WO2024251132A1 - Préparation de combinaison de médicaments comprenant la préparation de lymphocytes car-t de bcma pg et la préparation d'anticorps pg et son utilisation - Google Patents

Préparation de combinaison de médicaments comprenant la préparation de lymphocytes car-t de bcma pg et la préparation d'anticorps pg et son utilisation 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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Chinese (zh)
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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

L'invention concerne une préparation de combinaison de médicaments comprenant une préparation de lymphocytes CAR-T de BCMA PG et une préparation d'anticorps PG et son utilisation. L'invention concerne une préparation de combinaison de médicaments. La préparation comprend la préparation d'une préparation A et d'une préparation B, la préparation A comprenant une quantité thérapeutiquement efficace d'une cellule effectrice immunitaire exprimant un polypeptide de récepteur antigénique chimérique (CAR) régulateur de commutateur moléculaire et un excipient pharmaceutique I ; et la préparation B comprend une quantité thérapeutiquement efficace d'un anticorps ou d'un fragment de liaison à l'antigène qui se lie spécifiquement à une molécule BCMA et comprend une mutation P329G et un excipient pharmaceutique II. Une prescription de préparation stable de la cellule CAR-T et une prescription de préparation stable de l'anticorps monoclonal anti-BCMA recombinant sont déterminées par criblage et optimisation. La prescription de la préparation de combinaison de médicaments peut assurer un stockage stable à long terme d'une combinaison de médicaments et présente les avantages d'ingrédients simples et d'une facilité d'utilisation.
PCT/CN2024/097420 2023-06-06 2024-06-05 Préparation de combinaison de médicaments comprenant la préparation de lymphocytes car-t de bcma pg et la préparation d'anticorps pg et son utilisation Pending WO2024251132A1 (fr)

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