WO2024251132A1 - 一种包含bcma pg car-t细胞制剂和pg抗体制剂的药物组合制剂及其用途 - Google Patents

一种包含bcma pg car-t细胞制剂和pg抗体制剂的药物组合制剂及其用途 Download PDF

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WO2024251132A1
WO2024251132A1 PCT/CN2024/097420 CN2024097420W WO2024251132A1 WO 2024251132 A1 WO2024251132 A1 WO 2024251132A1 CN 2024097420 W CN2024097420 W CN 2024097420W WO 2024251132 A1 WO2024251132 A1 WO 2024251132A1
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cells
preparation
antibody
car
seq
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French (fr)
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张利红
安娜
刘懿
马一冬
何冰
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Innovent Cells Pharmaceuticals Su Zhou Co Ltd
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Innovent Cells Pharmaceuticals Su Zhou Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a drug combination preparation comprising a BCMA PG CAR-T cell preparation and a PG antibody preparation and uses thereof, and belongs to the technical field of antibody drugs.
  • BCMA B cell maturation antigen
  • TNFRSF17 B cell maturation antigen
  • BCMA is a member of the tumor necrosis factor receptor superfamily (TNFRSF).
  • 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.
  • BCMA's ligands include B cell activating factor (BAFF) and B cell proliferation inducing ligand (APRIL), of which B cell proliferation inducing ligand (APRIL) binds to BCMA with higher affinity and promotes 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, i.e. plasma cells, and is not expressed in normal hematopoietic stem cells and non-hematopoietic tissues.
  • BCMA signaling is indispensable for the survival of long-term bone marrow plasma cells, but is not necessary for overall B cell homeostasis.
  • BCMA on the membrane surface can be cleaved and shed by ⁇ -secretase, and the resulting soluble BCMA (sBCMA) may reduce the signal transduction of BCMA on the membrane surface by blocking BAFF/APRIL ligand binding.
  • BCMA myeloma
  • BCMA chimeric antigen receptor T cell
  • CAR-T cells As a new type of genetically engineered, highly efficient, and precisely targeted tumor cell immunotherapy drug, CAR-T cells have shown great therapeutic potential in the treatment of hematological tumors. Multiple products have been approved for marketing at home and abroad, but breakthroughs are urgently needed in the treatment of solid tumors.
  • CAR-T Traditional chimeric antigen receptor T cells directly target the surface antigens of tumor cells through chimeric antigen receptor (CAR) molecules on T cells, thereby achieving the purpose of identifying and killing tumors, wherein the N-terminus of the chimeric antigen receptor contains an antigen-binding domain that recognizes the antigen, for example, a single-chain antibody fragment (scFv) targeting the antigen.
  • scFv single-chain antibody fragment
  • CAR-T cells Since most of the tumor antigens targeted by CAR-T cells are not tumor-specific, in addition to being expressed on tumor cells, these tumor antigens are often expressed at low levels in many normal tissues, especially important tissues and organs. The recognition and killing of these normal tissues by CAR-T cells leads to "on-target/off tumor” toxicity, which may cause serious toxic side effects.
  • Drug stability is one of the important indicators to ensure drug effectiveness and safety. Obtaining a formulation formula that gives drugs good stability is a key condition for drugs to maintain their safety and effectiveness during their shelf life. In the prior art, there is a lack of formulation formulas for drug combinations containing PG CAR-T cells targeting BCMA and PG antibodies that can ensure long-term stable storage of the drug combination (e.g., 24 months) and have simple ingredients and ease of use.
  • the present invention provides a drug combination preparation comprising a BCMA PG CAR-T cell preparation and a PG antibody preparation and uses thereof.
  • the inventors have conducted in-depth research. Through research, the inventors have developed a group of "molecular switch” regulated chimeric antigen receptor cells, and by using Pro329Gly (the 329th proline of the antibody Fc segment according to the EU numbering is mutated to glycine, abbreviated as P329G or PG) mutant antibodies as "molecular switches” or adaptors, such CAR molecules are constructed, which can specifically bind to antibodies containing P329G mutant Fc domains without binding to antibodies that do not contain P329G mutant Fc domains (also referred to as "wild-type antibodies” herein), thereby, by combining immune effector cells (e.g., T cells) expressing the CAR with monoclonal antibodies of recombinant anti-BCMA molecules as "molecular switches", it is used to treat BCMA-related diseases in subjects, such as cancers that express or overexpress BCMA, such as relapse
  • the drug combination of the present invention includes two components: BCMA-specific P329G antibody and P329G CAR-T cells.
  • the BCMA-specific P329G antibody recognizes tumor cells expressing BCMA
  • the P329G CAR-T cells are redirected to tumor cells by recognizing the Fc domain of the P329G antibody, producing tumor recognition and killing effects (see Figure 1B).
  • the P329G antibody acts as a bridge connecting P329G CAR-T cells and tumor cells, plays the role of a "molecular switch", and regulates the activity of P329G CAR-T cells.
  • the inventors conducted forced tests at room temperature (22-26°C) and storage stability experiments in liquid nitrogen vapor phase ( ⁇ -150°C) to investigate the effects of different excipient contents on the viability and phenotype of chimeric antigen receptor cells, and optimized and screened out formulations that are beneficial to the stability of chimeric antigen receptor T cells.
  • the detection items during this study mainly include: cell viability, cell agglomeration rate, CD3 + CAR + and T cell phenotype.
  • the inventors studied the types and addition ratios of various excipients in the drug combination preparation containing BCMA PG CAR-T cells and PG antibodies, and screened out formulation prescriptions that are beneficial to the stability of chimeric antigen receptor T cells and formulation prescriptions that are beneficial to the stability of recombinant anti-BCMA monoclonal antibodies, thereby completing the present invention.
  • the first aspect of the present invention provides a pharmaceutical combination preparation, the preparation comprising preparation A and preparation B;
  • the preparation A comprises: a therapeutically effective amount of immune effector cells expressing a molecular switch-regulated chimeric antigen receptor (CAR) polypeptide and a pharmaceutical excipient I,
  • CAR chimeric antigen receptor
  • the pharmaceutical excipient I comprises: compound electrolyte injection, human serum albumin and CS10;
  • the compound electrolyte injection is Bomel A compound electrolyte injection;
  • the preparation B comprises: a therapeutically effective amount of an antibody or antigen-binding fragment that specifically binds to BCMA molecules and comprises a P329G mutation and a pharmaceutical excipient II,
  • the P329G mutated antibody that specifically binds to the BCMA molecule comprises a mutated Fc domain, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G), and the Fc ⁇ receptor binding of the mutated Fc domain is reduced compared to the Fc ⁇ receptor binding of the unmutated parent antibody Fc domain;
  • the pharmaceutical excipient II comprises: a pH regulator, an osmotic pressure regulator and a surfactant;
  • the pH regulator is histidine and/or L-histidine hydrochloride
  • the osmotic pressure regulator is sorbitol
  • the surfactant is polysorbate 80.
  • the pharmaceutical combination preparation of the first aspect of the present invention, wherein the molecular switch-regulated chimeric antigen receptor (CAR) polypeptide comprises:
  • a humanized anti-P329G mutant scFv sequence wherein the scFv sequence comprises the following sequence that can specifically bind to the Fc domain of an antibody comprising the P329G mutation, but cannot specifically bind to the Fc domain of an unmutated parent antibody:
  • CDR H2 represented by the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO:17);
  • CDR L1 represented by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19);
  • CDR L2 represented by the amino acid sequence GTNCRAP (SEQ ID NO: 20).
  • CDR L3 represented by the amino acid sequence ALWYSNHWV (SEQ ID NO: 21);
  • the (i) heavy chain variable region comprises the sequence of SEQ ID NO: 9, and (ii) light chain variable region comprises the sequence of SEQ ID NO: 10;
  • TM transmembrane region
  • CSD co-stimulatory signaling domain
  • a stimulatory signaling domain which is a CD3 ⁇ signaling domain shown in SEQ ID NO: 14;
  • the molecular switch-regulated CAR polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, and the nucleic acid molecule encoding the molecular switch-regulated CAR polypeptide comprises a nucleotide sequence as shown in SEQ ID NO: 31;
  • the immune effector cells expressing the molecular switch-regulated chimeric antigen receptor (CAR) polypeptide are T cells; preferably, the T cells are autologous T cells or allogeneic T cells; more preferably, the immune effector cells are prepared by isolating T cells from human PBMCs;
  • the immune effector cells expressing the molecular switch-regulated chimeric antigen receptor (CAR) polypeptide are HuR968B CAR-T cells.
  • the pharmaceutical combination preparation described in the first aspect of the present invention wherein the cell density of the immune effector cells expressing the molecular switch-regulated chimeric antigen receptor (CAR) polypeptide in the preparation A is 1E6 cells/mL to 1E8 cells/mL;
  • the contents of the components in the pharmaceutical excipient I in the preparation A are: 10.5% to 50.5% (v/v) of compound electrolyte injection, Human serum albumin 14.5 ⁇ 24.5% (v/v), CS10 50% ⁇ 70% (v/v);
  • the contents of the components in the pharmaceutical excipient I in the preparation A are respectively CS10 50.0% (v/v), compound electrolyte injection 30.5% (v/v), human serum albumin 19.5%;
  • the compound electrolyte injection is Bomel A compound electrolyte injection.
  • the drug combination preparation described in the first aspect of the present invention wherein the antibody or antigen-binding fragment that specifically binds to the BCMA molecule comprises 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:22) according to Kabat numbering; CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO:23); and CDR H3 shown in the amino acid sequence DRGDQILDV (SEQ ID NO:24); the light chain variable region comprises CDR L1 shown in the amino acid sequence RASQSISRYLN (SEQ ID NO:25) according to Kabat numbering, CDR L2 shown in the amino acid sequence AASSLQS (SEQ ID NO:26); and CDR L3 shown in the amino acid sequence QQKYFDIT (SEQ ID NO:27);
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 2
  • the light chain variable region comprises the sequence of SEQ ID NO: 3
  • mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody
  • mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody
  • mutant Fc domain is a mutant Fc domain of an IgG1 antibody
  • the antibody that specifically binds to the BCMA molecule with the P329G mutation is ADI-38497 PG Ab.
  • the content of the antibody or antigen-binding fragment that specifically binds to the BCMA molecule comprising the P329G mutation in the preparation B is 10 to 30 mg/mL; in some preferred embodiments, the content of the antibody or antigen-binding fragment that specifically binds to the BCMA molecule comprising the P329G mutation in the preparation B is 20.0 mg/mL;
  • the contents of the components in the pharmaceutical excipient II in the preparation B are: 10-25 mM pH regulator, 40-60 mg/mL osmotic pressure regulator and 0.1-0.3 mg/mL surfactant;
  • the contents of the components in the pharmaceutical excipient II in the preparation B are: 10 mM pH regulator, 50 mg/mL osmotic pressure regulator and 0.2 mg/mL surfactant;
  • the contents of the components in the pharmaceutical excipient II in the preparation B are: 0.76 g/ml histidine, 1.08 mg/mL L-histidine hydrochloride, 50.00 mg/mL sorbitol, and 0.2 mg/mL polysorbate 80;
  • the pH of the preparation B is 5.0-7.0, and preferably the pH of the preparation B is 6.0.
  • the pharmaceutical combination preparation of the first aspect of the present invention wherein the preparation A and preparation B are administered separately, simultaneously or sequentially.
  • the pharmaceutical combination preparation described in the first aspect of the present invention wherein the administration is parenteral administration; preferably, the administration is intravenous administration.
  • the second aspect of the present invention provides a use of the drug combination preparation according to the first aspect of the present invention in the preparation of a drug for treating and/or preventing cancer; preferably, the cancer is a cancer that expresses or overexpresses BCMA; more preferably, the cancer is relapsed/refractory multiple myeloma (RRMM).
  • the cancer is a cancer that expresses or overexpresses BCMA; more preferably, the cancer is relapsed/refractory multiple myeloma (RRMM).
  • the third aspect of the present invention provides a method for treating and/or preventing cancer, comprising administering to a subject in need thereof a drug combination preparation as described in the first aspect of the present invention; preferably, the cancer is a cancer that expresses or overexpresses BCMA; more preferably, the cancer is relapsed/refractory multiple myeloma (RRMM).
  • RRMM relapsed/refractory multiple myeloma
  • the method of the third aspect of the present invention is described, wherein the administration is parenteral administration; preferably, the administration is intravenous administration.
  • the final formulation of the stable recombinant anti-BCMA monoclonal antibody was determined to be: 20.0 mg/ml recombinant anti-B cell maturation antigen (BCMA) monoclonal antibody, 0.76 mg/ml histidine, 1.08 mg/ml L-histidine hydrochloride, 50.00 mg/ml sorbitol, 0.2 mg/ml polysorbate 80, pH 6.0.
  • BCMA recombinant anti-B cell maturation antigen
  • FIG1A shows the expression of CAR in CD3 + cells, CD4 + , and CD8 + T cell subsets after T cells were transduced with HuR968B and Blue21 CAR 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 antibodies.
  • SP represents signal peptide
  • TMD represents transmembrane domain
  • SSD represents costimulatory domain
  • the extracellular domain comprises an antigen binding portion that can specifically bind to a mutant Fc domain containing a P329G mutation
  • the antigen binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL).
  • Figure 2 shows the binding activity of P329G BCMA antibody to BCMA-expressing positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1.
  • FIG. 3A shows the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human H929 tumor cells that highly express BCMA.
  • cCAR-T represents traditional CAR-T, i.e., Blue21 CAR-T.
  • Figure 3B shows the changes in mouse body weight when immunodeficient tumor-bearing mice were treated with different doses of PG antibody combined with PG CAR-T cells inoculated subcutaneously with human H929 tumor cells that highly expressed BCMA.
  • Figure 3C shows the expansion of PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human H929 tumor cells that highly express BCMA, when treated with different doses of PG antibodies combined with PG CAR-T cells.
  • FIG. 4A shows the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human L363 low-expressing BCMA tumor cells.
  • cCAR-T represents traditional CAR-T, i.e., Blue21 CAR-T.
  • Figure 4B shows the changes in body weight of mice when they were treated with different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human L363 low-expressing BCMA tumor cells.
  • Figure 4C shows the expansion of PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human L363 low-expressing BCMA tumor cells and treated with different doses of PG antibodies combined with PG CAR-T cells.
  • Figure 5A shows the therapeutic effect of PG antibody combined with different doses of PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human H929 tumor cells.
  • Figure 5B shows the expansion of PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human H929 tumor cells when PG antibodies were combined with different doses of PG CAR-T cells for treatment.
  • FIG6 shows the “predicted value - actual value” of the survival rate at 0 h after resuscitation.
  • Figure 7 shows the “predicted value - actual value” of the survival rate 1 hour after resuscitation.
  • FIG8 shows the “predicted value - actual value” of the survival rate 2 hours after resuscitation.
  • FIG. 9 shows the “predicted value - actual value” of the survival rate 4 hours after resuscitation.
  • FIG. 10 shows the “predicted value - actual value” of the survival rate 8 hours after resuscitation.
  • FIG11 shows the importance of “alive rate 0h”, “alive rate 1h”, “alive rate 2h”, “alive rate 4h”, and “alive rate 8h” in the prediction profiler; the desirability function and the optimal condition.
  • Figure 12A shows the trend of the charge variant-acidic component (40°C, iCIEF method) in the prescription screening.
  • Figure 12B shows the trend of the charge variant-main component (40°C, iCIEF method) in the prescription screening.
  • Figure 12C shows the trend of the purity (40°C, SEC-HPLC method) in the prescription screening.
  • BCMA and “B cell maturation antigen” are used interchangeably and include variants, isoforms, species homologs, and analogs of human BCMA that have at least one identical epitope with BCMA (e.g., human BCMA).
  • BCMA protein may also include fragments of BCMA, such as the extracellular domain and fragments of the extracellular domain, such as fragments that retain the ability to bind to any of the antibodies of the present invention.
  • BCMA antibody As used herein, the terms “BCMA antibody”, “antibody against BCMA”, “antibody that specifically binds to BCMA”, “antibody that specifically targets BCMA”, “antibody that specifically recognizes BCMA” are used interchangeably and refer to an antibody that can specifically bind to B cell maturation antigen (BCMA).
  • antibody is used in the broadest sense herein to refer to a protein comprising an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, complete antibodies, and antibody fragments.
  • the antibody of the present invention is a single domain antibody or a heavy chain antibody.
  • Antibody fragment or "antigen-binding fragment” are used interchangeably herein and refer to a molecule different from an intact antibody, which comprises a portion of an intact antibody and binds to 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 continuously connected by means of a flexible short polypeptide linker and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • 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 the polypeptide), and the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.
  • CDR region or “CDR” or “hypervariable region” is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points").
  • the CDRs are primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2 and CDR H3, while the CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2 and CDR L3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the 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 the variability of antibody sequences (Kabat et al., Sequential ences of Proteins of Immunological Interest, 4th ed., U.S.
  • CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention).
  • a reference CDR sequence e.g., any of the CDRs exemplified in the present invention.
  • the present invention when referring to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), it refers to the numbering position according to the Kabat numbering system.
  • CDR is different from antibody to antibody, only a limited number of amino acid positions in CDR are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein.
  • the amino acid residues of the minimum 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 a portion thereof is changed, replaced or exchanged so that the antigen binding site is linked to a constant region of a different or altered class and/or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), etc. that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced antigenicity in humans as compared to the original mouse antibody.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region.
  • the human IgG heavy chain Fc region is generally defined as a segment from the amino acid residue at the Cys226 or Pro230 position 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.
  • a complete antibody composition may include an antibody group in which all K447 residues are eliminated, an antibody group in which no K447 residue is eliminated, or an antibody group in which antibodies with K447 residues and antibodies without K447 residues are mixed.
  • the Fc region of an immunoglobulin comprises two constant domains, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, CH2, CH3, and CH4.
  • IgG binding to Fc ⁇ receptors or C1q depends on residues located in the hinge region and the 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. It has been shown that substitution of residues 233-236 in human IgG1 and IgG2 and substitution of residues 327, 330 and 331 in human IgG4 can significantly 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).
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementary determining regions
  • binding means that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry 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 thereby mediates a signal transduction event, such as, but not limited to, signal transduction via a TCR/CD3 complex.
  • a stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation may mediate the expression of certain molecular changes, such as downregulation of TGF- ⁇ and/or reorganization of cytoskeletal structure, etc.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated, for example, by the binding of the TCR/CD3 complex to the MHC molecule loaded with a peptide and leads to mediating a T cell response, including but not limited to proliferation, activation, differentiation, etc.
  • the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, a primary signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as the protein provided by GenBank Accession No. BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as the amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain that are sufficient to functionally propagate the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to residue 164 of GenBank Accession No. BAG36664.1 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) that are functional homologs thereof.
  • the "CD3 ⁇ stimulatory signaling domain” is the sequence provided in SEQ ID NO:14 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 co-stimulatory response of the cell (e.g., but not limited to, proliferation).
  • 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), activated NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.
  • a "costimulatory molecule” is 4-1BB (i.e., CD137).
  • a co-stimulatory signaling domain refers to the intracellular portion of a co-stimulatory molecule.
  • 4-1BB refers to a TNFR superfamily member having an amino acid sequence provided as GenBank Accession No. AAA62478.2 or 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 No. AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • the "4-1BB co-stimulatory domain” is a sequence provided as SEQ ID NO: 13 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • signaling pathway refers to the biochemical relationships between multiple signaling molecules that play a role in propagating a signal from one part of a cell to another part of the cell.
  • cytokine is a generic term for proteins released by one cell population that act as intercellular mediators on another cell.
  • cytokines include lymphokines, monokines, interleukins (ILs), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including gamma-interferon.
  • an “isolated” antibody is one that has been separated from the components of its natural environment.
  • the antibodies of the invention are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed-phase HPLC
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • An isolated nucleic acid encoding an antibody of the invention refers to one or more nucleic acid molecules that encode a chain of an antibody of the invention or a fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.
  • Immuno effector function refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack on a target cell.
  • an immune effector function or response refers to a T cell or NK cell property that promotes killing of a target cell or inhibits growth or proliferation of a target cell.
  • 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 a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.
  • T cell activation refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • the chimeric antigen receptor of the present invention is 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 Retroviridae family. 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 all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, but are not limited to, those 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, exacerbated 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 rats).
  • 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 rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • tumor and cancer are used interchangeably herein and encompass both solid tumors and liquid tumors.
  • cancer and “cancerous” refer to the physiological condition in mammals in which cell growth is unregulated.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • Tumor immune escape refers to the process by which a tumor evades immune recognition and clearance.
  • tumor immunity is “treated” when such escape is weakened and the tumor is recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • half effective concentration refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% 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 Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).
  • pharmaceutically acceptable excipient refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, buffer or stabilizer, etc., which is administered together with the active substance.
  • adjuvant eg, Freund's adjuvant (complete and incomplete)
  • excipient eg, buffer or stabilizer, etc.
  • treat refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • the desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the antibody molecules of the present invention are used to delay the development of the disease or to slow the progression of the disease.
  • the term "effective amount” refers to an amount or dosage of an antibody or composition of the invention that produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses.
  • the effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of the mammal; body weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.
  • a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time.
  • the therapeutically effective amount of an antibody or antibody fragment or composition thereof may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious 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 at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%.
  • a measurable parameter e.g., tumor growth rate, tumor volume, etc.
  • the ability of a compound to inhibit a measurable parameter can be evaluated in an animal model system that is predictive of efficacy in human tumors.
  • drug combination refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition.
  • non-fixed combination means that the active ingredients (e.g., (i) P329G CAR-T cells, and (ii) P329G mutant antibodies against BCMA) are administered to a subject as separate entities simultaneously, without specific time limits, or at the same or different time intervals, sequentially, 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 are each administered to a patient simultaneously in the form of a specific single dose.
  • non-fixed combination means that the combination of the 329G mutant antibody against BCMA and the P329G CAR-T cells of the present invention are administered to a patient simultaneously, in parallel or sequentially as separate entities, without specific dosage and time limits, wherein such administration provides a therapeutically effective level of the drug combination of the present invention in the patient.
  • the drug combination is a non-fixed combination.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated 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 operatively attached. 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 offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity screened or selected in the initially transformed cells.
  • Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells.
  • Host cells include cultured cells, and also include cells inside transgenic animals, transgenic plants, or cultured plant tissues 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, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, 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 tumor samples.
  • treat or “treating” with reference to a disease means to alleviate the disease (ie, slow or arrest or reduce the development of the disease or at least one clinical symptom thereof), prevent or delay the onset or development or progression of the disease.
  • cell viability refers to the percentage of live cells in a total cell population.
  • cell clumping rate refers to the degree of cell aggregation and is an important indicator for evaluating the safety of cell therapy products.
  • MOI refers to the ratio of the number of viruses (bacteriophages) to the number of bacteria when the virus (bacteriophage) infects the bacteria, that is, the average number of viruses (bacteriophages) that infect each bacterium.
  • CE-SDS refers to the capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) with ultraviolet detection method, which quantitatively determines the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions by capillary electrophoresis.
  • SEC-HPLC refers to size exclusion chromatography-high performance liquid chromatography detection method.
  • main component refers to the target product. The higher the purity, the more target product is obtained.
  • charge variant is due to the fact that protein products undergo post-translational modification and degradation events in cells, resulting in heterogeneity in biophysical properties and differences in the charges they carry, hence the name charge variant.
  • acidic charge variant refers to charge variants that are earlier or later than the main peak in different analytical methods. There are many reasons for the formation of acidic charge variants, among which there may be modifications that affect the biological activity of the product. Therefore, the level of acidic charge variants is generally used as an indicator of the quality of antibody products.
  • CAR Molecular switch regulated chimeric antigen receptor
  • the present invention relates to a chimeric antigen receptor polypeptide capable of specifically binding to a mutant Fc domain of an antibody against a BCMA molecule.
  • the chimeric antigen receptor of the present invention comprises a humanized anti-P329G mutant scFv sequence, and the scFv sequence is capable of specifically binding to an antibody Fc domain comprising a P329G mutation, but is not specifically binding to an unmutated parent antibody Fc domain.
  • the binding of an antibody Fc domain comprising a P329G mutation to an Fc receptor is reduced.
  • the recombinant CAR construct of the present invention comprises a sequence encoding CAR, wherein CAR comprises a humanized anti-P329G mutation scFv sequence, and the scFv sequence specifically binds to the antibody Fc domain of the P329G mutation.
  • the scFv sequence in the CAR construct of the present invention comprises the following sequence:
  • CDR H2 represented by the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO:17);
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19);
  • CDR L2 represented by the amino acid sequence GTNCRAP (SEQ ID NO: 20).
  • the scFv can be connected to a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO:8, and the scFv can be connected to an optional hinge region sequence as provided in SEQ ID NO:11, a transmembrane region as provided in SEQ ID NO:12, a co-stimulatory signal domain as provided in SEQ ID NO:13 and an intracellular stimulatory signal domain comprising SEQ ID NO:14 or its variants at the C-terminus, for example, wherein the domains are adjacent to each other and are in the same reading frame to form a single fusion protein.
  • the scFv domain comprises (i) a heavy chain variable region comprising the sequence of SEQ ID NO:9, and (ii) a light chain variable region comprising the sequence of SEQ ID NO:10;
  • the scFv domain comprises (i) a heavy chain variable region set forth in SEQ ID NO: 9 and (ii) a light chain variable region set forth in SEQ ID NO: 10.
  • 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 chain variable region and the heavy chain variable region of the scFv can be, for example, 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 present invention comprises a signal peptide sequence, a humanized anti-P329G mutant scFv sequence, a hinge region/spacer, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular stimulatory 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 a CAR of the present invention, for example, comprising a nucleic acid molecule encoding an amino acid sequence shown in SEQ ID NO: 1.
  • the CAR construct encoding the present invention can be obtained using recombinant methods known in the art.
  • the target nucleic acid can be produced synthetically rather than by genetic recombination methods.
  • the present invention includes retroviral and lentiviral vector constructs that express CARs that can be directly transduced into cells.
  • the nucleic acid sequence of the CAR construct of the present 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.
  • the present invention provides nucleic acid molecules encoding CAR constructs described herein.
  • the nucleic acid molecule is provided as a DNA construct.
  • the present invention also provides a vector inserted with a CAR construct of the present invention.
  • a vector By effectively connecting a nucleic acid encoding a CAR polypeptide to a promoter and incorporating the construct into an expression vector, expression of a natural or synthetic nucleic acid encoding CAR is achieved.
  • the vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.
  • the present invention provides methods for expressing a CAR construct of the present invention in a mammalian immune effector cell (e.g., a mammalian T cell) and the immune effector cells (e.g., T cells) produced thereby.
  • a mammalian immune effector cell e.g., a mammalian T cell
  • the immune effector cells e.g., T cells
  • the present invention provides an antibody that binds to 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 by the amino acid sequence SSSYYWT (SEQ ID NO:22) according to the Kabat numbering; CDR H2 shown by the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO:23); and CDR H3 shown by the amino acid sequence DRGDQILDV (SEQ ID NO:24); the light chain variable region comprises CDR L1 shown by the amino acid sequence RASQSISRYLN (SEQ ID NO:25) according to the Kabat numbering; CDR L2 shown by the amino acid sequence AASSLQS (SEQ ID NO:26); and CDR L3 shown by the amino acid sequence QQKYFDIT (SEQ ID NO:27).
  • the antibodies of the invention that bind to BCMA molecules bind to mammalian BCMA, such as human, cynomolgus monkey, mouse, rat, and rabbit BCMA.
  • the antibodies of the invention that bind to BCMA molecules have one or more of the following properties:
  • BCMA-positive cancer cells can be killed by antibody-dependent cellular cytotoxicity and/or antibody-dependent cellular phagocytosis (ADCP).
  • ADCP antibody-dependent cellular cytotoxicity and/or antibody-dependent cellular phagocytosis
  • an antibody that binds to a BCMA molecule of the present invention comprises a heavy chain variable region and a light chain variable region that specifically bind to BCMA, 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 antibody that binds to the BCMA molecule provided herein comprises a mutant Fc domain, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G), and the Fc ⁇ receptor binding of the mutant Fc domain is reduced compared to the Fc ⁇ receptor binding of the unmutated parent antibody 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, 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 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 (antibody Fc segment according to the EU numbering of the 329th proline mutated to glycine, abbreviated as P329G) mutant antibody as a safety switch in the CAR treatment of the present invention.
  • P329G mutant antibody In the absence of the P329G mutant antibody, the CAR activity of the present invention is turned off; in the presence of the P329G mutant antibody, 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 regulated by the P329G mutant antibody.
  • the present invention provides a drug 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) a P329G mutant antibody that specifically binds to the BCMA molecule.
  • the immune effector cells are T cells expressing the molecular switch-regulated CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells, for example, the immune effector cells are T cells expressing the molecular switch-regulated CAR polypeptide of the present invention prepared from T cells isolated from human PBMC.
  • the P329G mutant antibody is ADI-38497 PG Ab.
  • the present invention provides a drug combination comprising (i) a nucleic acid molecule encoding a molecular switch-regulated CAR polypeptide of the present invention or a vector comprising the nucleic acid component; and (ii) a P329G mutant antibody that specifically binds to a BCMA molecule.
  • the pharmaceutical combination of the present invention optionally further comprises a pharmaceutically acceptable adjuvant of a suitable formulation.
  • a pharmaceutically acceptable adjuvant of a suitable formulation can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).
  • Pharmaceutically acceptable adjuvants can be exemplified by surfactants, excipients, colorants, spices, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, flavoring agents, etc.
  • other commonly used carriers can also be appropriately used, for example, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, Carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardened castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts and the like can be used as carriers, but are not limited thereto.
  • the drug combination of the present invention is used to treat a BCMA-related disease, such as a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma (RRMM).
  • a BCMA-related disease such as a cancer that expresses or overexpresses BCMA, such as relapsed/refractory multiple myeloma (RRMM).
  • RRMM relapsed/refractory multiple myeloma
  • Example 1 CAR gene synthesis, construction of viral expression vector, preparation of P329G CAR-T cells and detection of CAR expression
  • a P329G CAR molecule (SEQ ID NO:1), also known as HuR968B CAR, was constructed, which was composed of a fusion of the signal peptide (SP) shown in SEQ ID NO:8, a specific single-chain antibody fragment recognizing the P329G antibody (VH-linker-VL, having the VH shown in SEQ ID NO:9, the linker sequence shown in SEQ ID NO:30, and the VL shown in SEQ ID NO:10), the G4S hinge region shown in SEQ ID NO:11, the CD8 transmembrane domain (CD8TM) shown in SEQ ID NO:12, the 41BB co-stimulatory domain (41BB-CSD) shown in SEQ ID NO:13, and the CD3 ⁇ molecule intracellular activation domain (CD3 ⁇ SSD) shown in SEQ ID NO:14.
  • SP signal peptide
  • VH-linker-VL a specific single-chain antibody fragment recognizing the P329G antibody
  • Blue21 CAR (SEQ ID NO:7) directly targeting BCMA was constructed and used as a control. From the N-terminus to the C-terminus, Blue21 CAR contains the signal peptide shown in SEQ ID NO:8, an anti-BCMA single-chain antibody (from clone 11D53), the hinge region of the CD8 ⁇ molecule shown in SEQ ID NO:15, and the CD8 transmembrane domain shown in SEQ ID NO:12, the 4-1BB co-stimulatory domain shown in SEQ ID NO:13, and the CD3 ⁇ chain intracellular activation domain shown in SEQ ID NO:14.
  • SEQ ID NO:7 directly targeting BCMA was constructed and used as a control. From the N-terminus to the C-terminus, Blue21 CAR contains the signal peptide shown in SEQ ID NO:8, an anti-BCMA single-chain antibody (from clone 11D53), the hinge region of the CD8 ⁇ molecule shown in SEQ ID NO:15, and the CD8 transmembrane domain shown in S
  • the above-mentioned DNA fragments encoding the CAR polypeptide were respectively inserted into the downstream of the EF1 ⁇ promoter of the pRK lentiviral expression vector (which was modified by replacing the promoter and the resistance gene of the pRRLSIN.cPPT.PGK-GFP.WPRE vector (Addgene, 12252, purchased from BioWind)), replacing the EGFR sequence in the vector, and obtaining the CAR expression plasmids pRK-HuR968B and pRK-Blue21.
  • the CAR expression plasmid prepared in Example 1-1 was transfected into Lenti-X-293T cells (Takara) at a mass ratio of 3:3:2:2 using the PEI transfection method with structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) and envelope plasmid pMD2G (Addgene, 12259, purchased from Biowind) . After 16 hours of transfection, the medium was replaced with fresh DEME medium containing 2% fetal bovine serum (FBS). After 48 hours of continuous culture, the cell supernatant was collected and centrifuged to remove cell debris.
  • FBS fetal bovine serum
  • PEG8000 was added and incubated at 4°C for 16-64 hours to concentrate the lentivirus. After centrifugation again, the supernatant was removed and the lentivirus precipitate was resuspended in T cell culture medium to obtain a lentivirus concentrate, which was packaged and frozen at -80°C.
  • PBMC cells from multiple donors were obtained from ORiCELLS, and the specific information is shown in Table 1 below:
  • the resuscitated PBMCs of each donor were sorted using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to obtain T cells.
  • the T cells were resuspended to a certain density using T cell culture medium and activated by adding TransAct (Miltenyi, 130-111-160).
  • the lentivirus was a lentivirus encoding P329G CAR (SEQ ID NO: 1) or a control traditional CAR (SEQ ID NO: 7)
  • CAR-T cells were taken, washed once with FACS buffer (PBS + 2% FBS), resuspended and added with FACS buffer containing LIVE/DEAD Fixable Dead Cell Stain, stained at room temperature for 10-15 min, washed twice, and added with PerCP-Cy5.5-CD3, BUV805-CD, Biotin-F(ab') 2 Fragment goat anti-human IgG (Jackson ImmunoResearch, 109-066-006; PG CAR detection) or Biotin-F(ab') 2 Fragment goat anti-mouse IgG (Jackson ImmunoResearch, 115-066-006; Blue 21CAR detection) antibody combination, stained at 4°C for 30-45 min, washed twice, and then APC-Streptavidin was added and stained at 4°C for 30-45 min; the cells were washed twice and resuspended with FACS buffer and detected by flow cytometry.
  • FACS buffer PBS + 2% FBS
  • FIG1A shows the expression of CAR in CD3 + cells, CD4 + , and CD8 + T cell subsets after T cells were transduced with the two CARs constructed in Example 1-1, respectively.
  • the results show that the positive rate of CAR expression in these transduced T cells is about 18% to 29%.
  • the heavy and light chain variable region sequences of the BCMA parent antibody ADI-34861 (VH shown in SEQ ID NO: 28 and VL shown in SEQ ID NO: 29, respectively) were obtained from the international application number PCT/CN2019/074419 (BCMA antibody related patent).
  • the heavy and light chain variable region sequences of ADI-38497 (SEQ ID NO: 2 and SEQ ID NO: 3) were obtained by performing CDR region mutations on the basis of the parent antibody ADI-34861. Compared with the corresponding parent antibody, the affinity of the mutated antibody was significantly improved.
  • Table 2 The specific experimental data are shown in Table 2 below.
  • the light and heavy chain variable region sequences of the BCMA antibody clone J6M0 of GSK were obtained from patent US9273141B2 as a control antibody (GSK IgG).
  • the light and heavy chain variable region sequences of GSK IgG and ADI-38497 antibodies were synthesized by whole gene and loaded into pcDNA3.4 expression vectors (purchased from Shanghai Boying) containing WT human IgG1 heavy chain constant region (SEQ ID NO: 4) or human IgG1 heavy chain constant region containing P329G point mutation (SEQ ID NO: 5) and ⁇ light chain constant region (SEQ ID NO: 6).
  • the light and heavy chain expression vectors were co-transfected into HEK293 cells by PEI at a molar ratio of 2:3, and the culture supernatant was collected after 5-7 days of culture.
  • the supernatant culture medium containing antibodies was purified by Protein A column in one step, and then dialyzed with PBS.
  • the concentration was detected by reading the absorbance value at 280nm using NanoDrop instrument, and the sample purity was detected by SDS-PAGE and SEC-HPLC methods.
  • GSK WT antibody, GSK PG antibody; ADI-38497 WT antibody, ADI-38497 PG antibody were obtained.
  • Antibodies having the heavy chain variable region (SEQ ID NO: 2) and light chain variable region (SEQ ID NO: 3) sequences of the BCMA antibody clone ADI-38497 are also referred to as ADI-38497 antibodies in this application, including ADI-38497 PG antibodies and ADI-38497 WT antibodies (i.e., BCMA-specific WT antibodies).
  • Figure 2 shows the binding activity of different concentrations of P329G BCMA antibody with BCMA-expressing positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1
  • 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 Pronosai Life Science 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).
  • the ADI-38497 PG antibody can bind to BCMA-expressing positive tumor cells in a concentration-dependent manner.
  • BCMA-expressing positive tumor cells MM.1s cells expressed BCMA at the highest level, RPMI8226, U266 and H929 cells expressed BCMA at a moderate level, and L363 and AMO1 cells expressed BCMA at a low level.
  • Example 3 Effect of PG CAR-T cells combined with different doses of ADI-38497 PG antibody (hereinafter referred to as PG antibody or PG Ab) against BCMA-overexpressing tumors in vivo
  • H929 cells were resuspended in 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice aged 4-6 weeks, weighing 15-17g, female
  • NOG mice were shaved on the right back and subcutaneously injected with H929 cell suspension, with an injection volume of 0.2mL/mouse, that is, the inoculation amount was 1 ⁇ 10 6 cells/mouse.
  • mice with tumor volumes of 50.82-104.36mm 3 were divided into 7 groups, namely PBS vehicle group, PG Ab group, PG CAR-T group only, traditional CAR-T group, 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.
  • the antibodies were prepared at concentrations of 0.3 mg/mL, 0.1 mg/mL, and 0.03 mg/mL, respectively.
  • the antibodies were administered on the 7th day, with a volume of 10 mL/kg per mouse, and the administration frequency was once a week, and the administration method was intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended in 1 ⁇ PBS to prepare a cell suspension of 25 ⁇ 10 6 /mL of CAR + cells.
  • 0.2 mL/mouse of the cell suspension was injected into the tail vein, that is, 5 ⁇ 10 6 /mouse of CAR + cells were infused.
  • the weight of the mice, the maximum long axis (L) and the maximum wide axis (W) of the tumor tissue were monitored twice a week.
  • FIG3A shows the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human H929 tumor cells that highly express BCMA.
  • the results showed that in the BCMA high-expressing tumor model, the administration of PG CAR-T cells alone did not produce obvious anti-tumor effects, the administration of PG antibodies alone produced a certain anti-tumor effect, and only mice treated with PG CAR-T cells and PG antibodies simultaneously produced significant anti-tumor effects, and showed an antibody dose-dependent effect.
  • TGI tumor growth inhibition rates
  • Figure 3B shows the weight changes of mice in this experiment.
  • the results showed that the weight of mice treated with PG CAR-T cells combined with PG antibodies remained stable after treatment, and the average weight increased by 5.2%, 3.0%, and 7.6% after treatment with 0.3 mg/kg, 1 mg/kg, and 3 mg/kg of PG antibodies.
  • the results showed that PG CAR-T cells combined with PG antibodies produced a significant anti-tumor effect without obvious toxic side effects.
  • Figure 3C shows the expansion of PG CAR-T cells in mice in the experiment of Example 3-1.
  • the results showed that the expansion of PG CAR-T cells in vivo depended on PG antibodies, showing a certain dependence on antibody dose, and mice in the higher dose group had a higher level of PG CAR-T cell expansion.
  • PG CAR-T cells When only PG CAR-T cells were administered, they began to expand in the mice one week after being reinfused into the mice, expanded to 466 cells/100 ⁇ L peripheral blood after 2 weeks, and reached a higher level after 3 weeks (3644 cells/100 ⁇ L peripheral blood), and still maintained at a high level after 4 weeks (3214 cells/100 ⁇ L peripheral blood); when PG CAR-T cells were used in combination with different doses of PG antibodies of 0.3mg/kg, 1mg/kg, and 3mg/kg, After 2 weeks, the peak expansion levels of PG CAR-T cells in vivo reached 11428 cells/100 ⁇ L peripheral blood, 19299 cells/100 ⁇ L peripheral blood, and 35368 cells/100 ⁇ L peripheral blood, respectively.
  • Example 4 Effect of PG CAR-T cells combined with different doses of ADI-38497 PG antibody (hereinafter referred to as PG antibody or PG Ab) against BCMA low-expressing tumors in vivo
  • L363 cells were resuspended in 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 /mL.
  • NOG mice aged 4-6 weeks, weight 15-17g, female
  • L363 cell suspension at an injection volume of 0.2mL/mouse, i.e., an inoculation amount of 1 ⁇ 10 6 cells/mouse.
  • mice with tumor volumes ranging from 74.14 to 110.29mm 3 were divided into 7 groups, namely, vehicle group, PG Ab group, PG CAR-T group, traditional CAR-T group, 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.
  • the antibodies were prepared at concentrations of 0.3 mg/mL, 0.1 mg/mL, and 0.03 mg/mL, respectively.
  • the antibodies were administered on the 9th day, with a volume of 10 mL/kg per mouse, and the administration frequency was once a week, and the administration method was intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended in 1 ⁇ PBS to prepare a cell suspension of 25 ⁇ 10 6 /mL of CAR + cells.
  • 0.2 mL/mouse of the cell suspension was injected into the tail vein, that is, 5 ⁇ 10 6 /mouse of CAR + cells were infused.
  • the weight of the mice, the maximum long axis (L) and the maximum wide axis (W) of the tumor tissue were monitored twice a week.
  • Figure 4A shows the therapeutic effects of different doses of PG antibody combined with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously inoculated with human L363 low-expressing BCMA tumor cells.
  • the results showed that in the BCMA low-expressing tumor model, the administration of PG CAR-T alone did not produce anti-tumor effects, while the anti-tumor effect of the administration of PG antibody alone was not obvious, with a TGI of 21%. Only mice treated with PG CAR-T cells and PG antibodies at the same time produced significant anti-tumor effects, and showed an antibody dose-dependent effect.
  • PG CAR-T cells When PG CAR-T cells were combined with PG antibodies at a dose of 0.3 mg/kg, PG CAR-T cells induced significant anti-tumor effects with a TGI of 87%. When the combination increased the PG antibody dose to 1 mg/kg and 3 mg/kg, the maximum anti-tumor effect induced by PG CAR-T cells increased significantly, with TGIs of 103% and 103%, respectively, showing the same anti-tumor effect as traditional Blue21 CAR-T cells.
  • Figure 4B shows the weight changes of mice in this experiment.
  • the results showed that the weight of mice treated with PG CAR-T cells combined with PG antibodies remained steadily increased after treatment, and the average weight increase was 18.2%, 10.5%, and 8.5% after combined use of PG antibodies at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg.
  • the results showed that PG CAR-T cells combined with PG antibodies produced a significant anti-tumor effect and did not induce significant toxicity.
  • Figure 4C shows the expansion of PG CAR-T cells in mice in the experiment of Example 4-1.
  • the results showed that when only PG CAR-T cells were administered, they began to proliferate in the mice one week after being infused back into the body, and expanded to 919 cells/100 ⁇ L peripheral blood after 2 weeks, and rapidly decreased to 204 cells/100 ⁇ L peripheral blood after 3 weeks; when PG CAR-T cells were used in combination with different doses of PG antibodies of 0.3mg/kg, 1mg/kg and 3mg/kg, the peak proliferation level of PG CAR-T cells in vivo reached 4380 cells/100 ⁇ L peripheral blood, 8049 cells/100 ⁇ L peripheral blood and 3347 cells/100 ⁇ L peripheral blood after 2 weeks, and remained at a high level after 3 weeks, which were 2475 cells/100 ⁇ L peripheral blood, 4121 cells/100 ⁇ L peripheral blood and 1969 cells/100 ⁇ L peripheral blood, respectively, which were much higher than the group not given antibodies in the same period.
  • Blue21 CAR-T cells used as a positive control also expanded to reach a peak level (76,836 cells/100 ⁇ L peripheral blood) 2 weeks after being infused back into mice, and still maintained a high level (36,328 cells/100 ⁇ L peripheral blood) 3 weeks later.
  • Example 5 Anti-tumor effects of different doses of PG CAR-T cells combined with ADI-38497 PG antibody (hereinafter referred to as PG antibody or PG Ab) in vivo
  • H929 cells were resuspended in 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • NOG mice aged 4-6 weeks, weighing 15-17g, female
  • NOG mice were shaved on the right back and subcutaneously injected with H929 cell suspension, with an injection volume of 0.2mL/mouse, that is, the inoculation amount was 1 ⁇ 10 6 cells/mouse.
  • mice with tumor volumes of 59.50-105.82mm 3 were divided into 7 groups, namely, vehicle group, PG Ab group, PG CAR-T group only, PG Ab+PG CAR-T, 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 group, with 7 mice in each group.
  • the antibody concentration was 0.3 mg/mL. After grouping, the antibody was administered on the 9th day.
  • the volume of each mouse was 10 mL/kg.
  • the administration frequency was once a week and the administration method was intraperitoneal injection.
  • the CAR-T cells prepared by donor 4 were resuspended in 1 ⁇ PBS to prepare a cell suspension of 50 ⁇ 10 6 cells/mL of CAR + cells, followed by 10-fold gradient dilution to prepare a cell suspension of 5 ⁇ 10 6 , 0.5 ⁇ 10 6 and 0.05 ⁇ 10 6 /mL. On the 9th day, 0.2 mL/mouse of the cell suspension was injected into the tail vein. The weight of the mice, the maximum long axis (L) and the maximum wide axis (W) of the tumor tissue were monitored twice a week.
  • Figure 5A 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 when a very low dose of 0.01 ⁇ 10 6 CAR-T cells was given, CAR-T cells produced an anti-tumor effect similar to that of PG antibody alone, with TGIs of 49% and 50%, respectively.
  • Increasing the CAR-T cell dose to 0.1 ⁇ 10 6 , 1 ⁇ 10 6 , and 10 ⁇ 10 6 CAR-T cells the anti-tumor effect induced by PG CAR-T cells increased significantly, with TGIs of 91%, 104%, and 103%, respectively. No anti-tumor effect was shown when CAR-T cells were administered alone.
  • Figure 5B shows the expansion of PG CAR-T cells in mice in the experiment of Example 5-1.
  • the results showed that CAR-T cells were infused back into mice and began to expand under the induction of PG antibodies in 1 week, and the expansion reached a peak level after 2 weeks, and remained at a high level after 3 weeks.
  • PG antibodies were used in combination, the expansion of CAR-T cells in vivo depended on the dose of CAR-T cells. Mice in the higher CAR-T dose group had a higher level of CAR-T cell expansion.
  • the peak expansion levels of 0.01 ⁇ 10 6 , 0.1 ⁇ 10 6 , 1 ⁇ 10 6 , and 10 ⁇ 10 6 CAR-T cell dose groups 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, respectively.
  • the formulation prescriptions corresponding to the Hu968B CAR-T cell part and the anti-BCMA antibody part (i.e., ADI-38497 PG antibody) in the CAR-T cell drug (i.e., anti-BCMA chimeric antigen receptor T cells) were screened and optimized, and the formulation prescriptions that are beneficial to the stability of CAR-T cells (see Example 6 for details) and the formulation prescriptions that are beneficial to the stability of antibodies (see Example 7 for details) were screened out respectively.
  • test samples used in the study were produced by Innovent Biologics (Suzhou) Co., Ltd., and detailed information is shown in Table 5.
  • Bomeili A compound electrolyte injection
  • Table 6 The main components of Bomeili A (compound electrolyte injection) in Table 6 are: 5.26g sodium chloride, 5.02g sodium gluconate, 3.68g sodium acetate trihydrate (C 2 H 3 NaO 2 ⁇ 3H 2 O), 0.37g potassium chloride, and 0.30g magnesium chloride hexahydrate (MgCl 2 ⁇ 6H 2 O) per 1000mL.
  • Auxiliary materials appropriate amount of sodium hydroxide (for adjusting pH value).
  • This experiment mainly investigated the CS10( The effects of different ratios of CS10, Bomel A (compound electrolyte injection), and human serum albumin (HSA) on the stability of HuR968B CAR-T cells were studied to obtain an optimal mixing ratio range.
  • the cell viability of the 12 formulations was >70% within 2 hours after cell recovery at a cell density of 1E6 cells/mL, and the viability at a freezing density of 1E8 cells/mL was slightly lower than that of the 1E6 cells/mL group. As the room temperature storage time increased, the cell viability of the experimental group showed a downward trend (see Table 10 for specific results).
  • CD3 + CAR + and T cell phenotypes including TE , TN , TCM and TEM .
  • the detection values of CAR + in the 1E6 cell density group were more dispersed (21.40% to 31.70%), and the detection values of CAR + in the 1E8 cell density group were more concentrated (23.0% to 28.7%); the detection values of TN in the 1E6 cell density group were more concentrated and the overall values were higher (48.2% to 57.2%), and the detection values of TN in the 1E8 cell density group were lower overall (42.3% to 55.0%) (see Table 12 for specific results).
  • the results of the prescription research experiment showed that under the same preparation freezing conditions, the viability of the 12 experimental groups with a cell density of 1E8 cells/mL after resuscitation was lower than that of the cell density of 1E6 cells/mL. Therefore, in the prescription confirmation experiment, the worst case scenario, i.e., a cell density of 1E8 cells/mL, was taken for prescription confirmation.
  • Hu968B CAR-T cells which consists of: 1E6/mL to 1E8/mL Hu968B CAR-T cells, CS10 50%-70% (v/v), Bolali A compound electrolyte injection 10.5%-50.5% (v/v), human serum albumin 14.5%-24.5% (v/v).
  • the optimal formulation for the chimeric antigen receptor T cell part in the recombinant anti-BCMA monoclonal antibody preparation and chimeric antigen receptor T cell preparation (drug combination) was confirmed.
  • the finished product specification of the chimeric antigen receptor T cell preparation is 40mL/bag, the dosage form is injection, and the administration method is intravenous injection.
  • test samples used in the study were produced by Innovent Biologics (Suzhou) Co., Ltd., and detailed information is shown in Table 17.
  • Table 21 Pre-prescription experimental plan Note: (1) ⁇ indicates sampling at this time point. (2) After sampling at the above time points, place the samples in a -70°C freezer and thaw and send for inspection as needed.
  • This experiment mainly investigated the effects of different excipients (including histidine, sorbitol, methionine, arginine hydrochloride, edetate disodium and polysorbate 80) on the stability of ADI-38497 PG antibody. Detailed prescription information is shown in Table 24.
  • the results of the mandatory stability study are detailed in Table 26.
  • the trends of the main components and acidic components of the charge variants are shown in Figures 12A and 12B, respectively, and the trends of the purity-monomer content (SEC-HPLC method) are shown in Figure 12C.
  • the results show that after being placed at 40°C ⁇ 2°C for 4 weeks, the appearance and visible foreign matter of the samples of all prescriptions are qualified; the protein content and pH value have not changed significantly; the purity of the samples of prescription 2 (SEC-HPLC method) has changed significantly, and the other prescriptions have not changed; the purity of all prescription samples (nrCE-SDS method) has decreased, the trend of change is basically the same, and there is no significant difference between the prescriptions.
  • the acidic components of the charge variants of all prescription samples increased significantly, the main components decreased significantly, and the alkaline components did not change significantly.
  • the performance of the sample of prescription 2 was slightly better than that of other prescription samples.
  • the content of polysorbate 80 in all prescriptions has not changed.
  • the biological activity of all prescriptions is within an acceptable range.
  • formulation 1 was finally selected as the final formulation of ADI-38497 PG antibody.
  • the buffer system was adjusted to histidine and histidine hydrochloride. That is, the final formulation of ADI-38497 PG antibody is: 20.0mg/ml recombinant anti-B cell maturation antigen (BCMA) monoclonal antibody, 0.76mg/ml histidine, 1.08mg/ml L-histidine hydrochloride, 50.00mg/ml sorbitol, 0.2mg/ml polysorbate 80, pH 6.0.
  • BCMA recombinant anti-B cell maturation antigen
  • the optimal formulation for the recombinant anti-BCMA monoclonal antibody part in the recombinant anti-BCMA monoclonal antibody preparation and chimeric antigen receptor T cell preparation (drug combination) was confirmed.
  • the finished product specification of the antibody preparation is 60 mg (3 mL)/bottle, the dosage form is injection, and the administration method is intravenous injection.

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Abstract

一种包含BCMA PG CAR-T细胞制剂和PG抗体制剂的药物组合制剂及其用途。一种药物组合制剂，所述制剂包含制剂A和制剂B；其中，所述制剂A包含治疗有效量的表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞和药用辅料I；所述制剂B包含治疗有效量的包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段和药用辅料II。通过筛选和优选确定了CAR-T细胞稳定的制剂处方和重组抗BCMA单克隆抗体稳定的制剂处方。该药物组合制剂处方能够保证药物组合长期稳定储存并且成分简单、具有易用性的优势。

Description

一种包含BCMA PG CAR-T细胞制剂和PG抗体制剂的药物组合制剂及其用途
优先权和相关申请
本发明要求2023年6月6日提交的名称为“一种包含BCMA PG CAR-T细胞制剂和PG抗体制剂的药物组合制剂及其用途”的中国专利申请202310664272.0的优先权，该申请包括附录在内的全部内容作为参考并入本发明。
技术领域
本发明涉及一种包含BCMA PG CAR-T细胞制剂和PG抗体制剂的药物组合制剂及其用途，属于抗体药物技术领域。
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。
CAR-T细胞作为一种基因工程修饰的新型高效、精准靶向的肿瘤细胞免疫治疗药物，已经在血液肿瘤治疗中展现巨大治疗潜力，国内外有多个产品获批上市，但在实体肿瘤治疗亟待突破。
传统嵌合抗原受体T细胞(CAR-T)是通过T细胞上的嵌合抗原受体(CAR)分子直接靶向肿瘤细胞的表面抗原，从而达到识别和杀伤肿瘤的目的，其中所述嵌合抗原受体的N端包含识别抗原的抗原结合结构域，例如，针对抗原的单链抗体片段(scFv)。传统嵌合抗原受体T细胞的缺陷在于，所述CAR-T细胞的活性缺乏控制，只要所述CAR-T细胞针对的抗原阳性细胞存在，则CAR-T细胞就会持续识别并杀伤这些抗原阳性细胞。
由于CAR-T细胞靶向的绝大部分肿瘤抗原不具有肿瘤特异性，这些肿瘤抗原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，这可能引起严重的毒副效应。
药品稳定性是保证药品有效性和安全性的重要指标之一。获得赋予药品良好稳定性的制剂处方是药品在货架期内保持其安全性和有效性的关键条件。在现有技术中，针对包含靶向BCMA的PG CAR-T细胞和PG抗体的药物组合缺乏能保证所述药物组合长期稳定储存(例如24个月)并且成分简单和具有易用性优势的制剂处方。
发明内容
发明要解决的问题
针对现有技术中存在的问题，本发明提供了一种包含BCMA PG CAR-T细胞制剂和PG抗体制剂的药物组合制剂及其用途。
用于解决问题的方案
鉴于上述现有技术中存在的问题，发明人进行了深入的研究。本发明人通过研究，开发了一组“分子开关”调控型嵌合抗原受体细胞，通过将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或PG)突变抗体作为“分子开关”或适体(adaptor)，构建了这样的CAR分子，其能够特异结合包含P329G突变Fc结构域的抗体而不结合不包含P329G突变Fc结构域的抗体(本文中也称为“野生型抗体”)，由此，通过将表达所述CAR的免疫效应细胞(例如，T细胞)与作为“分子开关”的重组抗BCMA分子的单克隆抗体进行组合，用于在受试者中治疗与BCMA相关的疾病，例如表达或过表达BCMA的癌症，所述癌症是例如复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
有别于传统BCMA靶向CAR-T细胞直接识别杀伤多发性骨髓瘤(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。
在此基础上，本发明人基于平台经验，通过室温(22～26℃)强制试验和液氮气相(≤-150℃)储存稳定性实验，考察不同辅料的含量对嵌合抗原受体细胞的活率与表型的影响，优化和筛选出有利于嵌合抗原受体T细胞稳定的制剂处方，该研究过程中的检测项目主要包括：细胞活率、细胞结团率、CD3+CAR+和T细胞表型。另外，通过高温稳定性实验，考察不同缓冲液pH值、不同辅料和不同缓冲体系对重组抗BCMA单克隆抗体稳定性的影响，优化和筛选出有利于重组抗BCMA单克隆抗体的制剂处方，该研究过程中检测项目主要包括：外观、可见异物、蛋白含量、pH、纯度、电荷变异体、聚山梨酯80含量和生物学活性(具体为BCMA相对结合活性)。
即本发明人对包含BCMA PG CAR-T细胞和PG抗体的药物组合制剂中的各种辅料的种类和添加比例进行了研究，分别筛选出有利于嵌合抗原受体T细胞稳定的制剂处方和有利于重组抗BCMA单克隆抗体稳定的制剂处方，从而完成了本发明。
本发明的第一方面提供了一种药物组合制剂，所述制剂包含制剂A和制剂B；
其中，所述制剂A包含：治疗有效量的表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞和药用辅料I，
所述药用辅料I包含：复方电解质注射液、人血清白蛋白和CS10；优选的，所述复方电解质注射液为勃脉力A复方电解质注射液；
所述制剂B包含：治疗有效量的包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段和药用辅料II，
所述P329G突变的特异性结合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；
所述药用辅料II包含：pH调节剂、渗透压调节剂和表面活性剂；
在一些优选的实施方案中，所述pH调节剂为组氨酸和/或L-盐酸组氨酸，所述渗透压调节剂为山梨醇，所述表面活性剂为聚山梨酯80。
在一些实施方案中，本发明在第一方面所述的药物组合制剂，其中所述分子开关调控型嵌合抗原受体(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:16)所示的CDR H1；
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:17)所示的CDR H2；和
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:18)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:19)所示的CDR L1；
(e)氨基酸序列GTNKRAP(SEQ ID NO:20)所示的CDR L2；和
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:21)所示的CDR L3；
在一些优选的实施方案中，所述(i)重链可变区，其包含SEQ ID NO:9的序列，和(ii)轻链可变区，其包含SEQ ID NO:10的序列；
(2)铰链区/间隔区，其选自SEQ ID NO:11或SEQ ID NO：30所示的序列；
(3)跨膜区(TM)，其为SEQ ID NO:12所示的CD8跨膜结构域；
(4)共刺激信号结构域(CSD)，其为SEQ ID NO:13所示的4-1BB共刺激结构域；
(5)刺激信号结构域(SSD)，其为SEQ ID NO:14所示的CD3ζ信号传导结构域；
(6)信号肽序列，其为SEQ ID NO：8所示的信号肽序列；
在一些优选的实施方案中，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，编码所述分子开关调控型CAR多肽的核酸分子包含SEQ ID NO:31所示的核苷酸序列；
所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞为T细胞；优选的，所述T细胞是自体T细胞或同种异体T细胞；更优选的，所述免疫效应细胞是自人PBMC分离T细胞后制备的；
在一些最优选的实施方案中，所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞为HuR968B CAR-T细胞。
在一些实施方案中，本发明在第一方面中所述的药物组合制剂，其中所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞在所述制剂A中的细胞密度为1E6个/mL～1E8个/mL；
所述药用辅料I中的各成分在所述制剂A中的含量分别为：复方电解质注射液10.5％～50.5％(v/v)、 人血清白蛋白14.5～24.5％(v/v)、CS10 50％～70％(v/v)；
在一些优选的实施方案中，所述药用辅料I中的各成分在所述制剂A中的含量分别为CS10 50.0％(v/v)、复方电解质注射液30.5％(v/v)、人血清白蛋白19.5％；
在一些更优选的实施方案中，所述复方电解质注射液为勃脉力A复方电解质注射液。
在一些实施方案中，本发明在第一方面中所述的药物组合制剂，其中所述特异性结合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，其中所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:22)所示的CDR H1；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:23)所示的CDR H2；和氨基酸序列DRGDQILDV(SEQ ID NO:24)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:25)所示的CDR L1氨基酸序列AASSLQS(SEQ ID NO:26)所示的CDR L2；和氨基酸序列QQKYFDIT(SEQ ID NO:27)所示的CDR L3；
在一些优选的实施方案中，所述重链可变区包含SEQ ID NO:2的序列，且所述轻链可变区包含SEQ ID NO:3的序列
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，在一些优选地实施方案中，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；在一些更优选地实施方案中，所述突变Fc结构域是IgG1抗体的突变Fc结构域；
在一些最优选的实施方案中，所述P329G突变的特异性结合BCMA分子的抗体是ADI-38497 PG Ab。
在一些实施方案中，本发明在第一方面中所述的药物组合制剂，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在所述制剂B中的含量为10～30mg/mL；在一些优选的实施方案中，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在所述制剂B中的含量为20.0mg/mL；
所述药用辅料II中的各成分在所述制剂B中的含量分别为：10～25mM pH调节剂，40～60mg/mL渗透压调节剂和0.1～0.3mg/mL表面活性剂；
在一些优选的实施方案中，所述药用辅料II中的各成分在所述制剂B中的含量分别为：10mM pH调节剂，50mg/mL渗透压调剂和0.2mg/mL表面活性剂；
在一些更优选的实施方案中，所述药用辅料II中的各成分在所述制剂B中的含量分别为：0.76g/ml组氨酸、1.08mg/mL L-盐酸组氨酸、50.00mg/mL山梨醇、0.2mg/mL聚山梨酯80；
在一些任选的实施方案中，所述制剂B的pH为5.0～7.0，优选的所述制剂B的pH为6.0。
在一些实施方案中，本发明在第一方面中所述的药物组合制剂，其中所述制剂A和制剂B分开、同时或依次施用。
在一些实施方案中，本发明在第一方面中所述的药物组合制剂，其中所述施用的方式为胃肠外施用；优选的，所述施用的方式为静脉内施用。
本发明的第二方面提供了一种根据本发明第一方面所述的药物组合制剂在制备治疗和/或预防癌症的药物中的用途；优选的，所述癌症是表达或过表达BCMA的癌症；更优选的，所述癌症是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
本发明的第三方面提供了一种治疗和/或预防癌症的方法，所述方法包括向有需要的受试者施用如本发明第一方面中所述的药物组合制剂；优选的，所述癌症是表达或过表达BCMA的癌症；更优选的，所述癌症是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
在一些实施方案中，本发明在第三方面所述的方法，其中所述施用的方式为胃肠外施用；优选的，所述施用的方式为静脉内施用。
发明的效果
由本发明的技术方案可见，本发明的技术方案与现有技术相比，具有以下有益效果：
(1)为开发出稳定的CAR-T细胞制剂处方，对CAR-T细胞制剂的各辅料的种类和添加比例进行了研究，筛选出有利于CAR-T细胞稳定的制剂处方。最终确定CAR-T细胞稳定的制剂处方为：1E6个/mL～1E8个/mL Hu968B嵌合抗原受体(CAR)-T细胞，复方电解质注射液10.5％～50.5％(v/v),人血清白蛋白14.5～24.5％(v/v)，CS10 50％～70％(v/v)。
在液氮气相(≤-150℃)条件下放置4周，该制剂处方细胞活率均>70％、无细胞结团现象发生、CD3+CAR+和T细胞表型均无明显变化趋势；室温(22～26℃)条件放置4小时，该制剂处方细胞活率均>70％、无细胞结团现象发生。
(2)为开发出稳定的重组抗BCMA单克隆抗体制剂处方，对重组抗BCMA单克隆抗体制剂的各辅料的种类和添加比例进行了研究，筛选出有利于重组抗BCMA单克隆抗体稳定的制剂处方。最终确定重组抗BCMA单克隆抗体稳定的制剂处方为：20.0mg/ml重组抗B细胞成熟抗原(BCMA)单克隆抗体、0.76mg/ml组氨酸、1.08mg/ml L-盐酸组氨酸、50.00mg/ml山梨醇、0.2mg/ml聚山梨酯80，pH 6.0。
40℃±2℃条件下放置4周，pH 6.0条件下的样品外观、可见异物均合格，并且该pH条件下样品的纯度(SEC-HPLC)无明显差异。另外，pH 6.0条件下样品纯度表现优于其他pH条件样品。因此，确定在pH 6.0条件下展开处方确定实验。
40℃±2℃条件下放置4周后，上述稳定的制剂处方的样品外观、可见异物合格，蛋白含量和pH值均未发生明显变化，样品的纯度(SEC-HPLC法)未发生变化，电荷变异体的酸性组分显著上升，主成分显著下降，聚山梨酯80含量未发生变化，生物学活性在可接受范围内。
(3)上述两种药物制剂处方配合使用，具有对肿瘤细胞的杀伤作用，并促进细胞因子释放。
附图说明
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。2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)；“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(VL)。
图2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，Blue21 CAR-T。
图3B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3C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4A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，Blue21 CAR-T。
图4B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4C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5A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5B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6示出了复苏后0h活率“预测值-实际值”。
图7示出了复苏后1h活率“预测值-实际值”。
图8示出了复苏后2h活率“预测值-实际值”。
图9示出了复苏后4h活率“预测值-实际值”。
图10示出了复苏后8h活率“预测值-实际值”。
图11示出了预测刻画器中“活率0h”、“活率1h”、“活率2h”、“活率4h”、“活率8h”的重要度；意愿函数和最优条件。
图12A示出了处方筛选电荷变异体-酸性组分(40℃，iCIEF法)变化趋势。图12B示出了处方筛选电荷变异体-主成分(40℃，iCIEF法)变化趋势。图12C示出了处方筛选纯度(40℃，SEC-HPLC法)变化趋势。
具体实施方式
以下将详细说明本发明的各种示例性实施例、特征和方面。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好地说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段、器材和步骤未作详细描述，以便于凸显本发明的主旨。
1.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。
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，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)。
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，6th 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:14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:13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。
“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的基因递送技术、来自Lentigen的LENTIMAXTM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(EC50)”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。
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。
术语“细胞活率”指总细胞群体中活细胞所占的百分比。
术语“细胞结团率”指细胞的凝集程度，是评价细胞治疗产品安全性的重要指标。
术语“MOI”指病毒(噬菌体)感染细菌时病毒(噬菌体)与细菌的数量比值，即平均每个细菌感染病毒(噬菌体)的数量。
术语“CE-SDS”是指十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛细管电泳法定量测定重组单克隆抗体产品的纯度。
术语“SEC-HPLC”是指体积排阻色谱-高效液相色谱检测方法。
术语“主成分”即目标产物，纯度越高，表明所得的目标产物越多。
术语“电荷变异体”，是由于蛋白质产物在细胞内会发生译后修饰和降解事件，导致和生物物理特性具有异质性，所带电荷有差异，故称为电荷变异体。
术语“酸性电荷变异体”，是在不同分析方法中早于或晚于主峰的电荷变异体。形成酸性电荷变异体的原因较多，其中可能存在影响产品生物活性的修饰，因此酸性电荷变异体的高低一般作为抗体产物质量的一个判断指标。
2.本发明的分子开关调控型嵌合抗原受体(CAR)、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:16)所示的重链互补决定区CDR H1；
(b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:17)所示的CDR H2；和
(c)氨基酸序列PYDYGAWFAS(SEQ ID NO:18)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:19)所示的轻链互补决定区(CDR L)1；
(e)氨基酸序列GTNKRAP(SEQ ID NO:20)所示的CDR L2；和
(f)氨基酸序列ALWYSNHWV(SEQ ID NO:21)所示的CDR L3。
进一步地，scFv可以在N端连接有信号肽序列，例如，SEQ ID NO:8所示的信号肽序列，并且scFv可以在C端连接有如SEQ ID NO:11中提供的任选铰链区序列、如SEQ ID NO:12中提供的跨膜区、如SEQ ID NO:13的共刺激信号结构域和包含SEQ ID NO:14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:9的序列，和(ii)轻链可变区，其包含SEQ ID NO:10的序列；
在一些实施方案中，scFv结构域包含(i)SEQ ID NO:9所示的重链可变区和(ii)SEQ ID NO:10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。
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)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(例如，T细胞)。
3.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：
所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:22)所示的CDR H1；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:23)所示的CDR H2；和氨基酸序列DRGDQILDV(SEQ ID NO:24)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:25)所示的CDR L1；氨基酸序列AASSLQS(SEQ ID NO:26)所示的CDR L2；和氨基酸序列QQKYFDIT(SEQ ID NO:27)所示的CDR L3。
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：
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。
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)。
4.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。
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)。可药用辅料可以例示例如表面活性剂、赋形剂、着色料、香料、防腐剂、稳定剂、缓冲剂、悬浮剂、等张剂、粘合剂、崩解剂、润滑剂、流动促进剂、矫味剂等。进一步地，也可合适地使用其他常用的载剂，例如，轻质无水硅酸、乳糖、结晶纤维素、甘露醇、淀粉、 羧甲基纤维素钙、羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、聚乙烯缩醛二乙氨基乙酸酯、聚乙烯吡咯啶酮、明胶、中链脂肪酸三甘油酯、聚氧乙烯硬化蓖麻油60、白砂糖、羧甲基纤维素、玉米淀粉、无机盐类等作为载剂，但不限于此。
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)。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
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:8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:9所示的VH、SEQ ID NO:30所示的接头序列、SEQ ID NO:10所示的VL)、SEQ ID NO:11所示的G4S铰链区、SEQ ID NO:12所示的CD8跨膜结构域(CD8TM)、SEQ ID NO:13所示的41BB共刺激结构域(41BB-CSD)以及SEQ ID NO:14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Blue21 CAR(SEQ ID NO:7)，用作对照。Blue21 CAR从N端到C端含有SEQ ID NO:8所示的信号肽、抗BCMA单链抗体(来自11D53克隆)、SEQ ID NO:15所示的CD8α分子的铰链区及SEQ ID NO:12所示的CD8跨膜结构域、SEQ ID NO:13所示的4-1BB共刺激结构域以及SEQ ID NO:14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
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:7)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℃CO2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2～8x108个细胞)进行细胞收获。离心去除培养基后，将CAR-T细胞采用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')2Fragment山羊抗人IgG(Jackson ImmunoResearch,109-066-006；PG CAR检测)或Biotin-F(ab')2Fragment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:28所示的VH、SEQ ID NO:29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)。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
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。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(即BCMA特异性WT抗体)。
(2-2)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')2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2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3 P329G BCMA抗体与表达BCMA的阳性肿瘤细胞的结合EC50值
实施例3、PG CAR-T细胞联合不同剂量ADI-38497 PG抗体(下文也简称PG抗体或PG Ab)在体内抗BCMA高表达肿瘤的效应
(3-1)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5×106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1×106个细胞/只小鼠。肿瘤细胞接种后7天，将小鼠肿瘤体积在50.82～104.36mm3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25×106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5×106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3A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Blue21 CAR-T细胞的抗肿瘤效应相当，TGI分别为101％和102％。
图3B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。
(3-2)小鼠体内CAR-T细胞检测：
取30μL实施例3-1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 FcXTM(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')2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3C显示实施例3-1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Blue21 CAR-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4、PG CAR-T细胞联合不同剂量ADI-38497 PG抗体(下文也简称PG抗体或PG Ab)在体内抗BCMA低表达肿瘤的效应
(4-1)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5×106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1×106个细胞/只小鼠。肿瘤细胞接种后9天，将小鼠肿瘤体积在74.14～110.29mm3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25×106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5×106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4A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Blue21 CAR-T细胞相同的抗肿瘤效应。
图4B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。
(4-2)小鼠体内CAR-T细胞检测：方法同实施例3-2。
图4C显示实施例4-1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Blue21 CAR-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5、不同剂量PG CAR-T细胞联合ADI-38497 PG抗体(下文也简称PG抗体或PG Ab)在体内的抗肿瘤效应
(5-1)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5×106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1×106个细胞/只小鼠。肿瘤细胞接种后9天，将小鼠肿瘤体积在59.50～105.82mm3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,10×106组、PG Ab+PG CAR-T,1×106个细胞组、PG Ab+PG CAR-T，0.1×106个细胞组和PG Ab+PG CAR-T,0.01×106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50×106个/mL细胞悬液，随后10倍梯度稀释，再制备成5×106、0.5×106和0.05×106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5A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0.01×106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0.1×106、1×106、10×106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。
(5-2)体内CAR-T细胞检测：方法同实施例3-2。
图5B显示实施例5-1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，0.01×106、0.1×106、1×106、10×106CAR-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。
在上述实施例1-5的基础上，对所述CAR-T细胞药物(即抗BCMA嵌合抗原受体T细胞)中的Hu968B CAR-T细胞部分和抗BCMA抗体部分(即ADI-38497 PG抗体)对应的制剂处方进行了筛选和优化，分别筛选出有利于CAR-T细胞稳定的制剂处方(具体参见实施例6)和有利于抗体稳定的制剂处方(具体参见实施例7)。
实施例6、HuR968B CAR-T细胞制剂处方的筛选和优化
【缩略词】
本实施例中涉及的缩略词与全称如下表4所示
表4缩略词与全称
【实验样品】
研究用供试品由信达生物制药(苏州)有限公司自制，详细信息见表5。
表5研究用供试品信息

注：“/”表示没有检测。
【其他关键信息】
表6研究用试剂耗材

注：“N/A”表示不适用。
表6中勃脉力A(复方电解质注射液)主要组份为：每1000mL中含：氯化钠5.26g；葡萄糖酸钠5.02g；三水合醋酸钠(C2H3NaO2·3H2O)3.68g；氯化钾0.37g；六水合氯化镁(MgCl2·6H2O)0.30g。辅料：适量氢氧化钠(用于调节pH值)。
【仪器设备】
表7研究用关键仪器设备信息
6.1处方研究(混料配比)
6.1.1实验步骤
本实验主要考察制剂溶液中CS10(CS10)、勃脉力A(复方电解质注射液)、人血清白蛋白(HSA)的不同配比对HuR968B CAR-T细胞稳定性的影响，以获得较优的混料比例范围。
实验考察研究因素和水平见表8，使用JMP15软件设计一个三因子2水平的实验，共需配置12组制剂冻存液(具体分组见表9)，将HuR968B CAR-T细胞离心后重悬于上述不同混料比例的冻存液中，调节细胞浓度至1E6个/mL和1E8个/mL并转移至1.8mL冻存管中。使用程序降温仪对CAR-T细胞制剂进行冷冻，待降温程序完成后转移至液氮罐中气相保存。冷冻一段时间(4周)后将上述样品于室温条件下进行稳定性考察(0H、1H、2H、4H、8H)，具体实验方案见表9。
表8 HuR968B CAR-T细胞制剂实验研究因素与水平
表9实验方案

注：“●”表示待检测项。
6.1.2实验结果
(1)细胞活率
气相液氮条件下储存4周，12组制剂配方在细胞密度1E6个/mL条件下细胞复苏后2H内细胞活率均>70％，1E8个/mL冻存密度条件下活率略低于1E6个/mL组。随着室温放置时间延长，实验组细胞活率呈下降趋势(具体结果参见表10)。
表10制剂冻存4周后细胞活率

(2)细胞结团率
气相液氮条件下储存4周，12组制剂配方组的细胞结团率均未发生显著变化，室温放置不同时间也不会影响细胞结团率(具体结果参见表11)。
表11制剂冻存4周后细胞结团率

(3)CD3+CAR+及T细胞表型
气相液氮条件下储存4周，12组制剂配方组细胞复苏后CD3+CAR+、T细胞表型(具体包括TE、TN、TCM和TEM)没有明显变化趋势。1E6个/mL细胞密度组CAR+的检测数值更分散(21.40％～31.70％)，1E8个/mL细胞密度组CAR+的检测数值分布更集中(23.0％～28.7％)；1E6个/mL细胞密度组TN的检测数值更集中且整体数值偏高(48.2％～57.2％)，1E8个/mL细胞密度组TN的检测数值整体偏低(42.3％～55.0％)(具体结果参见表12)。
表12制剂冻存4周后CAR阳性率及T细胞表型

注：“*”数据异常舍弃。
综上所述，处方研究实验结果表明，在相同制剂冻存条件下，12组实验分组的1E8个/mL的细胞密度复苏后活率低于1E6个/mL的细胞密度，所以在处方确认实验中，取最差状态(worst case scenario)即1E8个/mL的细胞密度进行处方确认。
6.2处方确认
6.2.1实验步骤
12组制剂处方的配置及检测时间点、检测项目参见本实施例“6.1.1实验步骤”部分。
6.2.2实验结果
(1)细胞活率
气相液氮条件下储存4周，12组制剂配方细胞复苏后细胞活率均>70％,随着室温放置时间延长， 所有组的细胞活率均有下降(具体参见表13)。
表13制剂冻存4周后细胞活率
(2)结团率
气相液氮条件下储存4周，12组制剂配方组的细胞结团率均未发生显著变化，室温放置不同时间也不会影响细胞结团率(具体参见表14)。
表14制剂冻存4周后细胞结团率
(3)CD3+CAR+及T细胞表型
气相液氮条件下储存4周，12组制剂配方组细胞复苏后CD3+CAR+、T细胞表型(包括TE、TN、TCM和TEM)均没有明显变化趋势(具体见表15)。
表15制剂冻存4周后CAR阳性率及T细胞表型
(4)结果分析
1.把处方确认实验1E8个/mL复苏后四周的0h、1h、2h、4h、8h的细胞活率输入JMP15数据表中最小二乘法拟合模型。其中，由图6、图10可知，“活率0h”、“活率8h”，P值>0.05，模型不显著。图7、图8、图9可知，“活率1h”、“活率2h”、“活率4h”的P值分别为0.0003、0.0016、0.0004，均<0.05，说明模型显著。
2.已知实际使用过程中，CAR-T细胞复苏之后会在2h之内回输，在预测刻画器中所以将“活率1h”、“活率2h”的重要度设置为1，“活率4h”的重要度设置为0.1。“活率0h”、“活率8h”由于模型不显著，故重要度设置为0。通过意愿函数得出最优条件，CS10 50.0％、勃脉力A 30.5％、HSA 19.5％，如图11。
6.2.3试验结论
根据上述实验结果及制剂处方开发的平台经验，最终选定Hu968B CAR-T细胞的稳定制剂处方，其组成为：1E6个/mL～1E8个/mL Hu968B CAR-T细胞、CS10 50％～70％(v/v)、勃脉力A复方电解质注射液10.5％～50.5％(v/v)、人血清白蛋白14.5％～24.5％(v/v)。
基于上述研究，确认了重组抗BCMA单克隆抗体制剂和嵌合抗原受体T细胞制剂(药物组合)中针对嵌合抗原受体T细胞部分的最佳制剂处方。在实际应用中，所述嵌合抗原受体T细胞制剂的成品规格为40mL/袋，剂型为注射剂，给药方式为静脉注射。
实施例7、ADI-38497 PG抗体制剂处方的筛选和优化
【缩略词】
本实施例中涉及的缩略词与全称如下表16所示
表16缩略词与全称
【实验样品】
研究用供试品由信达生物制药(苏州)有限公司自制，详细信息见表17。
表17样品信息
【其他关键信息】
具体信息见表18和表19：
表18试剂信息

注：N/A表示不涉及。
表19.材料信息
【仪器设备】
具体信息见表20
表20仪器设备信息
7.1 pH筛选
7.1.1实验步骤
配制含1.55mg/ml组氨酸，50.00mg/ml山梨醇的缓冲液，分别用稀盐酸溶液调节pH至5.0、5.5、6.0、6.5、7.0，后将ADI-38497 PG抗体超滤置换至不同pH值的缓冲液中，调节蛋白含量至约20mg/ml；加入聚山梨酯80使其终浓度为0.3mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40℃±2℃条件下进行稳定性考察，具体方案见表21。
表21处方前实验方案

注：(1)√表示该点取样。(2)上述时间点取样后放入-70℃中冻存，按需化冻送检。
7.1.2判断标准
表22质量未发生变化的判断标准

注：该标准仅适用于处方确定实验。
7.1.3实验结果
处方前研究结果详见表23。结果表明，40℃±2℃条件下放置4周，所有pH条件下的样品外观、可见异物均合格。不同pH条件下样品的纯度(SEC-HPLC)无明显差异。纯度(CD-SDS)发生了显著变化，pH 6.0样品纯度表现优于其他pH条件样品。电荷变异体均发生显著变化，变化趋势基本一致。因此确定在pH 6.0展开处方确定实验。
表23.处方前研究结果


注：N/A代表取样未送检
处方前研究结果详见表23。结果表明，40℃±2℃条件下放置4周，所有pH条件下的样品外观、可见异物均合格。不同pH条件下样品的纯度(SEC-HPLC)无明显差异。纯度(CD-SDS)发生了显著变化，pH 6.0样品纯度表现优于其他pH条件样品。电荷变异体均发生显著变化，变化趋势基本一致。因此确定在pH 6.0展开处方确定实验。
7.2处方确定实验
7.2.1实验步骤
本实验主要考察不同辅料(包括组氨酸、山梨醇、甲硫氨酸、盐酸精氨酸、依地酸二钠和聚山梨酯80)对ADI-38497 PG抗体稳定性的影响，详细处方信息见表24。
按照表24配制各个处方的缓冲液，将ADI-38497 PG抗体超滤置换至各自的处方溶液中。置换完成后，调节各处方蛋白含量至20mg/ml；加入聚山梨酯80，使其终浓度为0.2mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40℃±2℃条件下进行稳定性考察。具体稳定性考察方案参见表25。
表24备选处方信息表

注：用稀盐酸溶液调节pH。
表25稳定性考察方案

注：上述时间点取样后均先放入-70℃冰箱中冻存待检，按需化冻送检。
7.2.2判断标准
判断标准具体见表22。
7.2.3实验结果
强制稳定性研究结果详见表26，电荷变异体主成分和酸性组分变化趋势分别见图12A和图12B，纯度-单体含量(SEC-HPLC法)变化趋势见图12C。结果表明，40℃±2℃条件下放置4周后，所有处方的样品外观、可见异物均合格；蛋白含量和pH值均未发生明显变化；处方2的样品的纯度(SEC-HPLC法)发生明显变化，其它处方均未发生变化；所有处方样品纯度(nrCE-SDS法)均有所下降，变化趋势基本一致，处方间无显著差异。所有处方样品电荷变异体的酸性组分均显著上升，主成分均显著下降，碱性组分无明显变化，处方2样品表现稍优于其他处方样品。所有处方的聚山梨酯80含量未发生变化。所有处方的生物学活性都在可接受范围。
结果表明，40℃±2℃条件下放置4周后，所有处方的样品外观、可见异物均合格；蛋白含量和pH值均未发生明显变化；处方2的样品的纯度(SEC-HPLC法)发生明显变化，其它处方均未发生变化；所有处方样品纯度(nrCE-SDS法)均有所下降，变化趋势基本一致，处方间无显著差异。所有处方样品电荷变异体的酸性组分均显著上升，主成分均显著下降，处方2样品表现稍优于其他处方样品。 所有处方的聚山梨酯80含量未发生变化。所有处方的生物学活性都在可接受范围。
表26强制稳定性研究结果


注：N.A表示未检测。
7.2.4实验结论
综合上述实验结果及制剂处方开发平台经验，最终选定处方1作为ADI-38497 PG抗体最终制剂处方。为生产时避免使用盐酸调节pH，将缓冲体系调整为组氨酸和盐酸组氨酸。即ADI-38497 PG抗体最终制剂处方为：20.0mg/ml重组抗B细胞成熟抗原(BCMA)单克隆抗体，0.76mg/ml组氨酸，1.08mg/ml L-盐酸组氨酸，50.00mg/ml山梨醇，0.2mg/ml聚山梨酯80，pH 6.0。
基于上述研究，确认了重组抗BCMA单克隆抗体制剂和嵌合抗原受体T细胞制剂(药物组合)中针对重组抗BCMA单克隆抗体部分的最佳制剂处方。在实际应用中，所述抗体制剂的成品规格为60mg(3mL)/瓶，剂型为注射剂，给药方式为静脉注射。
需要说明的是，尽管以具体实例介绍了本发明的技术方案，但本领域技术人员能够理解，本发明应不限于此。
以上已经描述了本发明的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。本文中所用术语的选择，旨在最好地解释各实施例的原理、实际应用或对市场中的技术改进，或者使本技术领域的其它普通技术人员能理解本文披露的各实施例。
序列表



Claims (10)

  1. 一种药物组合制剂，所述制剂包含制剂A和制剂B；
    其中，所述制剂A包含：治疗有效量的表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞和药用辅料I，
    所述药用辅料I包含：复方电解质注射液、人血清白蛋白和CS10；优选的，所述复方电解质注射液为勃脉力A复方电解质注射液；
    所述制剂B包含：治疗有效量的包含P329G突变的特异性结合BCMA分子的抗体或抗原结合片段和药用辅料II，
    所述P329G突变的特异性结合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；
    所述药用辅料II包含：pH调节剂、渗透压调节剂和表面活性剂；
    优选的，所述pH调节剂为组氨酸和/或L-盐酸组氨酸，所述渗透压调节剂为山梨醇，所述表面活性剂为聚山梨酯80。
  2. 根据权利要求1所述的药物组合制剂，其特征在于，其中所述分子开关调控型嵌合抗原受体(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:16)所示的CDR H1；
    (b)氨基酸序列EITPDSSTINYAPSLKG(SEQ ID NO:17)所示的CDR H2；和
    (c)氨基酸序列PYDYGAWFAS(SEQ ID NO:18)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:19)所示的CDR L1；
    (e)氨基酸序列GTNKRAP(SEQ ID NO:20)所示的CDR L2；和
    (f)氨基酸序列ALWYSNHWV(SEQ ID NO:21)所示的CDR L3；
    优选的，所述(i)重链可变区，其包含SEQ ID NO:9的序列，和(ii)轻链可变区，其包含SEQ ID NO:10的序列；
    (2)铰链区/间隔区，其选自SEQ ID NO:11或SEQ ID NO：30所示的序列；
    (3)跨膜区(TM)，其为SEQ ID NO:12所示的CD8跨膜结构域；
    (4)共刺激信号结构域(CSD)，其为SEQ ID NO:13所示的4-1BB共刺激结构域；
    (5)刺激信号结构域(SSD)，其为SEQ ID NO:14所示的CD3ζ信号传导结构域；
    (6)信号肽序列，其为SEQ ID NO：8所示的信号肽序列；
    优选的，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，编码所述分子开关调控型CAR多肽的核酸分子包含SEQ ID NO:31所示的核苷酸序列；
    所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞为T细胞；优选的，所述T细胞是自体T细胞或同种异体T细胞；更优选的，所述免疫效应细胞是自人PBMC分离T细胞后制备的；
    最优选的，所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞为HuR968B CAR-T细胞。
  3. 根据权利要求1或2所述的药物组合制剂，其特征在于，其中所述表达分子开关调控型嵌合抗原受体(CAR)多肽的免疫效应细胞在所述制剂A中的细胞密度为1E6个/mL～1E8个/mL；
    所述药用辅料I中的各成分在所述制剂A中的含量分别为：复方电解质注射液10.5％～50.5％(v/v)、人血清白蛋白14.5～24.5％(v/v)、CS10 50％～70％(v/v)；
    优选的，所述药用辅料I中的各成分在所述制剂A中的含量分别为CS10 50.0％(v/v)、复方电解质注射液30.5％(v/v)、人血清白蛋白19.5％；
    更优选的，所述复方电解质注射液为勃脉力A复方电解质注射液。
  4. 根据权利要求1～3中任一项所述的药物组合制剂，其特征在于，其中所述特异性结合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，其中所述重链可变区包含根据Kabat编号的氨基酸序列SSSYYWT(SEQ ID NO:22)所示的CDR H1；氨基酸序列SISIAGSTYYNPSLKS(SEQ ID NO:23)所示的CDR H2；和氨基酸序列DRGDQILDV(SEQ ID NO:24)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:25)所示的CDR L1氨基酸序列AASSLQS(SEQ ID NO:26)所示的CDR L2；和氨基酸序列QQKYFDIT(SEQ ID NO:27)所示的CDR L3；
    优选的，所述重链可变区包含SEQ ID NO:2的序列，且所述轻链可变区包含SEQ ID NO:3的序列其中所述突变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；
    最优选的，所述P329G突变的特异性结合BCMA分子的抗体是ADI-38497PG Ab。
  5. 根据权利要求1～4中任一项所述的药物组合制剂，其特征在于，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在所述制剂B中的含量为10～30mg/mL；优选的，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在所述制剂B中的含量为20.0mg/mL；
    所述药用辅料II中的各成分在所述制剂B中的含量分别为：10～25mM pH调节剂，40～60mg/mL渗透压调节剂和0.1～0.3mg/mL表面活性剂；
    优选的，所述药用辅料II中的各成分在所述制剂B中的含量分别为：10mM pH调节剂，50mg/mL渗透压调剂和0.2mg/mL表面活性剂；
    更优选的，所述药用辅料II中的各成分在所述制剂B中的含量分别为：0.76g/ml组氨酸、1.08mg/mL L-盐酸组氨酸、50.00mg/mL山梨醇、0.2mg/mL聚山梨酯80；
    任选的，所述制剂B的pH为5.0～7.0，优选的所述制剂B的pH为6.0。
  6. 根据权利要求1～5中任一项所述的药物组合制剂，其中所述制剂A和制剂B分开、同时或依次施用。
  7. 根据权利要求6所述的药物组合制剂，其中所述施用的方式为胃肠外施用；优选的，所述施用的方式为静脉内施用。
  8. 根据权利要求1～7中任一项所述的药物组合制剂在制备治疗和/或预防癌症的药物中的用途；优选的，所述癌症是表达或过表达BCMA的癌症；更优选的，所述癌症是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
  9. 一种治疗和/或预防癌症的方法，所述方法包括向有需要的受试者施用如权利要求1～7中任一项所述的药物组合制剂；优选的，所述癌症是表达或过表达BCMA的癌症；更优选的，所述癌症是复发/难治性多发性骨髓瘤(relapsed/refractory multiple myeloma,RRMM)。
  10. 根据权利要求9所述的方法，其中所述施用的方式为胃肠外施用；优选的，所述施用的方式为静脉内施用。
PCT/CN2024/097420 2023-06-06 2024-06-05 一种包含bcma pg car-t细胞制剂和pg抗体制剂的药物组合制剂及其用途 Ceased WO2024251132A1 (zh)

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