链区、SEQ ID NO:15所示的CD8跨膜结构域(CD8TM)、SEQ ID NO:16所示的41BB共刺激结构域(41BB-CSD)以及SEQ ID NO:17所示的CD3ζ分子胞内激活结构域(CD3ζSSD)融合而成。
另外，构建了直接靶向BCMA的Blue21CAR(SEQ ID NO:8)，用作对照。Blue21CAR从N端到C端含有SEQ ID NO:11所示的信号肽、抗BCMA单链抗体(来自11D53克隆)、SEQ ID NO:18所示的CD8α分子的铰链区及SEQ ID NO:15所示的CD8跨膜结构域、SEQ ID NO:16所示的4-1BB共刺激结构域以及SEQ ID NO:17所示的CD3ζ链胞内激活结构域。
分别将上述编码CAR多肽的DNA片段插入pRK慢病毒表达载体(由pRRLSIN.cPPT.PGK-GFP.WPRE载体(Addgene，12252，购自生物风)通过替换启动子和抗性基因改构而成)的EF1α启动子下游，替换载体中的EGFR序列，获得了CAR表达质粒pRK-HuR968B、pRK-Blue21。
(1-2)慢病毒浓缩液的制备
将实施例1-1制备的CAR表达质粒与结构质粒pMDLg/pRRE(Addgene，12251，购自生物风)、调节质粒pRSV-rev(Addgene，12253，购自生物风)及包膜质粒pMD2G(Addgene，12259，购自生物风)以3:3:2:2的质量比例用PEI转染法转染Lenti-X-293T细胞(Takara公司)，转染16小时后，更换为含有2％胎牛血清(FBS)的新鲜DEME培养基，继续培养48小时后，收集细胞上清，离心去除细胞碎片，加入PEG8000 4℃孵育16-64小时进行慢病毒浓缩，再次离心后去上清，采用T细胞培养基重悬慢病毒沉淀物，获得慢病毒浓缩液，分装后-80℃冻存。
(1-3)P329G CAR-T细胞制备及CAR表达检测：
添加注射用重组人白介素-2(国药准字S20040020)至TexMACS GMP Medium(Miltenyi Biotec,170-076-309)中，配制成IL-2浓度为200IU/ml的T细胞培养基。
自ORiCELLS获得了多个供者PBMC细胞，具体信息如下表1所示：
表1.供者PBMC细胞的相关来源信息
PBMC细胞编号 目录号 批次号 供者ID号
供者3 PBMC FPB004F-C PCH2020110004 Z0052
供者4 PBMC FPB005F-C PCH20201200002 Z0066
供者5 PBMC FPB004F-C PCH20210100004 Z0086
供者6 PBMC FPB004F-C PCH20201200043 Z0084
供者7 PBMC FPB004F-C PCH20210100012 Z0091
第0天使用Pan T Cell Isolation Kit(human)(Miltenyi,130-096-535)对复苏后的各供者PBMC进行分选，获得T细胞，使用T细胞培养基将T细胞重悬至一定的密度并添加TransAct(Miltenyi,130-111-160)进行激活。
第1天分出一定量T细胞不添加慢病毒浓缩液继续培养，该部分细胞为未转导细胞(UNT,un-transduced T cells)，剩余的T细胞按MOI＝1～5添加自实施例1-2获得的慢病毒浓缩液(所述慢病毒为编码P329G CAR(SEQ ID NO:1)或对照传统CAR(SEQ ID NO:8)的慢病毒)并将T细胞吹打均匀；第2天离心去除病毒上清，重悬细胞至新鲜T细胞培养基。第3天将所有细胞转移至G-Rex(WILSONWOLF,货号80040S)中，并添加适量新鲜T细胞培养基，放置于37℃CO 2培养箱中静置培养；每隔2～3天，将细胞以培养基的半量更换为新鲜培养基或直接补加IL-2，其中，添加IL-2至细胞培养基中IL-2浓度为200IU/ml。当细胞数量扩增至约为20-80倍时，满足需求后(一般达到2～8x10 8个细胞)进行细胞收获。离心去除培养基后，将CAR-T细胞采用
Figure PCTCN2022137265-appb-000003
CS10(Stemcell,07930)重悬后分装，程序降温至-80℃进行冻存。
取适量CAR-T细胞，FACS缓冲液(PBS+2％FBS)洗涤一次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain的FACS缓冲液，室温染色10-15min，洗涤两次，加入PerCP-Cy5.5-CD3、BUV805-CD、Biotin-F(ab')Fragment山羊抗人IgG(Jackson ImmunoResearch,109-066-006；PG CAR检测)或Biotin-F(ab')Fragment山羊抗小鼠IgG(Jackson ImmunoResearch,115-066-006；Blue 21CAR检测)抗体组合，4℃染色30～45min，洗涤两次后再加入APC-Streptavidin，4℃染色30～45min；细胞洗涤两次后用FACS缓冲液重悬，采用流式细胞仪进行检测。
图1A显示了分别用实施例1-1构建的2种CAR转导T细胞后，CD3 +细胞、CD4 +、CD8 +T细胞亚群中CAR的表达，结果表明这些经转导的T细胞中CAR表达阳性率约为18％～29％。
实施例2、BCMA特异P329G突变抗体与抗原结合活性检测
(2-1)BCMA特异性抗体的合成
从国际申请号PCT/CN2019/074419(BCMA抗体相关专利)获得BCMA亲本抗体ADI-34861的重轻链可变区序列(分别为SEQ ID NO:37所示的VH、SEQ ID NO:38所示的VL序列)、ADI-34857的重轻链可变区序列(分别为SEQ ID NO:39所示的VH、SEQ ID NO:40所示的VL序列)，在亲本抗体ADI-34861的基础上进行CDR区突变获得ADI-38497的重轻链可变区序列(SEQ ID NO:2、SEQ ID NO:3)，在亲本抗体ADI-34857的基础上进行CDR区突变获得ADI-38484的重轻链可变区序列(SEQ ID NO:9、SEQ ID NO:10)。与相应的亲本抗体相比，突变后的抗体亲和力显示显著提高，具体实验数据见下表2。
表2亲本和突变抗体与BCMA结合亲和力
Figure PCTCN2022137265-appb-000004
从US9273141B2专利中获得GSK公司BCMA抗体克隆J6M0轻重链可变区序列，作为对照抗体(GSK IgG)。
采用全基因合成GSK IgG、ADI-38497、ADI-38484抗体轻重链可变区序列，装入含有WT的人源IgG1重链恒定区(SEQ ID NO:4)或含有P329G点突变的人源IgG1重链恒定区(SEQ ID NO:5)和κ轻链恒定区(SEQ ID NO:6)的pcDNA3.4表达载体(购自上海伯英)上。将轻重链表达载体按照2:3摩尔比通过PEI共转染到HEK293细胞中，培养5～7天后收集培养基上清。含有抗体的上清培养基通过Protein A柱进行一步纯化，之后用PBS透析。采用NanoDrop仪器读取280nm吸光度值检测浓度，并用SDS-PAGE和SEC-HPLC方法检测样品纯度。获得了GSK WT抗体、GSK PG抗体；ADI-38497WT抗体、ADI-38497 PG抗体；以及ADI-38484WT抗体、ADI-38484 PG抗体。具有BCMA抗体克隆ADI-38497重链可变区(SEQ ID NO:2)和轻链可变区(SEQ ID NO:3)序列的抗体在本申请中也称为ADI-38497抗体，包括ADI-38497 PG抗体和ADI-38497WT抗体；具有BCMA抗体克隆ADI-38484重链可变区(SEQ ID NO:9)和轻链可变区(SEQ ID NO:10)序列的抗体在本申请中也称为ADI-38484抗体，包括ADI-38484 PG抗体和ADI-38484WT抗体。
(2-2)抗体的亲和力检测
通过Biacore T200测定了ADI-38497 PG抗体与不同种属BCMA的亲和力，图2A显示了采用表面等离子共振法(SPR)测定抗体亲和力的方法示意图。
具体方法如下：将抗人Fc IgG(Ab97221,Abcam)偶联到CM5芯片(29149603,Cytiva)表面后，将ADI-38497 PG抗体捕获在芯片表面，通过检测芯片表面抗体与流动相中的BCMA抗原之间的结合与解离获得亲和力及动力学常数。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。亲和力检测中的每个循环包括捕获ADI-38497 PG抗体、结合一种浓度抗原及芯片再生。将梯度稀释后的抗原(抗原浓度梯度为1.25-40nM，2倍稀释)，以30μl/分钟的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，设定合适的解离时间(900s或600s或60s)。最后使用10mM甘氨酸-HCl,pH 1.5(BR-1003-54, Cytiva)对芯片进行再生。
数据结果通过Biacore T200分析软件(版本号3.1)，使用1:1结合模型进行分析。
图2B展示了采用SPR测定ADI-38497 PG抗体与重组人、食蟹猴、小鼠、大鼠和兔BCMA蛋白的代表性亲和力图谱。结果显示，ADI-38497 PG抗体与上述不同种属来源的BCMA蛋白均可结合，其中结合活性的高低顺序依次为人BCMA>猴BCMA>小鼠BCMA>大鼠BCMA>兔BCMA。
表3 ADI-38497 PG抗体与来自不同种属的BCMA蛋白的结合亲和力
Figure PCTCN2022137265-appb-000005
(2-3)P329G BCMA抗体与不同种属来源BCMA抗原结合活性检测
首先，制备了表达不同种属来源BCMA抗原的CHO GS细胞。具体而言，将人、小鼠、食蟹猴源的BCMA基因合成并克隆至慢病毒载体中，随后包装含不同种属来源BCMA基因的慢病毒，并用此慢病毒感染CHO GS细胞，后续经流式细胞术分选，得到表达不同种属来源BCMA抗原的CHO GS细胞系，即hBCMA-CHO GS、mBCMA-CHO GS和cynoBCMA-CHO GS细胞。
然后，用FACS缓冲液将ADI-38497 PG抗体及GSK来源的BCMA抗体(即，GSK PG IgG用作Benchmark)配制成10倍梯度稀释的不同浓度抗体溶液，分别与1E5个所制备的表达不同种属来源BCMA抗原的CHO GS细胞4 oC孵育30分钟，用FACS缓冲液清洗后，再与Fcγ片段特异的APC-山羊抗人IgG(Jackson ImmunoResearch,109-136-098)在4 oC孵育30分钟。采用流式细胞术检测与细胞结合的P329G抗体，分析APC通道MFI，以抗体浓度为X轴，APC通道MFI为Y轴进行绘图并计算结合的EC50。
图2C显示了不同浓度P329G BCMA抗体与稳定表达人、食蟹猴及小鼠BCMA的CHO-GS细胞的结合能力。由图2C可见，ADI-38497 PG IgG抗体能够与细胞表面表达的不同种属BCMA结合，而GSK来源的BCMA抗体(Benchmark)具有较高的种属BCMA特异性，其不识别小鼠BCMA，该结果与SPR检测结果一致。
表4 P329G BCMA抗体与表达不同种属BCMA的CHO-GS细胞的结合EC50值
Figure PCTCN2022137265-appb-000006
(2-4)P329G BCMA抗体与肿瘤细胞表面BCMA抗原结合活性检测
取适量处于对数生长期的肿瘤细胞，FACS缓冲液洗涤2次，加入ADI-38497 PG抗体、ADI-38484 PG抗体及用作Benchmark的GSK PGIgG，对作为染色对照的细胞加入同种型hIgG1抗体，4℃染色30分钟，洗涤两次，加入APC-F(ab')片段的山羊抗人IgG抗体，4℃染色30分钟，细胞洗涤两次后FACS缓冲液重悬，采用流式细胞仪进行检测。
图2D显示不同浓度P329G BCMA抗体与表达BCMA的阳性多发性骨髓瘤细胞系MM.1s、RPMI8226、U266、H929、L363及AMO1(MM.1s购自南京科佰生物科技有限公司，CBP60239；RPMI8226购自南京科佰生物科技有限公司，CBP60244；U266购自武汉普诺赛生命科技有限公司，CL-0510；H929购自南京科佰生物科技有限公司，CBP60243；L363南京科佰生物科技有限公司，CBP6024；AMO1购自南京科佰生物科技有限公司，CBP60242)的结合活性，ADI-38497 PG抗体、ADI-38484 PG抗体能够与表达BCMA的阳性肿瘤细胞结合并呈现浓度依赖性。在所述表达BCMA的阳性肿瘤细胞中，MM.1s细胞有最高水平BCMA表达，RPMI8226、U266和H929细胞以中等水平表达BCMA，L363、AMO1细胞以低水平表达BCMA。
表5 P329G BCMA抗体与表达BCMA的阳性肿瘤细胞的结合EC50值
Figure PCTCN2022137265-appb-000007
实施例3、BCMA特异性P329G突变抗体与P329G CAR胞外结构域的结合活性检测
(3-1)P329G突变抗体与P329G CAR胞外结构域结合的亲和力检测
通过Biacore T200测定ADI-38497分子(P329G突变抗体或野生型抗体)与抗P329G突变特异性单链抗体-兔Fc融合蛋白(也称为“抗PG scFv融合蛋白”或简称为“抗PG scFv”)的结合亲和力。图3A显示采用表面等离子共振法(SPR)测定抗P329G突变的特异性单链抗体-兔Fc融合蛋白与ADI-38497 P329G突变抗体亲和力的检测示意图。
具体方法如下：将合成的抗PG scFv融合蛋白(SEQ ID NO:7)偶联到C1芯片(BR100535,Cytiva)表面后，通过检测芯片表面抗体与流动相中的ADI-38497分子结合与解离获得亲和力及动力学常数。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。亲和力检测的每个循环包括结合一种浓度的抗体及芯片再生。将梯度稀释后的ADI-38497分子(浓度梯度为3.125nM－100nM，2倍稀释)，以30μl/分钟的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，解离时间300s。最后使用10mM Glycine-HCl,pH 1.5(BR-1003-54,Cytiva)对芯片进行再生。
数据结果通过Biacore T200分析软件(版本号3.1)，使用1:1结合模型进行分析。
图3B展示了采用SPR测定ADI-38497 PG抗体及野生型抗体与抗PG scFv融合蛋白结合的代表性亲和力图谱。结果显示，只有ADI-38497 PG抗体可特异性的与作为P329G CAR胞外结构域的抗PG scFv结合。
表6 SPR测定的P329G BCMA抗体及野生型抗体与抗PG scFv的亲和力
Figure PCTCN2022137265-appb-000008
(3-2)ADI-38497分子与抗PG scFv融合蛋白的结合亲合力检测
通过Biacore T200测定ADI-38497分子(P329G突变抗体或野生型抗体)与抗PG scFv融合蛋白的结合亲合力。图3C显示采用表面等离子共振法(SPR)测定抗体亲合力(Avidity)的方法示意图。
具体方法如下：将抗Fab IgG(I5260，Sigma)偶联到HLC芯片(HLC30M,Xantec)表面后，将ADI-38497分子捕获在芯片表面，通过检测芯片表面抗体与流动相中的抗PG scFv融合蛋白结合与解离获得亲合力及动力学常数。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。亲合力检测的每个循环包括捕获ADI-38497分子、结合一种浓度的抗PG scFv及芯片再生。将梯度稀释后的抗PG scFv(浓度梯度为1.25nM－40nM，2倍稀释)，以30μl/分钟的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，解离时间300s。最后使用10mM甘氨酸-HCl,pH 1.5(BR-1003-54,Cytiva)对芯片进行再生。
数据结果通过Biacore T200分析软件(版本号3.1)，使用1:1结合模型进行分析。
图3D展示了采用SPR测定ADI-38497 PG抗体及野生型抗体与作为P329G CAR胞外结构域的抗PG scFv的代表性亲合力图谱。结果显示，只有ADI-38497 PG抗体可特异性地与P329G CAR结合。
表7 SPR测定的P329G BCMA抗体及野生型抗体与抗PG scFv的亲合力
Figure PCTCN2022137265-appb-000009
(3-3)P329G BCMA抗体与P329CAR结合活性检测
复苏实施例1制备的P329G CAR-T细胞，用含有10％FBS的RPMI 1640培养基重悬，并在37℃培养稳定24小时。用FACS缓冲液将ADI-38497 PG抗体以及野生型ADI-38497WT抗体分别配制成5倍梯度稀释的不同浓度抗体溶液，分别与1E5个CAR阳性细胞4℃孵育30分钟，用FACS缓冲液清洗后，再与Fcγ片段特异的APC山羊抗人IgG(Jackson ImmunoResearch,109-136-098)4℃孵育30分钟。采用流式细胞术检测与细胞结合的抗体，分析APC通道MFI，以抗体浓度为X轴，APC通道MFI为Y轴进行绘图并计算结合的EC50。
图3E显示了ADI-38497WT抗体和ADI-38497 PG抗体与P329G CAR-T细胞的结合能力。结果表明，只有P329G突变抗体显示与CAR结合，而WT抗体不与CAR结合，该结果与SPR结果一致。
表8总结了ADI-38497 PG抗体和WT抗体与不同供者来源的P329G CAR-T细胞的结合EC50值和EC90值。
表8 BCMA IgG抗体与CAR-T细胞结合的EC50值和EC90值
Figure PCTCN2022137265-appb-000010
实施例4、BCMA特异性P329G突变抗体的Fc结构域功能检测
(4-1)ADI-38497 PG抗体的ADCC效应功能检测
复苏供者3的PBMC细胞(Peripheral Blood Mononuclear Cells，外周血单个核细胞)，用含有RPMI 1640培养基重悬，并在37℃稳定1-2小时。按照效靶比25:1混合PBMC和靶细胞，并与不同浓度的BCMA抗体进行混合，37℃分别继续培养4小时和24小时，用LDH检测试剂盒(Promega，G1780)检测抗体介导的PBMC对靶细胞的杀伤效应，并以抗体浓度为X轴，细胞裂解比例为Y轴进行绘图和分析。同时收集细胞，用FACS缓冲液洗涤2次，加入CD3、CD56、CD16及CD107a抗体，其中CD107a抗体需提前加入，与细胞在37℃共孵育1小时。上述细胞抗体混合液于4℃染色30分钟，洗涤两次，FACS缓冲液重悬，采用流式细胞仪进行检测。
图4A显示ADI-38497WT抗体和ADI-38497 PG抗体介导ADCC杀伤的能力，结果表明，当测试不同的孵育时间(4小时、24小时)和使用不同的检测指标(对靶细胞的细胞毒性、对CD3、CD56、CD16及CD107a表达的影响)，均显示只有WT抗体介导了对表达BCMA的阳性H929肿瘤细胞的ADCC细胞毒杀伤效应，而P329G突变抗体缺乏诱导ADCC效应的能力。
(4-2)ADI-38497 PG抗体的ADCP效应功能检测
取对数生长期的ADCP报告细胞系(Promega,G9871)与H929细胞，按照效靶比2:1、5:1混合ADCP报告细胞和H929靶细胞，并与不同浓度的BCMA抗体进行混合，37℃继续培养20小时，用萤光素酶检测试剂盒(Promega，E2620)检测抗体介导的依赖靶细胞的报告细胞激活效应，并以抗体浓度为X轴，荧光读值变化为Y轴进行绘图和分析。
图4B显示ADI-38497WT抗体和ADI-38497 PG抗体介导ADCP杀伤的能力。结果表明，当测试不同的效靶比(2:1或5:1)，均显示只有ADI-38497WT抗体介导了对表达BCMA的阳性H929肿瘤细胞的ADCP杀伤效应，而P329G突变抗体缺乏对表达BCMA的阳性H929肿瘤细胞诱导ADCP杀伤效应的能力。
(4-3)仅用ADI-38497 PG抗体是否介导靶细胞裂解的功能检测
取对数生长期H929细胞和L363细胞，按一定数量铺于孔板中；并将一部分对数生长期H929细胞和L363细胞作为靶细胞经丝裂霉素C处理，用作阴性对照。随后加入不同浓度的ADI-38497 PG抗体进行混合，37℃继续分别培养48小时、72小时及120小时，用CellTiter-Glo(Promega,G9242)检测活细胞比例，以共孵育时间为X轴，荧光读值为Y轴进行绘图并分析。
图4C显示了ADI-38497 PG抗体介导靶细胞裂解的能力，结果表明，当测试的不同孵育时间(48小时、72小时及120小时)与不同ADI-38497 PG抗体浓度(5μg/ml、50μg/ml)，均显示仅用ADI-38497 PG抗体缺乏诱导靶细胞裂解的能力。
实施例5、P329G CAR-T体外功能研究
(5-1)CAR-T细胞激活检测
复苏实施例1中供者5制备的CAR-T细胞并在37℃培养稳定过夜。将H929或L363肿瘤靶细胞和CAR-T细胞按照E:T为2:1进行混合，并添加5倍或10倍梯度稀释的不同浓度ADI-38497 PG抗体溶液至总体积为200μL，放于37℃培养约24小时后离心，收集细胞，用FACS缓冲液洗涤2次，重悬后加入含LIVE/DEAD Fixable Dead Cell Stain、Biotin-F(ab')Fragment山羊抗人IgG(Jackson ImmunoResearch,109-066-006)的FACS缓冲液，4℃染色30分钟，洗涤两次，加入CD4、CD8、CD25、CD69和APC-Streptavidin抗体组合，上述细胞抗体混合液于4℃染色30分钟，洗涤两次，FACS缓冲液重悬，采用流式细胞仪进行检测。
图5A显示了针对H929细胞只有含P329G突变的ADI-38497 PG抗体能特异性介导CAR +-T的激活，上调CD25、CD69的表达水平，且对CD4 +CAR +和CD8 +CAR +细胞无差异地激活，并呈现ADI-38497 PG抗体浓度梯度依赖性。
图5B显示不同BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、GSK PG IgG)诱导L363靶细胞对HuR968B CAR-T细胞的激活效应。由图5B可见，含P329G突变的BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、GSK PG IgG)能特异性介导CAR+T细胞的激活，显著上调CD25、CD69的表达水平。CAR阴性细胞群中CD25、CD69的表达也有微弱上调，表明有较低水平的激活，但该激活程度不显著，可忽略不计。
因此，不论是在高表达BCMA的H929细胞(图5A)还是在低表达BCMA的L363细胞(图5B)作为靶细胞的情况下，只有含P329G突变的BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、GSK PG IgG)能特异性介导CAR +-T的激活，显著上调CD25、CD69的表达水平，并具有P329G突变的BCMA抗体浓度梯度依赖性。
(5-2)CAR-T细胞增殖实验
复苏实施例1中供者4制备的UNT细胞和CAR-T细胞并在37℃培养稳定过夜。使用PBS稀释ADI-38497WT抗体或ADI-38497 PG抗体，加入96孔板并于4℃孵育过夜来包被板。将经过夜培养恢复后的CAR-T细胞添加至包被有抗体的96孔板内，作为阳性对照，在CAR-T细胞中直接加入CD3/CD28偶联磁珠(磁珠:细胞比例3:1)；37℃分别共孵育72小时、120小时，采用
Figure PCTCN2022137265-appb-000011
Luminescent Cell Viability Assay(Promega,G7572)检测细胞的发光值。
图5C显示HuR968B CAR-T细胞受包被的ADI-38497WT抗体或ADI-38497 PG抗体刺激后的增殖情况。HuR968B CAR-T细胞在包被的ADI-38497 P329G抗体刺激下产生增殖，与使用ADI-38497WT抗体刺激相比较，在刺激3天或5天后，ADI-38497 PG抗体使得 HuR968B CAR-T细胞分别增殖了约7倍或43.5倍；而UNT细胞在ADI-38497 PG抗体刺激下，与使用ADI-38497WT抗体刺激相比较，使用ADI-38497 PG抗体刺激未导致UNT细胞产生显著增殖。HuR968B CAR-T细胞和UNT细胞在CD3/CD28抗体偶联磁珠刺激情况下均产生了显著增殖。
(5-3)细胞因子检测
复苏实施例1制备的CAR-T细胞并在37℃培养稳定过夜。将肿瘤靶细胞和CAR-T细胞按照E:T为2:1进行混合，并添加5倍或10倍梯度稀释的不同浓度BCMA抗体(ADI-38497PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或作为Benchmark的GSK PG IgG)溶液至总体积为200μL，放于37℃培养约24小时后离心，收集上清。使用BD TM Cytometric Bead Array(CBA)Human Th1/Th2Cytokine Kit II((BD,551809))对细胞因子进行检测。将试剂盒内Capture Beads等体积混匀后，以25μL/孔进行布板。添加等体积的上清液或上清稀释液或标准品。混匀后添加25μL等体积的人Th1/Th2 PE检测试剂，室温避光孵育3小时。使用洗涤缓冲液洗涤两次后重悬，采用流式细胞仪进行检测，通过PE通道MFI值计算细胞因子浓度。
图5D显示来自实施例1的供者4的HuR968B CAR-T细胞与H929细胞、RPMI8226细胞共培养，在加入不同浓度BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或作为阳性对照的GSK PG IgG)后的CAR-T细胞分泌效应细胞因子的释放结果。
图5E显示来自供者5(购自ORiCELLS，Cat NO.:FPB004F-C，Lot NO.:PCH20210100004，Donor ID:Z0086)的HuR968B CAR-T细胞与不同肿瘤细胞共培养，在加入不同浓度BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或Benchmark GSK PG IgG)后CAR-T细胞分泌效应细胞因子的释放结果。
从图5D和图5E可以看出，在加入WT BCMA抗体(ADI-38497WT抗体)的情况下，HuR968B CAR-T细胞没有被激活，不分泌IL-2、IFN-γ、TNFα等效应细胞因子，只有在加入含P329G突变的BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、或作为阳性对照的GSK PG IgG)的情况下，HuR968B CAR-T细胞被激活，分泌效应细胞因子。且依据不同靶细胞条件下的细胞因子分泌结果来看，效应细胞分泌细胞因子的水平与靶细胞的BCMA表达水平无明显相关性。
(5-4)杀伤效率检测
复苏实施例1制备的CAR-T细胞并在37℃培养稳定过夜。将肿瘤靶细胞和CAR-T细胞按照E:T为2:1进行混合，并添加10倍梯度稀释的不同浓度BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或Benchmark GSK PG IgG)溶液至总体积为200μL，放于37℃培养约24小时后离心，转移细胞上清液至96孔白底酶标板中。使用CytoTox  96 Non-Radioactive Cytotoxicity Assay(Promega,G1780)以及酶标仪(Molecular Devices，SpectraMax i3x)测量上清液中的LDH值，计算杀伤效率。
图5F显示了不同BCMA抗体(ADI-38497 PG抗体、ADI-38484 PG抗体、ADI-38497WT抗体、或Benchmark GSK PG IgG)诱导HuR968B CAR-T细胞对不同BCMA表达水平肿瘤细胞(H929 ++细胞、RPMI8226 +++细胞、AMO1 +细胞和L363 +细胞)的杀伤效应。在加入WT BCMA抗体的情况下，HuR968B细胞没有被激活，不产生对肿瘤细胞的杀伤效应，只有在加入含P329G突变的BCMA抗体的情况下，P329G CAR-T细胞被激活，从而杀伤靶细胞，且具有抗体浓度依赖性，与本实施例中的上述CAR-T细胞激活及效应细胞因子结果相一致。此外，在以高表达BCMA的H929、RPMI8226细胞作为靶细胞的情况下，P329G BCMA抗体介导HuR968B CAR-T细胞的杀伤效应高于低表达BCMA的AMO1、L363靶细胞。
图5G显示了不论是P329G BCMA抗体还是WT BCMA抗体，均不会介导CAR-T对BCMA阴性靶细胞(例如BCMA-KO-H929细胞)的杀伤效应，而针对表达BCMA的H929细胞(H929)，WT BCMA抗体也无法介导杀伤效应，只有P329G BCMA抗体可诱导CAR-T产生对肿瘤细胞的杀伤效应。
表9 P329G BCMA抗体介导HuR968B CAR-T细胞对H929靶细胞的杀伤效应的EC50值
抗体 EC50(nM)
ADI-38497 PG 0.3502至0.7433
ADI-38497 WT 非常宽
实施例6、游离BCMA蛋白对PG CAR-T功能的影响
(6-1)杀伤效率检测
复苏实施例1中供者6制备的CAR-T细胞并在37℃培养稳定过夜。将肿瘤靶细胞和CAR-T细胞按照E:T为2:1进行混合，加入一定浓度的PG BCMA抗体，对于传统Blue21CAR-T则不需要加入BCMA抗体。均添加10倍梯度稀释的不同浓度BCMA蛋白溶液作为游离BCMA蛋白至总体积为200μL，放于37℃培养约24小时后离心，转移细胞上清液至96孔白底酶标板中。使用CytoTox 96 Non-Radioactive Cytotoxicity Assay(Promega,G1780)以及酶标仪(Molecular Devices，SpectraMax i3x)测量上清液中的LDH值，计算杀伤效率。
图6显示不同浓度的游离BCMA蛋白对HuR968B CAR-T及Blue21CAR-T细胞杀伤效应的影响。在加入不同浓度的游离BCMA蛋白的情形下，一旦传统CAR T细胞与游离BCMA蛋白结合，则其功能就会被阻断(由于传统CAR分子对BCMA的高亲和力)，而当PG BCMA抗体与可溶性BCMA结合，PG CAR-T仍然能够发挥正常的杀伤效应。因此，PG CAR-T细胞的优势在于其功能受可溶性配体(例如可溶性BCMA)的影响低。
实施例7、ADI-38497 PG抗体的体内药代动力学研究
(7-1)抗体注射及取样
将BALB/c小鼠(年龄4-6周，体重15-17g，雌性)分成3组，即ADI-38497 PG抗体，1mg/kg抗体组；ADI-38497 PG抗体，10mg/kg抗体组；和ADI-38497 PG抗体，200mg/kg抗体组，每组9只小鼠；用1×PBS将抗体分别稀释至0.1mg/mL,1mg/mL和20mg/mL，每只小鼠给药体积10mL/kg，即抗体给药剂量分别为1mg/kg，10mg/mL和200mg/mL；给药方式为静脉注射，给药频率为单次。抗体给药后5分钟、30分钟、2h、6h、24h、48h、96h、168h、336h和504h小鼠眼眶后静脉丛采集血样100μL，3000g离心，吸取上清液用于血药浓度测定。
(7-2)ADI-38497 PG抗体检测
提前一天包被96孔酶标板。用包被液(取一包碳酸盐(Thermo,28382)粉末，溶解于400mL超纯水，定容至500mL，混匀即为包被液)将BCMA抗原稀释至1μg/ml，每孔100μL，封板膜封板，室温过夜。倒掉包被溶液，在吸水纸上拍干，然后每孔加入300μL洗液，振荡混匀10秒，拍干洗液后，重复洗涤3次。排枪加入封闭液，每孔200μL，封板膜封板，室温孵育2h。随后洗板1次。将稀释好的标准曲线(由已知浓度的BCMA抗体梯度稀释，制备标准曲线(例如，使用已知浓度的ADI-38497 PG抗体制备标准曲线)、质量控制样品和待测样本每孔100μL，室温孵育2h。倒掉预包被溶液，在吸水纸上拍干，然后每孔加入300μL洗液，振荡混匀10秒，拍干洗液后，重复洗涤3次。重复一次。将山羊抗人IgG-Fc-HRP抗体(BETHYL)1:10万稀释，每孔加入100μL，室温避光孵育1h。随后洗板1次。将TMB底物加入到96孔酶标板中，每孔100μL，室温下避光显色5分钟。每孔加入50μL ELISA终止液，震荡10秒，30分钟内读取OD450nm和OD620nm值。
图7A和7B显示小鼠中ADI-38497 PG抗体(下文小鼠体内实验中也简称为PG Ab)药代动力学实验结果。小鼠静脉注射1mg/kg、10mg/kg、200mg/kg的ADI-38497 PG抗体后，血清中ADI-38497 PG抗体暴露量(Cmax和AUClast)呈现剂量依赖效应，其他药代动力学参数无明显差异，如表10所示，ADI-38497 PG抗体1mg/kg：AUC0-inf，Cmax，CL，T1/2分别为2480μg×h/mL，30ug/ml，0.40ml/kg/h、145h；ADI-38497 PG抗体10mg/kg：AUC0-inf，Cmax，CL，T1/2分别为24720μg×h/mL，187ug/ml，0.32ml/kg/h、219h；ADI-38497 PG抗体200mg/kg：AUC0-inf，Cmax，CL，T1/2分别为397734μg×h/mL，3895ug/ml，0.43ml/kg/h、197h，ADI-38497 PG抗体1mg/kg半衰期略短于ADI-38497 PG抗体10mg/kg和200mg/kg。
表10 ADI-38497 PG抗体药代动力学实验结果
Figure PCTCN2022137265-appb-000012
实施例8、PG CAR-T细胞联合不同剂量PG抗体在体内抗BCMA高表达肿瘤的效应
(8-1)小鼠肿瘤接种及处理
用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射H929细胞悬液，注射体积0.2mL/只，即接种量为1×10 6个细胞/只小鼠。肿瘤细胞接种后7天，将小鼠肿瘤体积在50.82～104.36mm 3的小鼠分成7组，分别为PBS载剂组、PG Ab组、仅PG CAR-T组、传统CAR-T组、PG Ab+PG CAR-T，3mg/kg抗体组、PG Ab+PG CAR-T，1mg/kg抗体组和PG Ab+PG CAR-T，0.3mg/kg抗体组，每组7只小鼠。分别配置浓度为0.3mg/mL，0.1mg/mL和0.03mg/mL的抗体，分组完成后，于第7日进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。用1×PBS重悬供者4制备的CAR-T细胞，制备成CAR +细胞为25×10 6个/mL细胞悬液，于第7日尾静脉注射细胞悬液0.2mL/只，即回输CAR +细胞5×10 6个/只小鼠。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。
图8A显示在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞的治疗效应。结果显示，在BCMA高表达肿瘤模型中，仅施用PG CAR-T细胞未产生明显抗肿瘤效应，仅施用PG抗体产生一定的抗肿瘤效应，只有同时接受PG CAR-T细胞和PG抗体治疗的小鼠产生显著抗肿瘤效应，并呈现抗体剂量依赖效应。PG CAR-T细胞联用0.3mg/kg抗体、1mg/kg抗体和3mg/kg抗体的处理使得肿瘤生长抑制率(TGI)分别为92％、88％和101％，而仅施用PG CAR-T细胞或仅施用PG抗体处理的小鼠中抗肿瘤药效TGI分别为25％和53％。此外，PG CAR-T细胞联用PG抗体3mg/kg产生的抗肿瘤效应与同等给药剂量的传统Blue21CAR-T细胞的抗肿瘤效应相当，TGI分别为101％和102％。
图8B显示了该实验中小鼠的体重变化。结果显示，经PG CAR-T细胞联用PG抗体治疗的小鼠，其体重在接受处理后保持稳定，在联用PG抗体0.3mg/kg、1mg/kg和3mg/kg处理后体重平均升高5.2％、3.0％、7.6％。结果表明，PG CAR-T细胞联合PG抗体治疗产生了显著抗肿瘤效应，并且无明显毒副效应。
(8-2)小鼠体内CAR-T细胞检测：
取30μL实施例8-1的小鼠血样，加入96孔V孔板中，标注为样本检测孔；取10μL小鼠血样，加入96孔V孔板中，标注为对照孔。向所有孔内加入100μL含有LIVE/DEAD Fixable Dead Cell Stain以及TruStain FcX TM(抗mouse小鼠CD16/32)(Biolegend)的FACS缓冲液，轻轻混匀，4℃避光孵育15分钟；随后向样本检测孔内加入Biotin-F(ab')片段Fragment山羊抗人IgG抗体，4℃避光孵育30分钟；随后向样本检测孔中各加入100μL FACS缓冲液，400g离心，弃上清；向所有孔中各加入100μL含有APC-Cy7抗人CD45(Biolegend)、PerCP-Cy5.5-CD3(BD Biosciences)和APC-链亲和素(Biolegend)的FACS缓冲液，轻轻混匀，4℃避光孵育30分钟；然后向所有孔中加入200μL/孔FACS缓冲液，400g离心，弃上清；加入250μL/孔1×RBC Lysis/Fixation solution(Biolegend),混匀,室温避光孵育20分钟；400g离心，弃上清；用100μL FACS缓冲液重悬细胞后，每孔加入10μL 123count ebeads，使用流式细胞仪检测。
图8C显示实施例8-1的实验中PG CAR-T细胞在小鼠体内扩增情况。结果显示，PG CAR-T细胞体内扩增依赖PG抗体，呈现一定抗体剂量依赖性，更高剂量组小鼠有更高水平的PG CAR-T细胞扩增。仅施用PG CAR-T细胞，其在回输小鼠体内1周开始扩增，2周后扩增到466个细胞/100μL外周血，3周后扩增达到较高水平(3644个细胞/100μL外周血)，4周后仍维持在高水平(3214个细胞/100μL外周血)；在联用PG CAR-T细胞时给予不同剂量PG抗体0.3mg/kg、1mg/kg、3mg/kg的情况下,2周后PG CAR-T细胞体内峰值扩增水平分别达到11428个细胞/100μL外周血、19299个细胞/100μL外周血、35368个细胞/100μL外周血，4周后仍维持在较高水平，分别为15486个细胞/100μL外周血、25073个细胞/100μL外周血和27666个细胞/100μL外周血,远高于同期未联合给予抗体组。此外，作为阳性对照的传统Blue21CAR-T细胞，显示相似的扩增动力学，同样在回输小鼠体内1周开始扩增，2周后扩增达到峰值水平(174769个细胞/100μL外周血)，4周后仍维持在极高水平(131963个细胞/100μL外周血)。
实施例9、PG CAR-T细胞联合不同剂量PG抗体在体内抗BCMA低表达肿瘤的效应
(9-1)小鼠肿瘤接种及处理
用1×PBS重悬L363细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄 4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射L363细胞悬液，注射体积0.2mL/只，即接种量为1×10 6个细胞/只小鼠。肿瘤细胞接种后9天，将小鼠肿瘤体积在74.14～110.29mm 3的小鼠分成7组，分别为载剂组、PG Ab组、PG CAR-T组、传统CAR-T组、PG Ab+PG CAR-T,3mg/kg抗体组、PG Ab+PG CAR-T,1mg/kg抗体组和PG Ab+PG CAR-T,0.3mg/kg抗体组，每组7只小鼠。分别配置浓度为0.3mg/mL，0.1mg/mL和0.03mg/mL的抗体，分组完成后，于第9日进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。用1×PBS重悬供者4制备的CAR-T细胞，制备成CAR +细胞为25×10 6个/mL细胞悬液，于第9日尾静脉注射细胞悬液0.2mL/只，即回输CAR +细胞5×10 6个/只小鼠。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。
图9A显示在皮下接种人L363低表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞的治疗效应。结果显示，在BCMA低表达肿瘤模型中，仅施用PG CAR-T未产抗肿瘤效应，而仅施用PG抗体抗肿瘤效应不明显，TGI为21％，只有同时接受PG CAR-T细胞和PG抗体治疗的小鼠才产生显著抗肿瘤效应，并呈现抗体剂量依赖效应。PG CAR-T细胞联用0.3mg/kg剂量的PG抗体情况下，PG CAR-T细胞诱导了显著的抗肿瘤效应，TGI为87％，当该联用增加PG抗体剂量至1mg/kg和3mg/kg时，PG CAR-T细胞诱导的抗肿瘤最大效应显著增加，TGI分别为103％和103％，显示出与传统Blue21CAR-T细胞相同的抗肿瘤效应。
图9B显示了该实验中小鼠的体重变化。结果显示，经PG CAR-T细胞联用PG抗体治疗的小鼠，其体重在接受处理后保持平稳上升，在联用PG抗体0.3mg/kg、1mg/kg、3mg/kg后体重平均升高18.2％、10.5％、8.5％。结果表明，PG CAR-T细胞联合PG抗体治疗产生了显著抗肿瘤效应，并且未诱导明显毒性。
(9-2)小鼠体内CAR-T细胞检测：方法同实施例8-2。
图9C显示实施例9-1的实验中PG CAR-T细胞在小鼠体内扩增情况。结果显示，仅施用PG CAR-T细胞，其在回输小鼠体内1周开始扩增，2周后扩增到919个细胞/100μL外周血，3周时快速下降到204个细胞/100μL外周血；在联用PG CAR-T细胞时给予不同剂量PG抗体0.3mg/kg、1mg/kg和3mg/kg的情况下,2周后PG CAR-T细胞体内峰值扩增水平达到4380个细胞/100μL外周血、8049个细胞/100μL外周血和3347个细胞/100μL外周血，3周后仍维持在较高水平，分别为2475个细胞/100μL外周血、4121个细胞/100μL外周血和1969个细胞/100μL外周血，远高于同期未联合给予抗体组。此外，作为阳性对照的传统Blue21CAR-T细胞同样在回输小鼠体内2周后扩增达到峰值水平(76836个细胞/100μL外周血)，3周后仍维持在高水平(36328个细胞/100μL外周血)。
实施例10、不同剂量PG CAR-T细胞联合PG抗体在体内的抗肿瘤效应
(10-1)小鼠肿瘤接种及处理
用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射H929细胞悬液，注射体积0.2mL/只，即接种量为1×10 6个细胞/只小鼠。肿瘤细胞接种后9天，将小鼠肿瘤体积在59.50～105.82mm 3的小鼠分成7组，分别为载剂组、PG Ab组、仅施用PG CAR-T组、PG Ab+PG CAR-T,10×10 6组、PG Ab+PG CAR-T,1×10 6个细胞组、PG Ab+PG CAR-T，0.1×10 6个细胞组和PG Ab+PG CAR-T,0.01×10 6个细胞组，每组7只小鼠。配置浓度为0.3mg/mL的抗体，分组完成后，于第9日进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。用1×PBS重悬供者4制备的CAR-T细胞，制备成CAR +细胞为50×10 6个/mL细胞悬液，随后10倍梯度稀释，再制备成5×10 6、0.5×10 6和0.05×10 6个/mL的细胞悬液，于第9日尾静脉注射细胞悬液0.2mL/只。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。
图10A显示在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合不同剂量PG CAR-T细胞的治疗效应。结果显示，在给予极低剂量0.01×10 6个CAR-T细胞的情况下，CAR-T细胞产生与仅施用PG抗体相似的抗肿瘤效应，TGI分别为49％和50％。增加CAR-T细胞剂量至0.1×10 6、1×10 6、10×10 6个CAR-T细胞，PG CAR-T细胞诱导的抗肿瘤效应显著增加，TGI分别为91％、104％、103％。仅施用CAR-T细胞未显示抗肿瘤效应。
(10-2)体内CAR-T细胞检测：方法同实施例8-2。
图10B显示实施例10-1的实验中PG CAR-T细胞在小鼠体内扩增情况。结果显示，CAR-T细胞回输小鼠体内在PG抗体诱导下，1周开始扩增，2周后扩增达到峰值水平，3周后仍保持高水平，此外在联用PG抗体时，CAR-T细胞体内扩增依赖CAR-T细胞剂量，更高CAR-T剂量组小鼠有更高水平的CAR-T细胞扩增，0.01×10 6、0.1×10 6、1×10 6、10×10 6CAR-T细胞剂量组峰值扩增水平分别为6个细胞/100μL外周血、338个细胞/100μL外周血、3640个细胞/100μL外周血、12895个细胞/100μL外周血。
实施例11、PG CAR-T细胞联合PG抗体不同给药频率体内抗肿瘤效应研究
(11-1)小鼠肿瘤接种及处理
用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射H929细胞悬液，注射体积0.2mL/只，即接种量为1×10 6个细胞/只小鼠。肿瘤细胞接种后7天，将小鼠肿瘤体积在51.25～94.97mm 3的小鼠分成7组，分别为载剂组、仅施用PG CAR-T组、PG Ab+PG CAR-T，Q3/4×2组、 PG Ab+PG CAR-T，QW×4组、PG Ab+PG CAR-T，Q2W×2组、PG Ab+PG CAR-T，Q3W×2组和传统CAR-T组，每组7只小鼠。配置浓度为0.1mg/mL的PG Ab抗体，分组完成后，抗体给药，每只小鼠给药体积10mL/kg，给药方式为腹腔注射。用1×PBS重悬供者4制备的CAR-T细胞，制备成CAR +细胞10×10 6个/mL细胞悬液，尾静脉注射细胞悬液0.2mL/只。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。
图11A显示实施例11-1的实验中PG抗体的给药频率。Q3-4D×2：以4周为周期，PG抗体在第1周给药2次，给药间隔3-4天；Q3W×2：PG抗体给药2次，给药频率为3周/次；Q2W×2：PG抗体给药2次，给药频率为2周/次；QW×4：PG抗体给药4次，给药频率为1周/次。
图11B显示在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中，联用PG CAR-T细胞时PG抗体不同给药时频率的治疗效应。结果显示，联用PG CAR-T细胞时PG抗体不同给药频率均能够诱导PG CAR-T细胞产生显著抗肿瘤效应，并且增加PG抗体给药频率，PG CAR-T细胞诱导的抗肿瘤效应显著增加，QW×4组CAR-T细胞诱导的抗肿瘤效应最显著，显示出与传统Blue21CAR-T细胞相同的抗肿瘤效应。
(11-2)体内CAR-T细胞检测：方法同实施例8-2。
图11C显示实施例11-1的实验中PG CAR-T细胞在小鼠体内扩增情况。结果显示，联用PG CAR-T细胞时，在PG抗体不同给药频率情况下，CAR-T细胞扩增动力学相似,CAR-T细胞回输小鼠体内1周开始扩增，2周后扩增达到峰值水平，3周后下降到基线水平。此外，传统Blue21CAR-T细胞同样在回输小鼠体内2周后扩增达到峰值水平，3周后仍维持在高水平。
实施例12、PG CAR-T细胞联合不同剂量PG抗体在体内抗系统性肿瘤效应研究
(12-1)小鼠肿瘤接种及处理
首先，制备了H929-luc细胞。具体而言，用H929细胞(购自南京科佰生物科技有限公司)包装含GFP-萤光素酶基因的慢病毒，并用获得的慢病毒感染H929细胞，后续经流式细胞术分选，得到GFP和萤光素酶双表达的H929-luc细胞系。
然后，用1×PBS重悬H929-luc细胞，制备成细胞浓度为25×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)尾静脉注射H929-luc细胞悬液，注射体积0.2mL/只。肿瘤细胞接种后14天，腹腔注射底物D-Luciferin(15mg/mL)，注射体积为10mL/kg/只小鼠，底物注射10分钟后用IVIS spectrum成像分析。将荧光信号在1.17×10 7～1.43×10 8photons/sec的小鼠分成7组，分别为载剂组、PG Ab，0.3mg/kg组、PG Ab，3mg/kg组、PG CAR-T组、PG Ab+PG CAR-T，0.3mg/kg+2×10 6组、PG Ab+PG CAR-T，3mg/kg+2×10 6 组和Blue21CAR-T组，每组6-7只小鼠。分别配置浓度为0.03mg/mL和0.3mg/mL的抗体，分组完成后，于第14日开始进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。用1×PBS重悬供者4制备的CAR-T细胞，制备成CAR +细胞10×10 6个/mL细胞悬液，于第7日尾静脉注射细胞悬液0.2mL/只。
图12A显示在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞治疗效应的荧光图像。结果显示，在系统性肿瘤模型中，PG抗体联合PG CAR-T细胞同样产生显著抗肿瘤效应，第18天，PG抗体0.3mg/kg联合PG CAR-T细胞组小鼠荧光分布及荧光强度相比对照组显著减少，同时给予PG抗体3mg/kg联合PG CAR-T细胞组小鼠无明显荧光信号，而传统Blue21CAR-T治疗组仍有大量荧光分布，显示PG CAR-T细胞组比Blue21CAR-T更快地诱导抗肿瘤效应；PG抗体3mg/kg联合PG CAR-T细胞组小鼠无明显荧光信号一直维持到第56天，至第63天部分小鼠肿瘤略微显示荧光信号，而此时Blue21CAR-T治疗组超过一半的小鼠肿瘤明显复发并转移至腹腔，表明PG CAR-T细胞联用PG抗体产生更持久的抗肿瘤效应。
图12B显示在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体联合PG CAR-T细胞的治疗效应。结果显示，在给予PG抗体0.3mg/kg情况下，PG CAR-T细胞产生一定的抗肿瘤效应，增加PG抗体剂量至3mg/kg，PG CAR-T细胞诱导的抗肿瘤效应显著增加，并且抗肿瘤效应维持的更持久，停止给予PG抗体，约4周左右肿瘤生长开始复发，而给予同等细胞剂量的传统Blue21CAR-T肿瘤复发更快，显示出PG CAR-T细胞比传统Blue21CAR-T更持久的抗肿瘤效应。仅施用PG CAR-T细胞组、PG抗体组未显示明显抗肿瘤效应。
图12C显示上述实验中小鼠体重变化。结果显示，治疗期间各处理组小鼠体重平稳上升，提示在血液肿瘤模型中不同剂量PG抗体联合PG CAR-T细胞处理未诱导显著毒性。
实施例13、PG CAR-T细胞联合PG抗体在体内毒理研究
(13-1)小鼠肿瘤接种及处理
用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射5×10 6个/mL的H929细胞悬液，注射体积0.2mL/只。肿瘤细胞接种后6天，将小鼠肿瘤体积在38.49～104.77mm 3的小鼠分成8组，如表11所示，分别为未荷瘤的载剂组、PG Ab+PG CAR-T，10mg/kg+10×10 6组；以及荷瘤的载剂组、PG CAR-T组、PG Ab组、PG Ab+PG CAR-T，10mg/kg+10×10 6组、PG Ab+PG CAR-T，3mg/kg+10×10 6组和PG Ab+PG CAR-T，3mg/kg+1×10 6组，每组24只小鼠。分别配置浓度为1mg/mL和0.3mg/mL的抗体，分组完成后，抗体给药，每只小鼠给药体积 10mL/kg，给药频率为每周1次，给药次数3次，给药方式为腹腔注射。第1次抗体给药后1天，用1×PBS重悬供者4制备的CAR-T细胞，分别制备CAR +细胞50×10 6个/mL和5×10 6个/mL细胞悬液。尾静脉注射细胞悬液0.2mL/只。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。第1次抗体给药前、CAR-T回输后、第3次抗体给药前及实验终点取外周血，每组4只小鼠，用于血液学和血生化检测。
表11各组的处理和给药剂量
组别 细胞系 处理 浓度
1 无肿瘤负荷 载剂 N/A
2 无肿瘤负荷 PG Ab+PG CAR-T 10mg/kg(QW×3)+10x 10 6个细胞/小鼠(SD)
3 H929 载剂 N/A
4 H929 PG Ab 10mg/kg(QW×3)
5 H929 PG CAR-T 10x 10 6个细胞/小鼠(SD)
6 H929 PG Ab+PG CAR-T 10mg/kg(QW×3)+10x 10 6个细胞/小鼠(SD)
7 H929 PG Ab+PG CAR-T 3mg/kg(QW×3)+10x 10 6个细胞/小鼠(SD)
8 H929 PG Ab+PG CAR-T 3mg/kg(QW×3)+1x 10 6个细胞/小鼠(SD)
图13A显示在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体联合PG CAR-T细胞的治疗效应。结果显示，PG抗体联合PG CAR-T细胞处理组均产生显著抗肿瘤效应，而仅施用PG抗体处理组小鼠抗肿瘤效应较弱，仅施用PG CAR-T未产生抗肿瘤效应。
图13B显示本实验中小鼠体重变化。结果显示，治疗期间荷瘤和非荷瘤处理组小鼠体重平稳上升，且体重相较对照小鼠无明显变化，提示不同剂量PG抗体联合不同剂量PG CAR-T细胞治疗未诱导明显毒性反应。
图13C和图13D显示上述实验中小鼠血液学和血生化检测结果。结果显示，治疗期间荷瘤和非荷瘤处理组小鼠血液学和血生化指标较对照小鼠无明显变化，表明PG抗体联合PG CAR-T细胞治疗未产生毒性反应。
实施例14、临床研究
1.临床研究目的：
1)评价输注“PG 38497抗体+PG CAR-T细胞”治疗复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)的安全性和耐受性；
2)评价输注“PG 38497抗体+PG CAR-T细胞”治疗RRMM的临床疗效。
3)评价“PG 38497抗体+PG CAR-T细胞”给药后的药代动力学(pharmacokinetics,PK) 和药效动力学(pharmacodynamic,PD)特征。
4)评价“PG 38497抗体+PG CAR-T细胞”给药后的免疫原性。
2.受试药物：
本实施例所述受试药物的活性组分为：P329G BCMA抗体(ADI-38497 PG抗体)和P329G CAR-T细胞(患者自体PBMC细胞改造获得CAR-T细胞)。
P329G BCMA抗体的制剂处方为20.0mg/ml P329G BCMA抗体，0.76mg/ml组氨酸，1.08mg/ml盐酸组氨酸，50.00mg/ml山梨醇，0.20mg/ml聚山梨酯80，pH 6.0；P329G BCMA抗体的规格：60mg(3mL)/瓶。
P329G CAR-T细胞的规格：每袋含有溶解于7.5％DMSO冻存保护剂中的90-140×10 6抗P329G CAR-T阳性细胞。
P329G BCMA抗体的生产企业为信达生物制药(苏州)有限公司；P329G CAR-T细胞的生产企业为信达细胞制药(苏州)有限公司。
3.入选标准
1)18周岁及以上，性别不限。
2)根据国际骨髓瘤工作组(International Myeloma Working Group,IMWG)多发性骨髓瘤的诊断标准，有初次诊断为多发性骨髓瘤的检查证明资料。
3)既往经过至少3线治疗，每线治疗有至少1个完整治疗周期，(除非最佳疗效记录为疾病进展(progressive disease,PD)，根据IMWG标准)；且必须包含蛋白酶体抑制剂和免疫调节剂。
4)在最近的抗骨髓瘤治疗期间或之后12个月内出现有检查资料证明的疾病进展。
5)根据以下任一标准确定筛选时存在可测量病灶：
①骨髓细胞学、骨髓活检组织或流式检测单克隆浆细胞比例≥5％；
②血清单克隆蛋白(M-蛋白)水平≥0.5g/dL；
③尿M蛋白水平≥200mg/24小时；
④血清或尿液中无可测量病灶的轻链型多发性骨髓瘤：血清免疫球蛋白游离轻链≥10mg/dL且血清免疫球蛋白κ/λ游离轻链比异常。
6)ECOG评分为0或1分。
7)预期生存时间≥12周。
8)受试者必须有适当的器官功能，入组前符合下列所有实验室检查结果：
①血常规：绝对嗜中性粒细胞计数(absolute neutrophil count,ANC)≥1×109/L；绝对淋巴细胞计数(absolute lymphocyte count,ALC)≥0.3×109/L；血小板≥50×109/L；血红蛋白≥60g/L；
②肝功能：ALT和AST≤2.5×正常值上限(upper limit of normal,ULN)；血清总胆红素≤1.5×ULN；
③肾功能：血清肌酐≤2.5×ULN；或根据Cockcroft-Gault公式计算的肌酐清除率(creatinine clearance,CrCl)≥40ml/min；
④凝血功能：纤维蛋白原≥1.0g/L；活化的部分凝血活酶时间，活化的部分凝血活酶时间≤1.5×ULN，凝血酶原时间≤1.5×ULN；
⑤静息状态下动脉血氧饱和度>91％；
⑥左心室射血分数(left ventricular ejection fraction,LVEF)≥50％。
9)受试者及其配偶同意在受试者签署知情同意书(informed consent form,ICF)后至最后一次接受“P329G BCMA抗体+PG CAR-T细胞”治疗(该治疗中的任一组分)给药一年内采取有效的工具或者药物避孕措施(不包括安全期避孕)。
10)受试者必须签署ICF，表明其理解本研究的目的和程序并且愿意参加研究。须在开始任何一项研究相关但不属于受试者疾病标准治疗的检查或程序前获得知情同意。
4.临床方案
本研究是一项评估输注“P329G BCMA和P329G CAR-T细胞”治疗RRMM受试者的安全性、耐受性、药代动力学和初步疗效的临床研究。
入组的受试者将接受外周血单个核细胞(peripheral blood mononuclear cell,PBMC)单采(-28～-21天)。之后，利用受试者自身的T细胞制备P329G CAR-T细胞制剂(以下简称PG CAR-T细胞)。成功制备PG CAR-T细胞后，受试者将会首先接受环磷酰胺和氟达拉滨的清淋预处理方案(-5～-3天)，预处理结束休息评估1天后接受1剂P329G BCMA抗体(以下简称ADI-38497 PG抗体)输注，隔天(0天)接受PG CAR-T细胞输注，之后自PG CAR-T细胞输注日算起，每21天周期性输注ADI-38497 PG抗体，最多输注24个月，直至疾病进展、出现不可耐受的毒性、受试者撤回知情同意或出现其他需停止研究治疗的原因(以先发生者为准)。
本研究分为剂量递增和剂量扩展两部分。
1)在剂量递增阶段，采用经典“3+3”方式，固定PG CAR-T细胞剂量，充分探索ADI-38497 PG抗体的最优剂量。ADI-38497 PG抗体剂量共设3个剂量组：0.3mg/kg、1mg/kg、3mg/kg。PG CAR-T细胞剂量为2*10 6个/kg。DLT观察窗为ADI-38497 PG抗体首剂给药后21天，DLT观察期内包含1次PG CAR-T细胞给药。
2)固定PG CAR-T细胞进行ADI-38497 PG抗体剂量递增。每个剂量组先入组3例受试者，如果先入组的3例受试者在DLT观察窗内未观察到DLT，可开始下一剂量组给药；如果3例中有≥2例发生DLT，则终止剂量递增；如果某剂量先入组的3例受试者中有1例出现 DLT，同一剂量组需增加3例受试者(此时该剂量组总共6例受试者)，若补充的3例受试者无DLT发生，则可开始下一剂量组给药，若补充的3例受试者中有≥1例发生DLT，不允许继续进行剂量递增。同时，需在上一剂量组补足6例受试者进行试验直至确定最大耐受剂量(maximum tolerated dose,MTD)，因此MTD剂量组至少需要6例受试者方能确认。若抗体起始剂量0.3mg/kg组前3例受试者中有≥2例受试者出现DLT，则退至更低的抗体剂量0.1mg/kg进行剂量探索；如果先入组的3例受试者中有1例出现DLT，该剂量组需增加3例受试者(此时该剂量组总共6例受试者)，若补充的3例受试者无DLT发生，则可开始下一剂量组给药，若补充的3例受试者中有≥1例发生DLT，则退至更低的抗体剂量0.1mg/kg进行剂量探索。
研究者可根据研究中累积的数据，与赞助方讨论一致后选择预测可能具有更高获益/风险比的剂量继续研究。例如，若上述试验整体安全性良好，预测进一步提高PG CAR-T细胞剂量有增加受试者获益的可能，则继续探索5*10 6个/kg PG CAR-T细胞剂量的安全性。在剂量递增阶段，将由研究者审查安全性数据，并结合PK数据，决定是否需要进行下一剂量组的探索并在必要时调整剂量递增方案。前3例受试者将采用交错入组策略。第1和第2例受试者、以及第2和第3例受试者之间必须间隔一个至少2周的观察期。
在研究药物剂量递增阶段，若受试者完成DLT观察期并且未出现DLT，将继续接受当前剂量组的治疗，直至疾病进展、不可耐受的毒性、受试者撤回知情同意或出现其他需停止研究治疗的原因(以先发生者为准)，最长治疗期24个月。
3)剂量扩展阶段：
为充分探索“P329G BCMA抗体和P329G CAR-T细胞”的有效性和安全性，在研究进行过程中或整体爬坡结束后，研究者可根据前期获得的安全性和疗效信号，PG CAR-T细胞及抗体PK等数据，选择1～2个剂量组进行剂量扩展，每个剂量组可补充3～6例受试者。
5.临床治疗给药方案
P329G BCMA和P329G CAR-T细胞制剂的给药信息见下表12错误！未找到引用源。。输注P329G CAR-T细胞制剂后受试者应至少住院2周，具体时间应由研究者根据受试者病情的评估决定出院时间。
表12 P329G BCMA和P329G CAR-T细胞输注给药
Figure PCTCN2022137265-appb-000013
Figure PCTCN2022137265-appb-000014
6.实验结果
本实施例为“P329G BCMA和P329G CAR-T细胞”治疗方法首次在人体开展的药物临床研究。从前述的体内外实验结果表明，P329G CAR-T细胞具有良好抗肿瘤效应，在足量P329G BCMA抗体存在情况下，极低剂量P329G CAR-T细胞就能够诱导完全抗肿瘤效应，效应至少与同等剂量传统CAR-T细胞相当；P329G CAR-T细胞活性受P329G BCMA抗体调控并呈现抗体剂量依赖性，不同剂量P329G BCMA抗体诱导不同抗肿瘤效应。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。
示例性序列
Figure PCTCN2022137265-appb-000015
Figure PCTCN2022137265-appb-000016
Figure PCTCN2022137265-appb-000017
Figure PCTCN2022137265-appb-000018

Claims (15)

  1. 药物组合，其包含
    (i)第一组分，其选自表达分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)、编码所述CAR多肽的核酸分子、包含所述核酸分子的载体、和它们的任意组合；和
    (ii)第二组分，其是包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段(也称为P329G突变抗体)，例如，所述P329G突变抗体包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；以及
    任选地可药用辅料；
    其中，所述分子开关调控型CAR多肽包含
    (1)人源化抗P329G突变scFv序列，其中所述scFv序列包含能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域的如下序列：
    (i)重链可变区，其包含根据Kabat编号的
    (a)氨基酸序列RYWMN(SEQ ID NO:19)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:20)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (c)氨基酸序列PYDYGAWFAS(SEQ ID NO:21)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (ii)轻链可变区，其包含根据Kabat编号的
    (d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:22)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (e)氨基酸序列GTNKRAP(SEQ ID NO:23)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (f)氨基酸序列ALWYSNHWV(SEQ ID NO:24)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    其中所述氨基酸变化是氨基酸的添加、缺失或取代；
    (2)铰链区/间隔区，其选自
    (i)(G 4S) n、(SG 4) n或G 4(SG 4) n肽接头，其中“n”是1至10的整数，例如1至4的整数；例如SEQ ID NO:14所示的序列；
    (ii)CD8α铰链区或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:18所示的序列或其具有1-2个氨基酸修饰的变体；
    (3)跨膜区(TM)，其选自CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:15所示的序列或其具有1-2个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其选自4-1BB共刺激结构域或其具有1-5个氨基酸修饰的 变体，例如，SEQ ID NO:16所示的序列或其具有1-2个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，SEQ ID NO:17所示的序列或其具有1-10个、1-5个氨基酸修饰的变体；
    优选地，所述CAR多肽包含
    (1)人源化抗P329G突变scFv序列，其中所述scFv序列包含能够特异性结合包含P329G突变的抗体Fc结构域，但不能特异性结合未突变的亲本抗体Fc结构域的如下序列：
    (i)重链可变区，其包含根据Kabat编号的
    (a)氨基酸序列RYWMN(SEQ ID NO:19)所示的CDR H1；
    (b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:20)所示的CDR H2；和
    (c)氨基酸序列PYDYGAWFAS(SEQ ID NO:21)所示的CDR H3；和
    (ii)轻链可变区，其包含根据Kabat编号的
    (d)氨基酸序列RSSTGAVTTSNYAN(SEQ ID NO:22)所示的CDR L1；
    (e)氨基酸序列GTNKRAP(SEQ ID NO:23)所示的CDR L2；和
    (f)氨基酸序列ALWYSNHWV(SEQ ID NO:24)所示的CDR L3；
    例如，(i)重链可变区，其包含SEQ ID NO:12的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
    (ii)轻链可变区，其包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，(i)重链可变区，其包含SEQ ID NO:12的序列，和(ii)轻链可变区，其包含SEQ ID NO:13的序列；
    (2)铰链区/间隔区，其选自
    (i)(G 4S) n、(SG 4) n或G 4(SG 4) n肽接头，其中“n”是1至4的整数，例如SEQ ID NO:14所示的序列；
    (ii)SEQ ID NO:18所示的CD8α铰链区序列或其具有1个氨基酸修饰的变体；
    (3)跨膜区(TM)，其选自SEQ ID NO:15所示的CD8跨膜结构域或其具有1个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其选自SEQ ID NO:16所示的4-1BB共刺激结构域或其具有1个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，其选自SEQ ID NO:17所示的CD3ζ信号传导结构域或其具有1个氨基酸修饰的变体；
    其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
  2. 根据权利要求1所述的药物组合，其中所述包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段包含重链可变区和轻链可变区，其中：
    (a)所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:25)所示的 CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:26)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列DRGDQILDV(SEQ ID NO:27)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISRYLN(SEQ ID NO:28)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列AASSLQS(SEQ ID NO:29)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQKYFDIT(SEQ ID NO:30)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (b)所述重链可变区包含根据Kabat编号的氨基酸序列NDVIS(SEQ ID NO:31)所示的CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列VIIPIFGIANYAQKFQG(SEQ ID NO:32)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列GRGYYSSWLHDI(SEQ ID NO:33)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的氨基酸序列QASQDITNYLN(SEQ ID NO:34)所示的CDR L1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；氨基酸序列DASNLET(SEQ ID NO:35)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和氨基酸序列QQAFDLIT(SEQ ID NO:36)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    其中所述氨基酸变化是氨基酸的添加、缺失或取代；
    例如，其中：
    (a)所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:25)所示的CDR H1；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:26)所示的CDR H2；和氨基酸序列DRGDQILDV(SEQ ID NO:27)所示的CDR H3；所述轻链可变区包含根据Kabat编号的氨基酸序列RASQSISRYLN(SEQ ID NO:28)所示的CDR L1；氨基酸序列AASSLQS(SEQ ID NO:29)所示的CDR L2；和氨基酸序列QQKYFDIT(SEQ ID NO:30)所示的CDR L3；
    (b)所述重链可变区包含根据Kabat编号的氨基酸序列NDVIS(SEQ ID NO:31)所示的CDR H1；氨基酸序列VIIPIFGIANYAQKFQG(SEQ ID NO:32)所示的CDR H2；和氨基酸序列GRGYYSSWLHDI(SEQ ID NO:33)所示的CDR H3；所述轻链可变区包含根据Kabat编号的氨基酸序列QASQDITNYLN(SEQ ID NO:34)所示的CDR L1；氨基酸序列DASNLET(SEQ ID NO:35)所示的CDR L2；和氨基酸序列QQAFDLIT(SEQ ID NO:36)所示的CDR L3；
    例如，其中：
    (a)重链可变区包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    (b)重链可变区包含SEQ ID NO:9的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:10的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，其中：
    (a)重链可变区包含SEQ ID NO:2的序列，且轻链可变区包含SEQ ID NO:3的序列；
    (b)重链可变区包含SEQ ID NO:9的序列，且轻链可变区包含SEQ ID NO:10的序列；
    例如，所述抗体是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是IgG1抗体；
    例如，所述抗原结合片段是Fab、Fab’、F(ab’) 2、Fv、单链Fv、单链Fab、双体抗体(diabody)。
  3. 根据权利要求1或2所述的药物组合，其中所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；和SEQ ID NO:6所示的轻链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:5所示的重链恒定区序列和SEQ ID NO:6所示的轻链恒定区序列。
  4. 根据权利要求1-3中任一项所述的药物组合，其中所述分子开关调控型CAR多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:11所示的信号肽序列，
    优选地，所述分子开关调控型CAR多肽具有SEQ ID NO:1所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
  5. 根据权利要求1-4中任一项所述的药物组合，其中编码CAR多肽的核酸分子是编码权利要求1-4中任一项所述药物组合中所述的CAR多肽的核酸分子，例如，是编码SEQ ID NO:1所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的核酸分子。
  6. 根据权利要求1-4中任一项所述的药物组合，其中所述载体是包含编码权利要求1-4中任一所述药物组合中所述的CAR多肽的核酸分子的载体，例如，所述载体选自DNA载体、 RNA载体、质粒、慢病毒载体、腺病毒载体或逆转录病毒载体。
  7. 根据权利要求1-4中任一项所述的药物组合，其中所述免疫效应细胞是自自体T细胞、NK细胞或同种异体T细胞、NK细胞制备的表达权利要求1-4中任一项所述的药物组合中的分子开关调控型CAR多肽的T细胞、NK细胞，例如，所述免疫效应细胞是自人PBMC分离的T细胞、NK细胞制备的表达权利要求1-4中任一项所述的药物组合中的分子开关调控型CAR多肽的T细胞、NK细胞。
  8. 根据权利要求1-7中任一项所述的药物组合，其中
    (i)将表达权利要求1-4中任一项所述药物组合中所述的CAR多肽的免疫效应细胞以1×10 6个细胞/kg体重－10×10 6个细胞/kg体重，例如1×10 6个细胞/kg体重、2×10 6个细胞/kg体重、3×10 6个细胞/kg体重、5×10 6个细胞/kg体重、7×10 6个细胞/kg体重、9×10 6个细胞/kg体重、10×10 6个细胞/kg体重的剂量以单次或多次静脉内施用至受试者；和
    (ii)将权利要求1-4中任一项所述药物组合中所述的P329G突变抗体以0.1-10mg/kg、优选地0.1mg/kg、0.3mg/kg、0.5mg/kg、1mg/kg、3mg/kg、5mg/kg、7mg/kg、9mg/kg、10mg/kg的剂量单元的形式，优选地胃肠外、更优选地静脉内施用至受试者。
  9. 根据权利要求8所述的药物组合，其中(i)和(ii)分开、同时或依次施用，例如，第一天施用(ii)，同一天静脉内施用(i)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者
    第一天施用(i)，第二天静脉内施用(ii)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；或者
    第一天施用(ii)，第二天静脉内施用(i)，然后按照一定频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)；
    例如，(i)和(ii)各施用一次，然后每3－4天一次、每周一次、每两周一次、每三周一次或每四周一次的给药频率多次施用(ii)，同时通过监测(i)的体内PK浓度和期望的治疗功效终点，确定是否多次施用(i)。
  10. 根据权利要求1-9中任一项所述的药物组合的用途，用于在受试者中治疗与BCMA相关的疾病，包括向受试者施用治疗有效量的权利要求1-9中任一项所述的药物组合，优选地，所述与BCMA相关的疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
  11. 根据权利要求1-9中任一项所述的药物组合在制备用于治疗与BCMA相关的疾病的药物中的用途，所述与BCMA相关的疾病是例如表达或过表达BCMA的癌症,所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
  12. 用于治疗与BCMA相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1-9中任一项所述的药物组合，所述疾病是例如表达或过表达BCMA的癌症,所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
  13. 成套药盒，其包含权利要求1-9中任一项所述的药物组合，优选地所述药盒为药物剂量单元形式。
  14. 一种药物复合物，其是一种由
    (i)表达权利要求1-4中任一项所述药物组合中所述的分子开关调控型CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)；和
    (ii)包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段(也称为P329G突变抗体)，例如，所述P329G突变抗体包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；结合而产生的复合物，
    其中，所述免疫效应细胞通过CAR多肽的胞外结构域中的人源化抗P329G突变scFv序列与所述P329G突变抗体的Fc结构域结合而产生所述复合物；
    例如，其中所述免疫效应细胞是自自体T细胞、NK细胞或同种异体T细胞、NK细胞制备的表达权利要求1-4中任一项所述药物组合中所述的分子开关调控型CAR多肽的T细胞、NK细胞，例如，所述免疫效应细胞是自人PBMC分离的T细胞、NK细胞制备的表达权利要求1-4中任一项所述药物组合中所述的分子开关调控型CAR多肽的T细胞、NK细胞；
    例如，其中P329G突变抗体是ADI-38497 PG Ab和/或ADI-38484 PG Ab。
  15. 根据权利要求14所述的药物复合物的用途，用于在受试者中治疗与BCMA相关的疾病，优选地，所述与BCMA相关的疾病是例如表达或过表达BCMA的癌症，所述癌症例如是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
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