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WO2025077829A1 - Utilisation d'anticorps cldn18.2 et cellule car-t dans le traitement d'une tumeur solide cldn18.2-positive - Google Patents

Utilisation d'anticorps cldn18.2 et cellule car-t dans le traitement d'une tumeur solide cldn18.2-positive Download PDF

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WO2025077829A1
WO2025077829A1 PCT/CN2024/124141 CN2024124141W WO2025077829A1 WO 2025077829 A1 WO2025077829 A1 WO 2025077829A1 CN 2024124141 W CN2024124141 W CN 2024124141W WO 2025077829 A1 WO2025077829 A1 WO 2025077829A1
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cells
antibody
seq
amino acid
car
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Chinese (zh)
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孙丽萍
林恩萱
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Innovent Cells Pharmaceuticals Suzhou Co Ltd
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Innovent Cells Pharmaceuticals Suzhou Co Ltd
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    • 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
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention generally relates to the fields of antibody engineering and cellular immunology. Specifically, the present invention generally relates to a combination of immune effector cells (e.g., T cells) engineered to express a molecular switch-regulated chimeric antigen receptor and an antibody, and a therapeutic method using the combination, and to the use of the combination to treat diseases associated with CLDN18.2, such as cancers that express or overexpress CLDN 18.2.
  • immune effector cells e.g., T cells
  • a therapeutic method using the combination e.g., cancers that express or overexpress CLDN 18.2.
  • Claudin (CLDN) protein is a four-transmembrane protein with a molecular weight of about 20-27 kDa. It contains two extracellular domains (ECL1 and ECL2), and both the C-terminus and the N-terminus are convex to the cytoplasm. This protein constitutes one of the key components of tight junctions between epithelial cells or endothelial cells, and plays an important role in regulating physiological functions such as intercellular barrier permeability and molecular flow. At present, at least 27 members of the CLDN protein family have been discovered, among which Claudin18 (CLDN18) has two isoforms, CLDN18.1 and CLDN18.2.
  • CLDN18.2 is ectopically highly expressed in 60-90% of pancreatic cancers.
  • CLDN18.2 expression in esophageal cancer, lung cancer, and colorectal cancer has also been reported. Given that CLDN18.2 is restrictedly expressed in normal tissues and significantly highly expressed in gastric cancer and pancreatic cancer, CLDN18.2 has become an ideal therapeutic target for the development of immunotherapy for solid tumors such as gastric cancer and pancreatic cancer.
  • CAR-T chimeric antigen receptor T cells
  • CAR chimeric antigen receptor
  • CAR-T 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, these tumor antigens are often expressed at low levels in many normal tissues, especially important tissues and organs, in addition to being expressed on tumor cells. The recognition and killing of normal tissues by CAR-T cells leads to "on-target/off tumor” toxicity, which may cause serious side effects. For example, CAR-T cells targeting HER2 recognize lung epithelial cells that express low levels of HER2. After being reintroduced into the body, the CAR-T cells quickly cause pulmonary edema and respiratory distress, followed by multiple organ failure, leading to patients.
  • CAR-T cells In order to reduce the "on-target/off tumor” toxicity of CAR-T cells, the following strategies are usually adopted in the prior art.
  • One strategy is to design the antigen-binding domain contained in the CAR to target the target antigen that is highly expressed on the tumor surface but not expressed or lowly expressed in normal tissues.
  • Another strategy is to strictly control the dose of administered T cells, because too many CAR-T cells will increase exponentially after being stimulated by antigens, which is more likely to cause on-target/off-tumor effects.
  • Another strategy is to introduce an inducible suicide gene, such as the "inducible Caspase-9" (iCasp9) suicide gene, when constructing CAR.
  • AP1903 a dimerization chemical inducer that can activate iCasp9 is administered to induce apoptosis of CAR-T cells and reduce toxicity
  • HSV-TK herpes simplex virus thymidine kinase
  • Another benefit of using this adapter switch strategy is that by giving different or even dual-targeted adapter molecules simultaneously or sequentially, CAR-T cells can target different tumor cell antigens simultaneously or successively, thereby potentially overcoming problems such as tumor recurrence and drug resistance caused by tumor heterogeneity. In this case, only the adapter molecule needs to be changed, and the CAR molecule does not need to be changed. Therefore, this "conditionally activated" CAR-T cell has good versatility.
  • the inventors Based on the P329G mod-uCAR-T cell technology, the inventors designed and constructed P329G CAR-T cells that target Claudin18.2 by binding to the Fc domain of an antibody that specifically binds to Claudin18.2 molecules containing the P329G mutation, and verified its specific and controllable anti-tumor effect in vitro and in vivo.
  • the drug combination of the present invention includes two components: Claudin18.2-specific P329G antibody and P329G CAR-T cells.
  • the P329G antibody acts as an adaptor molecule to bridge the P329G CAR-T cells and tumor cells.
  • the P329G CAR-T cells are then directed to tumor cells by recognizing the Fc domain of the P329G antibody, producing target-specific tumor recognition and killing effects (see Figure 1B).
  • the P329G antibody acts as a bridge connecting the P329G CAR-T cells and tumor cells, playing the role of a "molecular switch" and regulating the activity of the P329G CAR-T cells.
  • the CAR-T cell products developed by this technology have the following significant features: 1. By adjusting the frequency, dosage, route and function of antibody administration, the acute and chronic toxic effects of traditional CAR-T cell therapy can be effectively avoided, and the continuous proliferation and survival of CAR-T cells in vivo can be promoted to achieve a lasting anti-tumor effect; 2. By combining and sequentially administering antibodies, clinical pain points such as primary tumor resistance and recurrence after treatment can be effectively overcome without changing CAR-T cells; 3. By combining antibodies targeting different types of tumor antigens, the purpose of treating different tumors can be achieved without changing CAR-T cells. As the antibody drug market is mature and the cost is low, this therapy will help reduce treatment costs and improve the accessibility of CAR-T cell therapy.
  • the present invention provides P329G mutant antibodies that can specifically bind to Claudin18.2 molecules and serve as "molecular switches" for immune effector cells (e.g., T cells, NK cells) expressing CAR polypeptides, including but not limited to HB37A6 PG Ab (also referred to herein as "A6 PG Ab", “A6 PG antibody”, “P329G A6 antibody”).
  • immune effector cells e.g., T cells, NK cells
  • CAR polypeptides including but not limited to HB37A6 PG Ab (also referred to herein as "A6 PG Ab”, “A6 PG antibody”, “P329G A6 antibody”).
  • the antibody that specifically binds to the CLDN18.2 molecule of the present invention comprises 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 SYVMS (SEQ ID NO:25) according to the Kabat numbering; CDR H2 shown by the amino acid sequence TISHSGGSTYYADSVKG (SEQ ID NO:26); and CDR H3 shown by the amino acid sequence DAPYYDILTGYRY (SEQ ID NO:27); the light chain variable region comprises CDR L1 shown by the amino acid sequence RASQSISSWLA (SEQ ID NO:28) according to the Kabat numbering; CDR L2 shown by the amino acid sequence KASSLES (SEQ ID NO:29); and CDR L3 shown by the amino acid sequence QQYNSYSYT (SEQ ID NO:30).
  • the present invention obtains an antibody that specifically binds to the CLDN18.2 molecule, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the sequence of SEQ ID NO:2 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises the sequence of SEQ ID NO:3 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the present invention obtains an antibody that specifically binds to the CLDN18.2 molecule, which comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the sequence of SEQ ID NO:2, and the light chain variable region comprises the sequence of SEQ ID NO:3.
  • the antibody of the present invention that specifically binds to the CLDN18.2 molecule is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody.
  • the amino acid at position P329 according to EU numbering is Mutation of an amino acid to a glycine (G) results in an antibody having a mutated Fc domain, wherein Fc ⁇ receptor binding of the mutated Fc domain is reduced compared to 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;
  • the antibody that specifically binds to the CLDN18.2 molecule comprises a heavy chain constant region sequence shown in SEQ ID NO: 5, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the amino acid at position P329 according to EU numbering is mutated to G;
  • the antibody comprises a heavy chain constant region sequence shown in SEQ ID NO:5, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and wherein the amino acid at position P329 according to EU numbering is mutated to G; and a light chain constant region sequence shown in SEQ ID NO:6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the antibody that specifically binds to the CLDN18.2 molecule comprises the heavy chain constant region sequence shown in SEQ ID NO:5 and the light chain constant region sequence shown in SEQ ID NO:6.
  • the present invention provides a nucleic acid encoding the antibody of the first aspect of the present invention, a vector comprising the nucleic acid encoding the antibody, a cell comprising the nucleic acid molecule or the vector, and a method for preparing the antibody, the method comprising culturing a host cell into which an expression vector encoding the nucleic acid encoding the antibody specifically binding to Claudin18.2 molecule of the first aspect of the present invention is introduced under conditions suitable for expressing the antibody encoding the antibody specifically binding to Claudin18.2 molecule of the first aspect of the present invention, isolating the antibody specifically binding to Claudin18.2 molecule, and optionally the method further comprises recovering the antibody specifically binding to Claudin18.2 molecule from the host cell.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof, and most preferably, the host cell is a HEK293 cell or a CHO cell.
  • the present invention provides a pharmaceutical combination comprising
  • a first component selected from immune effector cells (e.g., T cells, NK cells) expressing a molecular switch-regulated CAR polypeptide, a nucleic acid molecule encoding the CAR polypeptide, a vector comprising the nucleic acid molecule, and any combination thereof; and
  • immune effector cells e.g., T cells, NK cells
  • a second component which is an antibody that specifically binds to Claudin18.2 molecules and comprises a P329G mutation (also referred to as a P329G mutant antibody), for example, the P329G mutant antibody of the first aspect of the present invention, and
  • the molecular switch regulated 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:
  • a heavy chain complementary determining region CDR H1 represented by the amino acid sequence RYWMN (SEQ ID NO: 19), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR H3 represented by the amino acid sequence PYDYGAWFAS (SEQ ID NO:21), or a variant of said CDR H3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:22), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L3 represented by the amino acid sequence ALWYSNHWV (SEQ ID NO: 24), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • CD8 ⁇ hinge region or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:18 or a variant thereof having 1-2 amino acid modifications;
  • TM transmembrane region
  • a co-stimulatory signaling domain selected from a 4-1BB co-stimulatory domain or a variant thereof having 1-5 amino acid modifications, for example, a sequence as shown in SEQ ID NO: 16 or a variant thereof having 1-2 amino acid modifications;
  • a stimulatory signaling domain which is a CD3 ⁇ signaling domain or a variant thereof having 1-10 amino acid modifications, for example, a sequence as shown in SEQ ID NO: 17 or a variant thereof having 1-10 or 1-5 amino acid modifications;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the molecular switch-regulated CAR polypeptide in the pharmaceutical combination of the present invention 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:20).
  • CDR L1 represented by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22);
  • CDR L2 represented by the amino acid sequence GTNCRAP (SEQ ID NO:23);
  • CDR L3 represented by the amino acid sequence ALWYSNHWV (SEQ ID NO: 24);
  • a heavy chain variable region comprising the sequence of SEQ ID NO:12, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and
  • a light chain variable region comprising a sequence of SEQ ID NO: 13 or having at least 90%, 91%, 92%, 93%, sequences with 94%, 95%, 96%, 97%, 98% or 99% identity;
  • TM transmembrane region
  • a co-stimulatory signaling domain selected from the 4-1BB co-stimulatory domain shown in SEQ ID NO: 16 or a variant thereof having one amino acid modification;
  • a stimulatory signaling domain selected from the CD3 ⁇ signaling domain set forth in SEQ ID NO: 17 or a variant thereof having one amino acid modification;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the molecular switch-regulated CAR polypeptide described in the drug combination of the present invention further comprises a signal peptide sequence located at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO:11.
  • the molecular switch regulated CAR polypeptide described in the drug combination of the present invention has the amino acid sequence shown in SEQ ID NO:1 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the present invention provides a nucleic acid encoding the molecular switch regulated CAR polypeptide described in the drug combination of the present invention, a vector comprising a nucleic acid encoding the CAR polypeptide, and a cell comprising the CAR nucleic acid molecule or vector, or a cell expressing the CAR polypeptide, preferably, the cell is an autologous T cell or an allogeneic T cell.
  • the nucleic acid molecule encoding the molecular switch regulated CAR polypeptide described in the drug combination of the present invention is a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the vector comprising the nucleic acid molecule encoding the molecular switch-regulated CAR polypeptide described in the drug combination of the present invention is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.
  • the immune effector cells in the drug combination of the present invention are autologous T cells, NK cells or allogeneic T cells, NK cells prepared from the T cells and NK cells expressing the molecular switch regulated CAR polypeptide of the present invention.
  • the immune effector cells are T cells and NK cells separated from human peripheral blood mononuclear cells (PBMC) and prepared from the T cells and NK cells expressing the molecular switch regulated CAR polypeptide of the present invention.
  • PBMC peripheral blood mononuclear cells
  • the present invention utilizes primary P329G CAR-T cells prepared from human PBMCs from multiple different donor sources, and utilizes P329G mutated anti-Claudin18.2 humanized antibodies to evaluate the effector function of P329G CAR-T cells against tumor cells expressing Claudin18.2 through antibodies in an in vitro co-culture system.
  • the P329G mutated anti-Claudin18.2 antibodies can act as a "molecular switch" to regulate the effector function of P329G CAR-T cells against Claudin18.2-positive tumors.
  • the recognition and killing activity of tumor cells is comparable to that of traditional CAR-T cells that directly target Claudin18.2-positive tumor cells, but the activity of traditional CAR-T cells does not depend on the P329G mutant antibody.
  • the present invention verifies the anti-tumor effect of P329G CAR-T cells combined with P329G mutant antibodies in immunodeficient mice inoculated with tumors derived from human tumor cell lines that express Claudin18.2 positively, and studies the dosage and interval of antibody administration.
  • the immune effector cells expressing the CAR polypeptide of the present invention are administered to the subject as a single intravenous dose at about 50 ⁇ 10 6 cells to about 750 ⁇ 10 6 cells, for example, about 50 ⁇ 10 6 cells, 100 ⁇ 10 6 cells, 150 ⁇ 10 6 cells, 200 ⁇ 10 6 cells, 250 ⁇ 10 6 cells, 300 ⁇ 10 6 cells, 350 ⁇ 10 6 cells, 400 ⁇ 10 6 cells, 450 ⁇ 10 6 cells, 500 ⁇ 10 6 cells, 550 ⁇ 10 6 cells, 600 ⁇ 10 6 cells, 650 ⁇ 10 6 cells, 700 ⁇ 10 6 cells, 750 ⁇ 10 6 cells; and
  • the P329G mutant antibody described in the pharmaceutical combination of the present invention is administered to the subject in the form of a dosage unit of about 0.1-3 mg/kg, preferably about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, preferably parenterally, more preferably intravenously.
  • (i) and (ii) in the pharmaceutical combination of the present invention are administered separately, simultaneously or sequentially, for example, (ii) is administered on the first day, (i) is administered intravenously on the same day or the next day, and then (ii) is administered multiple times at a certain frequency.
  • (i) and (ii) in the pharmaceutical combination of the present invention are administered according to the following regimen:
  • (ii) and (i) are sequentially administered, with an interval of about 1-5 hours (e.g., an interval of about 1, 2, 3, 4 or 5 hours).
  • (ii) is administered on the first day, and (i) is administered with an interval of about 1-5 hours (e.g., an interval of about 1, 2, 3, 4 or 5 hours); or (ii) is administered on the first day and (i) is administered on the second day, with an interval of about 24 hours.
  • the second to nth cycles (ii) is administered every three weeks (Q3W) in each cycle.
  • each dosing cycle is 21 days or 28 days.
  • administration is for at least 2-3 cycles.
  • the drug is administered for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles or more.
  • the present invention provides a drug combination of the present invention or its use, which is used to treat a disease associated with Claudin18.2 in a subject, comprising administering to the subject a therapeutically effective amount of the drug combination defined in the aforementioned third aspect, wherein the disease associated with Claudin18.2 is, for example, a cancer that expresses or overexpresses Claudin18.2, and the cancer is, for example, gastric cancer or pancreatic cancer, for example, advanced gastric cancer or advanced pancreatic cancer.
  • the present invention provides a use of the pharmaceutical combination of the present invention in the preparation of a medicament for treating a disease associated with Claudin18.2, wherein the disease associated with Claudin18.2 is, for example, a cancer that expresses or overexpresses Claudin18.2.
  • the cancer is, for example, gastric cancer or pancreatic cancer, for example, advanced gastric cancer or advanced pancreatic cancer.
  • the present invention provides a method for treating a disease associated with Claudin18.2, the method comprising administering to a subject a therapeutically effective amount of the drug combination of the present invention, wherein the disease is, for example, a cancer that expresses or overexpresses Claudin18.2, such as gastric cancer or pancreatic cancer, such as advanced gastric cancer or advanced pancreatic cancer.
  • a cancer that expresses or overexpresses Claudin18.2 such as gastric cancer or pancreatic cancer, such as advanced gastric cancer or advanced pancreatic cancer.
  • the present invention provides a kit of parts comprising the drug combination as defined in the third aspect above, preferably the kit is in the form of a drug dosage unit.
  • the present invention first obtains a high-affinity Claudin18.2-specific P329G antibody through in vitro binding ability, affinity and Fc effector function detection.
  • the antibody can simultaneously bind to the Claudin18.2 antigen and the P329GCAR molecule to exert a bridging effect.
  • the present invention uses the constructed P329G CAR structural molecule and combines the P329G CAR-T cells prepared by the CAR molecule with the Claudin18.2-specific P329G antibody, and then co-cultures with Claudin18.2-positive MM cells in vitro to construct an in vitro co-culture system.
  • the effect of the P329G antibody as a "molecular switch" to regulate the activity of the P329G CAR-T cells is verified, that is, only in the presence of the P329G mutant antibody, the P329G CAR-T cells can be activated, proliferated, secrete effector cytokines and produce a killing effect, and these effects are P329G antibody dose-dependent. As the antibody dose increases, the recognition and killing effects of the P329G CAR-T cells are enhanced. However, the WT antibody that does not carry the P329G mutation cannot stimulate the effector function of the P329G CAR-T cells.
  • the probability of the subject developing CRS or ICANS is low, for example, less than 50% (CRS) or 20% (ICANS), for example, lower than the existing Claudin18.2 CAR-T product.
  • the therapies of the invention have a good safety and/or tolerability profile.
  • the second component eg, anti-Claudin18.2 antibody
  • the second component is capable of modulating the activity of the first component, in particular the cellular activity of CAR-T cells.
  • the therapy of the present invention can partially relieve the subject's disease.
  • Clinical studies have shown that a total of 4 patients were enrolled, among which the best tumor efficacy of 1 subject was partial response (PR), and the best tumor efficacy of 2 subjects was stable disease (SD).
  • PR partial response
  • SD stable disease
  • the ORR was 25%
  • the DCR was 75%.
  • the drug combination therapy of the present invention achieves relatively good effects in terms of safety: (1) CRS: As of the data cutoff date, no cytokine release syndrome (CRS) occurred in this study; (2) ICANS: As of the data cutoff date, no neurological toxicity events were observed in this study.
  • FIG1 shows the expression of CAR in CD3 + cells, CD8 - (CD4 + )T, and CD8 + T cell subsets detected by flow cytometry after T cells were transduced with 7 types of CAR.
  • Figure 2A shows the binding of WT A6 antibody (i.e., HB37A6 antibody) and P329G mutant A6 antibody to P329G CAR-T cells.
  • FIG2B shows the WT A6 antibody (ie, HB37A6 antibody) and the P329G mutant A6 antibody (ie, HB37A6 PG antibody). Whether the PBMCs can mediate cell killing function through ADCC.
  • Figure 3 shows whether WT A6 antibody (i.e., HB37A6 antibody) and P329G mutant A6 antibody (i.e., HB37A6 PG antibody) mediate the cell killing function of PBMCs through CDC.
  • WT A6 antibody i.e., HB37A6 antibody
  • P329G mutant A6 antibody i.e., HB37A6 PG antibody
  • Figure 4A shows the therapeutic effect of HuR968B CAR-T cells combined with different doses of P329GA6 antibody in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells.
  • Figure 4B shows the changes in mouse body weight in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells and treated with HuR968B CAR-T cells combined with different doses of P329G A6 antibodies.
  • Figure 4C shows the in vivo expansion of HuR968B CAR-T cells induced by the combination of HuR968B CAR-T cells and different doses of P329G A6 antibodies in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells.
  • Figures 5A-5B show the antitumor effects produced by different sequences of administration of HuR968B CAR-T cells and P329GA6 antibodies in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells.
  • FIG6 shows the curve of CAR-T cell PK changes over time.
  • FIG. 7 shows the PK curve of A6 antibody over time.
  • Claudins refers to a class of integrin membrane proteins present in epithelial and endothelial tight junctions. They are important components of tight junctions and were discovered by Shoichiro Tsukita et al. in 1998. The family has 24 members.
  • the human Claudin 18 gene has two optional exons 1, resulting in two protein subtypes, Claudin 18.1 (also referred to as “CLDN18.1” in this article) and Claudin 18.2 (also referred to as "CLDN18.2” in this article), which differ in the sequence of about 50 amino acids in the first extracellular domain by only 7 amino acid residues.
  • Claudin 18.2 There are significant differences in the expression of Claudin 18.2 in cancer tissues and normal tissues, which may be due to the fact that the CpG methylation of the CREB binding site in the promoter region of Claudin 18.2 is high in normal tissues, while the CpG methylation is high in the process of cell carcinogenesis. The level is reduced, and then CREB participates in activating the transcription of Claudin18.2.
  • CLDN18.2 antibody As used herein, the terms “CLDN18.2 antibody”, “antibody against CLDN18.2”, “antibody that specifically binds to CLDN18.2”, “antibody that specifically targets CLDN18.2”, “antibody that specifically recognizes CLDN18.2” are used interchangeably, and refer to antibodies that can specifically bind to the Claudin protein CLDN18.2. In particular, in some specific embodiments, it refers to an antibody that specifically binds to human CLDN18.2, in particular, an antibody that specifically binds to human CLDN18.2 but not to human CLDN18.1.
  • 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.
  • CDRs can also be identified 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.
  • CDRs can also be identified based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention).
  • antibody variable regions and specific CDR sequences including heavy chain variable region residues
  • 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.
  • Humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in the humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies.
  • the humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.
  • human antibody refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region includes at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain.
  • the C-terminal lysine (Lys447) in the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • effector function refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype.
  • immunoglobulin effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK cells are activated by binding of the Fc region of an antibody to an Fc receptor Fc ⁇ RIIIA (i.e., CD16a) expressed on, for example, NK cells. Play an ADCC role.
  • CD16a belongs to the transmembrane receptor member of the immunoglobulin superfamily.
  • CD16a has differential expression of valine or phenylalanine at position 158, so that there are CD16a-158V/V (about 15%), CD16a-158V/F (about 25%) and CD16a-158F/F (about 60%) subtypes in the population.
  • the chimeric antigen receptor of the present invention provides antibody-dependent cellular cytotoxicity of T lymphocytes and enhances the antibody-dependent cellular cytotoxicity of NK cells.
  • the chimeric antigen receptor of the present invention binds to antibodies (or other anti-tumor molecules containing the Fc portion) that bind to tumor cells, thereby inducing activation of T cells expressing the chimeric antigen receptor, continuous proliferation, and exerting specific cytotoxicity to target cancer cells mediated by the antibody (or other anti-tumor molecules containing the Fc portion).
  • complement dependent cytotoxicity refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen.
  • C1q first component of the complement system
  • an antibody of the appropriate subclass
  • a CDC assay can be performed, for example, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).
  • variable region refers to the 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 a protein provided by GenBank Accession No. BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain 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:13 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand and thereby mediates 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, immune Globulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activation NK cell receptor, OX40, CD40, GITR, 4-1BB (i.e. CD137), CD27 and CD28.
  • costimulatory molecule is CD28, 4-1BB (i.e. CD137).
  • Costimulatory signal domain refers to the intracellular part of costimulatory 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: 11 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.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used to determine the identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6.
  • a particularly preferred set of parameters is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).
  • nucleic acid sequences and protein sequences described herein can further use as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.
  • amino acid change and “amino acid modification” are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of the addition, deletion, substitution and other modifications of amino acids can be performed, provided that the final polypeptide sequence has the desired properties.
  • amino acid substitution of the antibody results in reduced binding of the antibody to the Fc receptor.
  • non-conservative amino acid substitutions are particularly preferred, i.e., replacing one amino acid with another amino acid having different structures and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid changes can be produced using genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification) may be useful. A variety of names may be used herein to represent the same amino acid changes. For example, a substitution from proline to glycine at position 329 of the Fc domain can be represented as 329G, G329, G329 , P329GPro329Gly, or simply "PG".
  • conservative sequence modifications and “conservative sequence changes” refer to amino acid modifications or changes that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • ⁇ -side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues in the interior of the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the functional assay described herein can be used to test the changed C
  • autologous refers to any substance that is derived from the same individual into which the substance is later reintroduced.
  • allogeneic refers to any material that is derived from an animal of the same species as the individual into which the material was introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species can be sufficiently genetically dissimilar to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • the term "apheresis” refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor's or patient's circulation, e.g., by retransfusion.
  • apheresis sample a sample obtained using apheresis is referred to.
  • immune effector cell refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector reaction.
  • immune effector cells include T cells, e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid derived phagocytes.
  • 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.
  • the term "disease associated with CLDN18.2” refers to any condition caused by, exacerbated by or otherwise associated with increased expression or activity of CLDN18.2 (such as human CLDN18.2).
  • 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 Pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all Pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all Pre-cancerous 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 targeting CLDN18.2 Mutant antibodies) are administered to subjects simultaneously, without specific time limits or at the same or different time intervals, sequentially as separate entities, wherein such administration provides effective treatment in the subject.
  • fixed combination refers to the combination of CLDN18.2 P329G mutant antibodies and P329G CAR-T cells of the present invention, each of which is administered to the patient simultaneously in the form of a specific single dose.
  • non-fixed combination means that the combination of CLDN18.2 P329G mutant antibodies and P329G CAR-T cells of the present invention is administered to the 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.
  • combination therapy refers to the administration of two or more components to treat cancer as described herein.
  • administration includes co-administration of these components in a substantially simultaneous manner.
  • administration includes co-administration or separate administration or sequential administration of each active ingredient in multiple or separate containers (e.g., capsules, powders, and liquids).
  • the powder and/or liquid can be reconstituted or diluted to the desired dose before administration.
  • administration also includes using the CLDN18.2 P329G mutant antibody and P329G CAR-T cells of the present invention at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • 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.
  • 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 CLDN18.2 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 the antibody Fc domain comprising the P329G mutation to an Fc receptor e.g., an Fc ⁇ receptor
  • 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 H heavy chain complementary determining region 1 represented by the amino acid sequence RYWMN (SEQ ID NO:19), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:22), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • the scFv may be connected to a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO:11, and the scFv may be connected to an optional hinge region/spacer region sequence as provided in SEQ ID NO:14 or SEQ ID NO:18, a transmembrane region as provided in SEQ ID NO:15, an intracellular stimulatory signal domain as provided in SEQ ID NO:17 or its variants, a co-stimulatory signal domain as provided in SEQ ID NO:16 at the C-terminus, for example, wherein the various 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 a sequence of SEQ ID NO: 12, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and (ii) a light chain variable region comprising a sequence of SEQ ID NO: 13, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;
  • the scFv domain comprises (i) a heavy chain variable region set forth in SEQ ID NO: 2 and (ii) a light chain variable region set forth in SEQ ID NO: 3.
  • the scFv domain further comprises a (Gly4-Ser)n (SEQ ID NO: 32) 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.
  • 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, a membrane-proximal intracellular 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.
  • the nucleic acid sequence of the CAR construct of the present invention is selected from 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 CAR polypeptides of the present invention can also be modified so as to change in the amino acid sequence, but not in terms of the desired activity.
  • additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues can be made to the CAR polypeptide.
  • a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family.
  • an amino acid fragment can be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, for example, a conservative substitution can be made in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • the present invention contemplates the production of functionally equivalent CAR polypeptide molecules, for example, the VH or VL of the humanized anti-P329G mutant scFv sequence contained in the CAR can be modified to obtain a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:12 and a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:3.
  • the transmembrane domain included in the CAR of the present invention is an anchored transmembrane domain, which is a component of a polypeptide chain that can be integrated into the cell membrane.
  • the transmembrane domain can be fused with other extracellular and/or intracellular polypeptide domains, wherein these extracellular and/or intracellular polypeptide domains will also be restricted to the cell membrane.
  • the transmembrane domain confers membrane attachment to the CAR polypeptide of the present invention.
  • the CAR polypeptide of the present invention comprises at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising dominant hydrophobic residues such as leucine and valine.
  • the domain may be derived from a membrane-bound protein or a transmembrane protein such as CD28, CD8 (e.g., CD8 ⁇ , CD8 ⁇ ) across Membrane domain.
  • the transmembrane domain comprises the amino acid sequence of SEQ ID NO:15.
  • the transmembrane domain in the CAR of the present invention is connected to the extracellular region of the CAR (i.e., the humanized anti-P329G mutant scFv sequence) by means of a hinge region/spacer region.
  • the hinge can be a human Ig (immunoglobulin) hinge region, for example, an IgG4 hinge region, or a CD8a hinge region, a CD28 hinge region.
  • the hinge region or the spacer region sequence comprises the amino acid sequence of SEQ ID NO: 18.
  • glycine-serine doublets also provide particularly suitable linkers as hinge/spacer regions.
  • the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 14).
  • the cytoplasmic domain included in the CAR of the present invention includes an intracellular signaling domain.
  • the intracellular signaling domain can activate at least one effector function of the immune cell introduced with the CAR of the present invention.
  • intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to the P329G mutated antibody Fc domain, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to the P329G mutated antibody Fc domain, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • the CAR of the present invention is also designed to generate a costimulatory signal domain (CSD).
  • the activation of T cells is mediated by two different cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation through TCR (primary intracellular signaling domains) and those sequences that act in an antigen-independent manner to provide costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).
  • the CAR of the present invention comprises a primary intracellular signaling domain, for example, a primary signaling domain of CD3 ⁇ , for example, a CD3 ⁇ signaling domain shown in SEQ ID NO:17.
  • the intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (ie, a co-stimulatory signaling domain).
  • the co-stimulatory signaling domain refers to the CAR portion of the intracellular domain comprising a co-stimulatory molecule.
  • Co-stimulatory molecules are cell surface molecules required for lymphocytes to effectively respond to antigens in addition to antigen receptors or their ligands.
  • co-stimulatory molecules include but are not limited to CD28, 4-1BB (CD137), and the co-stimulatory effect caused by them enhances the proliferation, effector function and survival of human CART cells in vitro and enhances the anti-tumor activity of human T cells in vivo.
  • Intracellular signaling sequences of the CAR of the present invention can be connected to each other in a random order or in a specified order.
  • short oligopeptide linkers or polypeptide linkers can form bonds between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid, for example, alanine, glycine can be used as a suitable linker.
  • the intracellular signaling domain of the CAR of the present invention is designed to include the costimulatory signaling domain of CD28 and the stimulatory signaling domain of CD3 ⁇ . In another embodiment, the intracellular signaling domain is designed to include the costimulatory signaling domain of 4-1BB and the stimulatory signaling domain of CD3 ⁇ . In another embodiment, the intracellular signaling domain is designed to also include the proximal intracellular signaling domain of the CD3 ⁇ chain.
  • 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 into which the CAR construct of the present invention is inserted.
  • Expression of a natural or synthetic nucleic acid encoding a CAR is achieved by operatively linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector.
  • 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.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • a lentiviral vector is used.
  • Retroviral vectors can also be, for example, ⁇ retroviral vectors.
  • ⁇ retroviral vectors can, for example, include promoters, packaging signals ( ⁇ ), primer binding sites (PBS), one or more (e.g., two) long terminal repeats (LTR) and target transgenics, for example, genes encoding CAR.
  • ⁇ retroviral vectors can lack viral structural genes such as gag, pol and env.
  • a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter.
  • the natural EF1a promoter drives the expression of the ⁇ subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of CAR from a transgene cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17 (8): 1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutive strong promoter sequence that can drive any polynucleotide sequence effectively connected thereto to express at a high level.
  • other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • the present invention provides methods for expressing the CAR constructs of the present invention in mammalian immune effector cells (e.g., mammalian T cells) and the immune effector cells produced thereby.
  • mammalian immune effector cells e.g., mammalian T cells
  • a cell source e.g., immune effector cells, e.g., T cells
  • T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood components collected from a subject using any technique known to those skilled in the art (e.g., Ficoll TM separation).
  • cells from circulating blood of an individual are obtained by apheresis.
  • the apheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis can be washed to remove plasma.
  • the cells are fractionated and placed in an appropriate buffer or medium for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Specific T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • the time period is between about 30 minutes and 36 hours or longer.
  • Longer incubation time can be used to separate T cells in any case where there is a small amount of T cells, such as for separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals.
  • TIL tumor infiltrating lymphocytes
  • the efficiency of capturing CD8+T cells can be increased using longer incubation time.
  • T cell subsets can be preferentially selected at the beginning of culture or at other time points during the culture process.
  • Enrichment of T cell populations can be accomplished by a negative selection process using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One approach is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.
  • the immune effector cell can be an allogeneic immune effector cell, e.g., a T cell.
  • the cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II).
  • TCR functional T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that make up a functional TCR (e.g., engineered so that it does not express or exhibits reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered so that it produces very few functional TCRs on its surface.
  • the T cells described herein can be engineered, for example, so that it does not express functional HLA on its surface.
  • the T cells described herein can be engineered so that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated.
  • HLA e.g., HLA class I and/or HLA class II
  • downregulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
  • the T cell may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
  • cells transduced with a nucleic acid encoding a CAR described herein are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.
  • the CAR-expressing immune effector cells obtained after in vitro proliferation can be tested for effector function as described in the examples.
  • CLDN18 belongs to the Claudins protein family. It is an important molecule that constitutes the tight junction of epithelial cells, determines the permeability of epithelial cells, and also plays a role in blocking the diffusion of proteins and lipids on the cell membrane surface (Gunzel, D. and AS Yu (2013). "Claudins and the modulation of tight junction permeability.” Physiol Rev 93 (2): 525-569).
  • the human CLDN18 gene has two different exons 1. After transcription, it undergoes alternative splicing to ultimately generate two protein subtypes, CLDN18.1 and CLDN18.2, which have different sequences only at the N-terminus.
  • CLDN18 subtypes are It is composed of 261 amino acids and has four transmembrane domains, but the two are distributed in different tissues.
  • CLDN18.1 is mainly expressed in lung tissue, and CLDN18.2 is only expressed in differentiated gastric mucosal epithelial cells, not in gastric stem cells (Sahin, Ugur et al., "Claudin-18splice variant 2 is a pan-cancer target suitable for therapeutic antibody development.” Clinical Cancer Research 14.23 (2008): 7624-7634).
  • CLDN18.2 is highly expressed in a variety of tumor tissues, such as non-small cell lung cancer (25%), gastric cancer (70%), pancreatic cancer (50%) and esophageal cancer (30%), but is almost not expressed in normal tissues (Kumar, V. et al., (2016) "Emerging Therapies in the Management of Advanced-Stage Gastric Cancer.” Front Pharmacol 9:404). Due to its differential expression in tumor cells and normal tissues, it has become a very potential target for anti-tumor drugs.
  • Zolbetuximab is a human-mouse chimeric IgG1 monoclonal antibody that specifically targets CLDN18.2. It binds to the first extracellular region of CLDN18.2 expressed on tumor cells and induces tumor cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the heavy chain variable region comprises CDR H1 shown in the amino acid sequence SYVMS (SEQ ID NO:25) according to Kabat numbering, or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR H2 shown in the amino acid sequence TISHSGGSTYYADSVKG (SEQ ID NO:26), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR H3 shown in the amino acid sequence DAPYYDILTGYRY (SEQ ID NO:27), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change.
  • a variant of said light chain variable region comprising a CDR L1 shown in the amino acid sequence RASQSISSWLA (SEQ ID NO:28) according to Kabat numbering, or a variant of said CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; a CDR L2 shown in the amino acid sequence KASSLES (SEQ ID NO:29), or a variant of said CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and a CDR L3 shown in the amino acid sequence QQYNSYSYT (SEQ ID NO:30), or a variant of said CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid changes are amino acid additions, deletions or conservative amino acid substitutions.
  • the antibodies of the invention that bind to CLDN18.2 molecules bind to mammalian CLDN18.2, such as human CLDN18.2.
  • the antibodies of the invention that bind to CLDN18.2 molecules have one or more of the following properties:
  • the antibodies of the present invention that bind to CLDN18.2 molecules include antibodies that specifically bind to CLDN18.2.
  • the heavy chain variable region comprises a sequence of SEQ ID NO:2, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • the light chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • amino acid changes in the sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are preferably amino acid substitutions, more preferably conservative amino acid substitutions, and preferably, the amino acid changes do not occur in the CDR regions.
  • the antibody of the present invention that binds to a CLDN18.2 molecule is an IgG1, IgG2, IgG3, or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody, for example, a human IgG1 antibody.
  • the antibody that binds to the CLDN18.2 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 CLDN18.2 molecules and contain a P329G mutant Fc domain cannot exert antibody-dependent cellular cytotoxicity by binding to Fc ⁇ receptors, nor can they exert complement-dependent cytotoxicity, and therefore cannot kill CLDN18.2-positive cancer cells.
  • the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any of their chains that bind to the CLDN18.2 molecule.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the nucleic acid of the present invention comprises a nucleic acid encoding an antibody that binds to a CLDN18.2 molecule of the present invention.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • the vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).
  • the vector is a pcDNA3.4 expression vector.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion the cells are cultivated in a culture medium and screened for suitable activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the specific expression vector and mammalian host cell used. Variation or optimization.
  • cells that have stably incorporated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.
  • the markers can, for example, provide prototrophy, biocidal resistance, or (e.g., antibiotics) or heavy metals (e.g., copper) resistance, etc.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.
  • a host cell comprising a polynucleotide of the present invention.
  • a host cell comprising an expression vector of the present invention is provided.
  • the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli.
  • the host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells, etc. Vertebrate cells can also be used as hosts.
  • a mammalian cell line modified to be suitable for suspension growth can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), Chinese hamster ovary cells (CHO cells), CHO-S cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc.
  • COS-7 SV40 transformed monkey kidney CV1 line
  • HEK293 or 293F cells 293 cells
  • BHK baby hamster kidney cells
  • CV1 African green monkey kidney cells
  • HELA human cervical carcinoma cells
  • MDCK buffalo rat liver cells
  • W138 human lung cells
  • HepG2 human liver cells
  • CHO cells
  • the host cell is a CHO cell or a HEK293 cell.
  • the present invention provides a method for preparing an antibody that binds to a CLDN18.2 molecule (including a P329G mutant antibody), wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody that binds to a CLDN18.2 molecule (including a P329G mutant antibody) or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the antibody that binds to a CLDN18.2 molecule (including a P329G mutant antibody), and optionally isolating the antibody that binds to a CLDN18.2 molecule (including a P329G mutant antibody).
  • the method further comprises recovering the antibody that binds to a CLDN18.2 molecule (including a P329G mutant antibody) from the host cell (or host cell culture medium).
  • Antibodies (including P329G mutant antibodies) that bind to CLDN18.2 molecules of the present invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art.
  • the purity of antibodies (including P329G mutant antibodies) that bind to CLDN18.2 molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.
  • the antibodies (including P329G mutant antibodies) provided herein that bind to CLDN18.2 molecules can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the antibodies (including P329G mutant antibodies) that bind to CLDN18.2 molecules of the present invention are tested for their antigen binding activity, for example, by known methods such as FACS, ELISA or Western blotting. Binding to CLDN18.2 can be determined using methods known in the art, and exemplary methods are disclosed herein.
  • FACS is used to determine the binding of antibodies (including P329G mutant antibodies) that bind to CLDN18.2 molecules of the present invention to cell surface CLDN18.2 (eg, human CLDN18.2).
  • the present invention also provides antibodies (including P329G
  • the biological activity may include, for example, ADCC effect, CDC effect, etc.
  • Cells used in any of the above in vitro assays include cell lines that naturally express CLDN18.2 or are engineered to express CLDN18.2.
  • the cell lines engineered to express CLDN18.2 are cell lines that do not normally express CLDN18.2 but express CLDN18.2 after transfection of DNA encoding CLDN18.2 into the cells.
  • the molecular switch regulated chimeric antigen receptor of the present invention is a regulatable CAR that can control the activity of CAR.
  • the present invention uses the Pro329Gly (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) an immune effector cell (e.g., T cell) expressing a molecular switch-regulated CAR polypeptide of the present invention; and (ii) a P329G mutant antibody that specifically binds to the CLDN18.2 molecule.
  • the immune effector cell is a T cell expressing a molecular switch-regulated CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells, for example, the immune effector cell is a T cell expressing a molecular switch-regulated CAR polypeptide of the present invention prepared from T cells isolated from human PBMC.
  • the P329G mutant antibody is HB37A6 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 the CLDN18.2 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, U.S.A).
  • Pharmaceutically acceptable adjuvants can be exemplified by surfactants, excipients, colorants, flavors, 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 triglycerides, polyoxyethylene hardened castor oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc. as carriers, but are not limited thereto.
  • the drug combination of the present invention is used to treat a disease associated with CLDN18.2, such as a cancer that expresses or overexpresses CLDN 18.2, such as a CLDN18.2-positive solid tumor.
  • the present invention provides the aforementioned pharmaceutical combination of the present invention for treating a disease associated with CLDN18.2 in a subject, wherein the disease associated with CLDN18.2 is, for example, a cancer expressing or overexpressing CLDN 18.2, and the cancer is, for example, a CLDN18.2-positive solid tumor.
  • the disease associated with CLDN18.2 is, for example, a cancer expressing or overexpressing CLDN 18.2
  • the cancer is, for example, a CLDN18.2-positive solid tumor.
  • the drug combination of the present invention is used to treat a cancer that expresses or overexpresses CLDN 18.2 (e.g., a CLDN18.2-positive solid tumor) in a subject and is capable of reducing the severity of at least one symptom or indication of the cancer or inhibiting the growth of cancer cells.
  • CLDN 18.2 e.g., a CLDN18.2-positive solid tumor
  • the present invention provides a method for treating a disease associated with CLDN18.2 (e.g., a cancer that expresses or overexpresses CLDN 18.2, e.g., the cancer is a CLDN18.2-positive solid tumor) in a subject, comprising administering a therapeutically effective amount of a drug combination of the present invention to an individual in need thereof.
  • a disease associated with CLDN18.2 e.g., a cancer that expresses or overexpresses CLDN 18.2, e.g., the cancer is a CLDN18.2-positive solid tumor
  • the present invention provides the use of the aforementioned drug combination of the present invention in the preparation of a drug for treating a disease associated with CLDN18.2 (e.g., a cancer that expresses or overexpresses CLDN 18.2, e.g., the cancer is a CLDN18.2-positive solid tumor).
  • a disease associated with CLDN18.2 e.g., a cancer that expresses or overexpresses CLDN 18.2, e.g., the cancer is a CLDN18.2-positive solid tumor.
  • the pharmaceutical combinations of the invention may also be administered to individuals whose cancer has been treated with one or more prior therapies but has subsequently relapsed or metastasized.
  • the drug combination of the present invention expresses the immune effector cells (e.g., T cells) of the molecular switch regulated CAR polypeptide of the present invention and (ii) specifically binds to the P329G mutant antibody of the CLDN18.2 molecule for parenteral, percutaneous, intracavitary, intraarterial, intravenous, intrathecal administration, or directly injected into a tissue or tumor.
  • the immune effector cells e.g., T cells
  • the P329G mutant antibody of the CLDN18.2 molecule specifically binds to the P329G mutant antibody of the CLDN18.2 molecule for parenteral, percutaneous, intracavitary, intraarterial, intravenous, intrathecal administration, or directly injected into a tissue or tumor.
  • the drug combination of the present invention (ii) specifically binds to the P329G mutant antibody of the CLDN18.2 molecule before, simultaneously with, or after (i) expressing the immune effector cells (e.g., T cells) of the molecular switch regulated CAR polypeptide of the present invention.
  • the immune effector cells e.g., T cells
  • the immune effector cell expressing the molecular switch-regulated CAR polypeptide of the present invention in the drug combination of the present invention is a T cell expressing the CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells;
  • the P329G mutant antibody that specifically binds to the CLDN18.2 molecule in the drug combination of the present invention is any antibody that specifically binds to the CLDN18.2 molecule, which contains the P329G mutation.
  • the P329G mutant antibody is HB37A6 PG Ab.
  • administering the drug combination of the present invention to an individual suffering from cancer results in the complete disappearance of a tumor. In some embodiments, administering the drug combination of the present invention to an individual suffering from cancer results in a reduction of tumor cells or tumor size by at least 85% or more.
  • the reduction of a tumor can be measured by any method known in the art, such as X-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.
  • the drug combination of the present invention can reduce the "on-target/off tumor" toxicity of CAR-T cells.
  • the present invention relates to a method of treating multiple myeloma in an individual, comprising administering to the individual a drug combination comprising
  • the first component for example, is a T cell expressing a molecular switch-regulated CAR polypeptide, i.e., a CAR-T cell.
  • a second component for example, an antibody or antigen-binding fragment that specifically binds to Claudin18.2 molecules and comprises a P329G mutation, wherein the P329G mutant antibody comprises a mutant Fc domain, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G).
  • the method comprises administering the first component at a dose of about 50 ⁇ 10 6 cells to about 750 ⁇ 10 6 cells, for example, about 50 ⁇ 10 6 cells, 100 ⁇ 10 6 cells, 150 ⁇ 10 6 cells, 200 ⁇ 10 6 cells, 250 ⁇ 10 6 cells, 300 ⁇ 10 6 cells, 350 ⁇ 10 6 cells, 400 ⁇ 10 6 cells, 450 ⁇ 10 6 cells, 500 ⁇ 10 6 cells, 550 ⁇ 10 6 cells, 600 ⁇ 10 6 cells, 650 ⁇ 10 6 cells, 700 ⁇ 10 6 cells, 750 ⁇ 10 6 cells are administered to a subject.
  • the administration is a single administration.
  • the administration is intravenous administration.
  • the method comprises administering the second component at a dose of about 0.1-3 mg/kg, preferably about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg.
  • the administration is parenteral, more preferably intravenous, to the subject.
  • the first component and the second component are administered in the following regimen:
  • (i) and (ii) in the pharmaceutical combination of the present invention are administered separately, simultaneously or sequentially, for example, (ii) is administered on the first day, (i) is administered intravenously on the same day or the next day, and then (ii) is administered multiple times at a certain frequency.
  • (i) and (ii) in the pharmaceutical combination of the present invention are administered according to the following regimen:
  • the second component is applied on the first day, and the first component is applied at an interval of about 1-5 hours (e.g., at an interval of about 1, 2, 3, 4 or 5); or the second component is applied on the first day, and the first component is applied on the second day, with an interval of about 24 hours;
  • the second component was administered every 3 weeks (Q3W) in the first cycle;
  • Cycle 2 to Cycle n Administer the second component every three weeks (Q3W) in each cycle.
  • each dosing cycle is 21 days or 28 days.
  • administration is for at least 2-3 cycles.
  • the drug is administered for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles or more.
  • a lymphodepletion (LD) regimen is administered to the individual.
  • a lymphodepletion (LD) regimen is administered to the individual prior to administration of the second component.
  • the lymphodepletion regimen is administered prior to administration of the second component, for example, 1, 2, 3, 4, 5, or 6 days prior to administration.
  • the duration of LD is about 1 to 5 days, for example, about 3 days.
  • the time window between the end of LD and the start of administration of the second component, such as a CLDN18.2 antibody is 2 days or 3 days or 4 days.
  • LD is started about 3 to 5 days prior to administration of a dose of a second component, such as a CLDN18.2 antibody.
  • LD is initiated about 3, 4, or 5 days prior to administration of a dose of the second component, such as an anti-Claudin18.2 antibody.
  • a dose of the second component, such as an anti-Claudin18.2 antibody is administered about 1, 2, or 3 days after the end of LD.
  • LD comprises fludarabine, cyclophosphamide, and nab-paclitaxel.
  • the first component such as CAR-T cells
  • the first component is prepared within about 20-40 days before the second component of the present invention is administered.
  • the first component such as CAR-T cells
  • the first component is prepared within about 25-35 days.
  • the first component comprises T cells.
  • T cells are autologous T cells, such as T cells obtained from blood components collected from the subject.
  • the present invention relates to a pharmaceutical combination for use in the methods of treatment of the present invention.
  • the subject after administration of the therapy of the present invention, the subject has a low probability of developing CRS or ICANS,
  • the therapies of the invention have a good safety and/or tolerability profile.
  • the second component e.g., an anti-CLDN18.2 antibody
  • the second component is capable of modulating the activity of the first component, particularly Don't
  • the therapy of the present invention can partially alleviate the disease in the subject.
  • Clinical studies have shown that a total of 5 cases
  • the best tumor response was partial response (PR) in one subject and stable disease (SD) in two subjects.
  • the present invention provides a kit comprising the drug combination of the present invention, preferably the kit is in the form of a drug dosage unit, so that the dosage unit can be provided according to the dosage regimen or the drug administration interval.
  • kit of parts of the present invention comprises in the same package:
  • an immune effector cell e.g., T cell
  • a molecular switch-regulated CAR polypeptide of the present invention e.g., T cell
  • a nucleic acid molecule encoding a molecular switch-regulated CAR polypeptide of the present invention
  • a vector comprising the nucleic acid, and any combination thereof
  • a P329G CAR molecule (SEQ ID NO: 1), also known as HuR968B CAR, was constructed, which was fused with a signal peptide (SP) as shown in SEQ ID NO: 11, a specific single-chain antibody fragment recognizing the P329G antibody (VH-linker-VL, having the VH shown in SEQ ID NO: 12, the linker sequence shown in SEQ ID NO: 33, and the VL shown in SEQ ID NO: 13), a (G4S) 4 hinge region as shown in SEQ ID NO: 14, a CD8 transmembrane domain (CD8TM) as shown in SEQ ID NO: 15, a 41BB co-stimulatory domain (41BB-CSD) as shown in SEQ ID NO: 16, and a CD3 ⁇ molecule intracellular activation domain (CD3 ⁇ SSD) as shown in SEQ ID NO: 17.
  • SP signal peptide
  • VH-linker-VL a specific single-chain antibody fragment recognizing the P329G antibody
  • Blue21 CAR (SEQ ID NO: 8) 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: 11, the anti-BCMA single-chain antibody (from the 11D53 clone), the hinge region of the CD8 ⁇ molecule shown in SEQ ID NO: 18, the CD8 transmembrane domain shown in SEQ ID NO: 15, the 4-1BB costimulatory domain shown in SEQ ID NO: 16, and the CD3 ⁇ chain intracellular activation domain shown in SEQ ID NO: 17.
  • the DNA fragments encoding the CAR polypeptide were inserted into the pRK lentiviral expression vector (pRRLSIN.cPPT.PGK-GFP.WPRE vector (Addgene, 12252, purchased from Biowind) by replacing the promoter and resistance
  • the EGFR sequence in the vector was replaced by the downstream of the EF1 ⁇ promoter of the vector (gene modified), and the CAR expression plasmids pRK-HuR968B and pRK-Blue21 targeting the P329G mutation or various controls were obtained.
  • the CAR expression plasmid prepared in 1-1 was transfected into Lenti-X-293T cells (Takara) with the structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), the regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) and the envelope plasmid pMD2G (Addgene, 12259, purchased from Biowind) at a mass ratio of 3:3:2:2 using the PEI transfection method. 16 hours after transfection, the medium was replaced with fresh DEME medium containing 2% fetal bovine serum (FBS). After 48 hours of 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 the lentivirus concentrate, which was packaged and frozen at -80°C.
  • the revived PBMCs were sorted using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to obtain T cells, which were resuspended to a certain density using T cell culture medium and activated by adding TransAct (Miltenyi, 130-111-160).
  • T cells On the first day, a certain amount of T cells were separated and cultured without adding lentivirus. These cells were untransduced cells (UNT, un-transduced T cells). Different types of lentivirus concentrates obtained from Example 2-1 were added to the remaining cells at an MOI of 1 to 5, and the T cells were blown evenly; on the second day, the virus supernatant was removed by centrifugation, and the cells were resuspended in fresh T cell culture medium.
  • lentivirus concentrates obtained from Example 2-1 were added to the remaining cells at an MOI of 1 to 5, and the T cells were blown evenly; on the second day, the virus supernatant was removed by centrifugation, and the cells were resuspended in fresh T cell culture medium.
  • UNT cells were not operated on; on the third day, all cells were transferred to G-Rex (WILSONWOLF, item number 80040S), and an appropriate amount of fresh T cell culture medium was added, and placed in a 37°C CO 2 incubator for static culture; every 2 to 3 days, the cells were replaced with fresh culture medium at half the amount of the culture medium or IL-2 was directly added, wherein IL-2 was added to the cell culture medium concentration of 200 IU/ml.
  • the number of cells expanded to about 20-80 times the cells were harvested after meeting the requirements (generally reaching 2 to 8x10 8 cells). After centrifugation to remove the culture medium, CAR-T cells were used CS10 (Stemcell, 07930) was resuspended and aliquoted, and then programmed to cool to -80°C for cryopreservation.
  • FIG 1 shows the expression of CAR in CD3+ T cells, CD8- (CD4+) T cells, and CD8+ T cell subsets after T cells were transduced with two types of CAR constructed by 1-1. The results showed that the positive rate of CAR expression in these transduced T cells was approximately 15% to 60%.
  • the CLDN18.2 antibody HB37A6 also referred to as A6 antibody in this article
  • cloned light and heavy chain variable regions (SEQ ID NO: 2, SEQ ID NO: 3) were obtained from the patent (patent application number: PCT/CN2021/100870).
  • the nucleotide sequences of the light and heavy chain variable regions of the antibody were synthesized by whole gene and loaded into the pcDNA3.4 expression vector (purchased from Shanghai Boying) containing the human IgG1 heavy chain constant region of WT (SEQ ID NO: 4) or the human IgG1 heavy chain constant region containing the P329G point mutation (SEQ ID NO: 5) and the ⁇ 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 the antibody was purified by a Protein A column and then dialyzed with PBS. The concentration was detected by reading the absorbance at 280 nm using a NanoDrop instrument, and the purity of the samples was detected by SDS-PAGE and SEC-HPLC.
  • P329G CAR-T cells prepared in Example 1 were resuscitated, resuspended in RPMI 1640 medium containing 10% FBS, and cultured at 37°C for 24 hours.
  • P329G A6 antibody and wild-type antibody were prepared into antibody solutions of different concentrations with 5-fold gradient dilution using FACS buffer, and incubated with 1E5 P329G CAR-positive cells at 4°C for 30 minutes, washed with FACS buffer, and then incubated with APC goat anti-human IgG, Fc ⁇ fragment specific (Jackson ImmunoResearch, 109-136-098) at 4°C for 30 minutes.
  • Flow cytometry was used to detect antibodies bound to P329G CAR-T cells, and the APC channel MFI was analyzed.
  • the antibody concentration was used as the X-axis and the APC channel MFI was used as the Y-axis to draw a plot and calculate the EC50 of the binding.
  • FIG. 2A shows the binding of WT and P329G mutant A6 antibodies to P329G CAR-T cells.
  • the results indicate that only the P329G mutant antibody (sometimes referred to as "PG antibody” or “PG Ab” in the text) showed binding to P329G CAR, while the WT antibody (WT Ab) did not.
  • PG antibody sometimes referred to as "PG antibody” or "PG Ab” in the text
  • PBMC cells Peripheral Blood Mononuclear Cells
  • PBMC and target cells DAN-G18.2 tumor cells
  • the antibody-mediated killing effect of PBMC on target cells was detected using an LDH detection kit (Promega, G1780), and the antibody concentration was used as the X-axis and the cell lysis ratio was used as the Y-axis for plotting and analysis.
  • Figure 2B shows whether WT and P329G mutant A6 antibodies mediate cell killing by PBMCs through ADCC.
  • PBMCs from donor 3 were tested, and the results showed that only WT antibodies mediated the ADCC killing effect of PBMCs on CLDN18.2-positive DAN-G18.2 tumor cells, while P329G mutant antibodies lacked the ability to induce PBMCs to exert ADCC effects on CLDN18.2-positive DAN-G18.2 tumor cells.
  • P329G A6 antibody and wild-type A6 antibody were incubated with target cells (DAN-G18.2 tumor cells) at 37°C for 30 minutes, and then 10 ⁇ l of human serum complement (Sigma, S1764-1ML) was added and cultured for another 3 hours.
  • the proportion of live cells was detected using CellTiter-Glo (Promega, G9242), and a graph was drawn with the antibody concentration as the X-axis and the proportion of live cells as the Y-axis to analyze the CDC effect of the antibody.
  • FIG. 2C shows whether WT and P329G mutant A6 antibodies mediate complement cell killing function through CDC.
  • the results showed that only WT antibody mediated the CDC killing effect on CLDN18.2-positive DAN-G18.2 tumor cells, while the P329G mutant antibody lacked the ability to induce CDC effect.
  • Example 3 Study on the anti-tumor effect of HuR968B CAR-T cells combined with A6 antibody after single administration in vivo
  • DAN-G18.2 cells were resuspended in 1x PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 /mL.
  • NOG mice were shaved on the right back and subcutaneously injected with 5 ⁇ 10 6 /mL DAN-G18.2 cell suspension, 0.2 mL/mouse, that is, the inoculation amount was 1 ⁇ 10 6 /mouse.
  • mice with tumor volumes ranging from 72.13 to 113.94 mm 3 were divided into 5 groups, including vehicle group, 8B CAR-T group only (HuR968B CAR-T is abbreviated as "8B CAR-T"), 8B CAR-T+A6-PG (0.03 mg/kg) group, 8B CAR-T+A6-PG (0.1 mg/kg) group and 8B CAR-T+A6-PG (0.3 mg/kg) group, with 7-14 mice in each group. Resuspend 8B CAR-T cells in 1x PBS to prepare a cell suspension of 2.5 ⁇ 10 7 /mL of 8B CAR + cells.
  • FIG. 4A shows the therapeutic effect of HuR968B CAR-T cells combined with different doses of P329G A6 antibody in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells.
  • the results showed that HuR968B CAR-T cells induced significant anti-tumor effects when a dose of P329G A6 antibody as low as 0.03 mg/kg was given at the same time.
  • the maximum anti-tumor effect induced by HuR968B CAR-T cells did not increase significantly when the dose of P329G A6 antibody was increased to 0.1 and 0.3 mg/kg, but the anti-tumor effect was maintained for a longer time.
  • TGI tumor growth inhibition rate
  • Figure 4B shows the changes in the weight of mice in this experiment.
  • the results showed that the weight of mice treated with HuR968B CAR-T cells and P329G A6 antibodies began to decrease after treatment, and dropped to the lowest point 9 days after the simultaneous administration of P329G A6 antibodies.
  • the weight of the 0.03, 0.1, and 0.3 mg/kg treatment groups decreased by an average of 13.5%, 20.6%, and 24.4%. Afterwards, the weight of the mice began to recover.
  • the weight of the mice in the 0.03 and 0.1 mg/kg treatment groups returned to normal within 4 days, and the weight of the mice in the 0.3 mg/kg treatment group also returned to normal levels after 1 week.
  • the results showed that HuR968B CAR-T cells combined with low-dose P329G A6 antibody treatment induced significant anti-tumor effects, but also produced short-term tolerable toxic side effects.
  • the method is the same as 3-1.
  • FIG. 4C shows the proliferation of HuR968B CAR-T cells in mice in the experiment of 3-1.
  • the results showed that HuR968B CAR-T cells began to proliferate one week after being infused back into the mice, reached a peak level after two weeks, and then rapidly declined, returning to the baseline level after three weeks.
  • the expansion of CAR-T cells in vivo depends on the P329G A6 antibody, and mice in the group not given the antibody at the same time did not show obvious cell proliferation; in addition, CAR-T cell proliferation showed a certain P329G A6 antibody dose dependence, and mice in the higher dose group had a higher level of CAR-T cell proliferation.
  • the peak proliferation levels of the 0.03, 0.1, and 0.3 mg/kg dose groups were 121783, 135765, and 180927 cells/100 ⁇ l peripheral blood, respectively, which were much higher than the group not given the antibody at the same time (12160 cells/100 ⁇ l peripheral blood).
  • Example 4 Study on the anti-tumor effect of HuR968B CAR-T cells combined with A6 antibody at different administration sequences in vivo
  • DAN-G18.2 cells were resuspended in 1x PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 /mL.
  • NOG mice were shaved on the right back and subcutaneously injected with 5 ⁇ 10 6 /mL DAN-G18.2 cell suspension, with an injection volume of 0.2 mL/mouse, that is, the inoculation amount was 1 ⁇ 10 6 /mouse.
  • mice with tumor volumes ranging from 62.05 to 122.04 mm 3 were divided into 6 groups, including vehicle group, 8B CAR-T group only, 8B CAR-T+A6-PG (0.03 mg/kg) group, 8B CAR-T+A6-PG (0.1 mg/kg) group, A6-PG (0.03 mg/kg)+8B CAR-T group, and A6-PG (0.1 mg/kg)+8BCAR-T group, with 7 mice in each group.
  • 8B CAR-T cells were resuspended in 1x PBS to prepare 8B CAR+ cells at 2.5 ⁇ 10 7 /mL cell suspension.
  • mice in the 8B CAR-T+A6-PG (0.03 mg/kg) group and the 8BCAR-T+A6-PG (0.1 mg/kg) group were injected with 0.2 mL/mouse of cell suspension via tail vein; mice in the A6-PG (0.03 mg/kg)+8B CAR-T group and the A6-PG (0.1 mg/kg)+8B CAR-T group were administered with antibodies at doses of 0.03 mg/kg and 0.1 mg/kg, respectively, by intraperitoneal injection.
  • mice in the 8BCAR-T+A6-PG (0.03 mg/kg) and 8B CAR-T+A6-PG (0.1 mg/kg) groups were given antibodies by intraperitoneal injection; the mice in the A6-PG (0.03 mg/kg)+8B CAR-T and A6-PG (0.1 mg/kg)+8B CAR-T groups were injected with 0.2 mL of cell suspension per mouse via the tail vein.
  • the body weight of the mice, the maximum long axis (L) and the maximum wide axis (W) of the tumor tissue were monitored twice a week.
  • Figures 5A and 5B show the anti-tumor effects of different HuR968B CAR-T cells and P329G A6 antibody administration sequences in immunodeficient tumor-bearing mice inoculated with human DAN-G18.2 tumor cells.
  • the active components of the test drug described in this example are: P329G Claudin18.2 antibody (HB37A6 PG antibody, which comprises the heavy chain variable region shown in SEQ ID NO:2 and the heavy chain constant region shown in SEQ ID NO:5, as well as the light chain variable region shown in SEQ ID NO:3 and the light chain constant region shown in SEQ ID NO:6) and P329G CAR-T cells (CAR-T cells obtained by transforming the patient's autologous PBMC cells).
  • HB37A6 PG antibody which comprises the heavy chain variable region shown in SEQ ID NO:2 and the heavy chain constant region shown in SEQ ID NO:5, as well as the light chain variable region shown in SEQ ID NO:3 and the light chain constant region shown in SEQ ID NO:6
  • P329G CAR-T cells CAR-T cells obtained by transforming the patient's autologous PBMC cells.
  • P329G Claudin18.2 antibody is 10.0mg/ml recombinant anti-tight junction protein 18.2 (Claudin 18.2) monoclonal antibody HB37A6, 0.53mg/ml histidine, 1.38mg/ml histidine hydrochloride, 29.00mg/ml sorbitol, 10.53mg/ml arginine hydrochloride, 0.01mg/ml edetate disodium, 0.5mg/ml polysorbate 80, pH 5.8; the specification of P329G Claudin18.2 antibody is 100mg (10mL)/bottle.
  • the manufacturer of P329G Claudin18.2 antibody is Innovent Biologics (Suzhou) Co., Ltd.
  • P329G CAR-T cells Each bag contains PG CAR-T cells dissolved in 5% to 7.5% DMSO cryoprotectant; the manufacturer of P329G CAR-T cells is Innovent Biologics (Suzhou) Co., Ltd.
  • Claudin18.2 PG CAR the drug combination comprising HB37A6 PG antibody and P329G CAR-T cells.
  • MMSE Mini-Mental Status Exam
  • ANC Routine blood test: ANC ⁇ 1.5 ⁇ 10 9 /L; platelet count ⁇ 75 ⁇ 10 9 /L; hemoglobin content ⁇ 90g/L; Note: The subject must not have received blood product (including red suspension, single platelet, cryoprecipitate, etc.) transfusion, erythropoietin or colony stimulating factor support within 7 days before blood sample collection;
  • blood product including red suspension, single platelet, cryoprecipitate, etc.
  • TBIL serum total bilirubin
  • UPN upper limit of normal value
  • ALT and AST ⁇ 2.5 ⁇ ULN.
  • Patients with confirmed Gilbert syndrome are required to have TBIL ⁇ 3 ⁇ ULN;
  • Glucocorticoids as a preventive medication for allergic reactions (such as medication before CT).
  • CMOS central nervous system
  • asymptomatic brain metastasis i.e., no neurological symptoms, no need for glucocorticoid treatment, and brain metastasis lesions ⁇ 1.5 cm
  • patients with stable symptoms after treatment of brain metastasis clinically stable for at least 4 weeks, with definite clinical evidence of no new or enlarged brain metastasis lesions, and cessation of corticosteroid and anticonvulsant treatment for at least 14 days before mononuclear cell collection
  • the brain needs to be regularly examined as a site of disease.
  • the central nervous system is not a measurable lesion.
  • Subjects with active autoimmune or inflammatory diseases include inflammatory bowel disease (such as ulcerative colitis, Crohn's disease, etc.), celiac disease, systemic lupus erythematosus, Sarcoidosis syndrome or Wegener syndrome (granulomatosis with polyangiitis), Graves' disease, rheumatoid arthritis, hypophysitis, uveitis, etc.], or a history of the disease in the past 2 years (subjects with vitiligo, psoriasis, alopecia or Graves' disease that do not require systemic treatment in the past 2 years, hypothyroidism that only requires thyroid hormone replacement therapy, and type 1 diabetes that only requires insulin replacement therapy can be included in the group).
  • Subjects with only positive autoimmune antibodies need to confirm whether they have autoimmune diseases based on the researcher's judgment.
  • Acute or chronic active hepatitis B (defined as positive hepatitis B surface antigen and/or hepatitis B core antibody and hepatitis B virus DNA copy number ⁇ 1 ⁇ 103 copies/ml or ⁇ 200 IU/ml or above the detection limit) or acute or Chronic active hepatitis C virus (HCV) antibody positive (HCV antibody positive but RNA negative subjects are allowed to enter the group); cytomegalovirus (CMV) DNA positive; syphilis positive.
  • HCV chronic active hepatitis C virus
  • HIV 1/2 antibody positive Human immunodeficiency virus
  • Symptomatic congestive heart failure (New York Heart Association grade II to IV), symptomatic or poorly controlled arrhythmias.
  • Any arterial thromboembolic event including myocardial infarction, unstable angina, cerebrovascular accident, or transient ischemic attack, occurred within 6 months before mononuclear cell collection.
  • the tumor invades surrounding important organs (such as large blood vessels in the mediastinum, superior vena cava, trachea, esophagus, etc.) or there is a risk of gastrointestinal/respiratory tract fistula.
  • Pleural effusion, ascites, and pericardial effusion with clinical symptoms that require drainage intervention h. Pleural effusion, ascites, and pericardial effusion with clinical symptoms that require drainage intervention.
  • Subjects with esophageal or gastric varices requiring immediate intervention e.g., banding or sclerotherapy
  • who are considered at high risk of bleeding based on the opinion of the investigator or consultation with a gastroenterologist or hepatologist, with evidence of portal hypertension (including splenomegaly on imaging studies), or with a history of variceal bleeding must undergo endoscopic evaluation within 3 months prior to enrollment.
  • Subjects at risk for intestinal obstruction or intestinal perforation including but not limited to history of acute diverticulitis, intra-abdominal abscess, and intra-abdominal cancer) or history of the following diseases: inflammatory bowel disease or extensive intestinal resection (partial colectomy or extensive small bowel resection complicated by chronic diarrhea), Crohn's disease, ulcerative colitis, or chronic diarrhea.
  • the subject is known to have had a severe allergic reaction to other monoclonal antibodies in the past, or is allergic to any ingredient of the preparation.
  • P329G CAR-T cells The subjects enrolled in the study will undergo peripheral blood mononuclear cell (PBMC) apheresis (D-33 to D-26 (-33 days to -26 days)), and then use the subjects' own T cells to prepare P329G CAR-T cell preparations (hereinafter referred to as P329G CAR-T cells).
  • PBMC peripheral blood mononuclear cell
  • P329G CAR-T cells P329G CAR-T cells preparations
  • the researcher is allowed to choose an appropriate regimen for bridging therapy (chemotherapy/radiotherapy), but it must be completed at least 14 days before the study administration.
  • the subjects will receive a lymphoproliferative conditioning regimen (D-5 to D-3 (-5 days to -3 days)).
  • the specific lymphoproliferative conditioning regimen is: cyclophosphamide 250mg/m 2 , administered for 3 days; fludarabine 25mg/m 2 , administered for 3 days; albumin paclitaxel 100mg,
  • the patients were given medication for 1 day. After two days of rest after the pretreatment, they received a dose of P329G Claudin18.2 antibody (also known as HB37A6 PG antibody) infusion (D1); the patients received P329G CAR-T cell infusion on the second day after the completion of P329G Claudin18.2 antibody infusion (D2); thereafter, the observation period was 21 days from the day of P329G Claudin18.2 antibody infusion. During the observation period, the antibody and cells were administered once each. After the observation period, HB37A6 PG antibody was periodically infused at a frequency of Q3W, and it was allowed to be administered earlier, later, or suspended according to the changes in the cells.
  • the classic "3+3" dose escalation method was adopted.
  • the dose of HB37A6 PG antibody was fixed at 1mg/kg to fully explore the optimal dose of P329G CAR-T.
  • the starting dose is 1mg/kg HB37A6 PG antibody and 50*10 ⁇ 6cells P329G CAR-T cells.
  • the DLT observation window is 21 days after the first dose of HB37A6 PG antibody.
  • P329G CAR-T cells does not reach the maximum tolerated dose (MTD)
  • MTD maximum tolerated dose
  • the dose of P329G CAR-T cells will be increased to 250*10 ⁇ 6 cells for further exploration, and so on, increasing to the maximum dose of 750*10 ⁇ 6 cells.
  • the antibody dose is directly increased to 3 mg/kg for cell dose escalation exploration. If the subject completes the DLT observation period and does not experience DLT, continue to receive treatment in the current dose group until disease progression, intolerable toxicity, withdrawal of informed consent, or other reasons for stopping the study treatment (whichever occurs first). If DLT occurs during the DLT observation period, the investigator will determine whether to continue to give the antibody. Safety after the DLT observation period is also included in the safety and tolerability observation.
  • the subject After termination of the study treatment, the subject will enter a 90-day antibody safety follow-up (90 ⁇ 7 days after the last antibody administration), a 2-year short-term safety follow-up of CAR-T cell therapy (every 3 months after the last medication until CAR-DNA is not amplified), and a 15-year long-term safety follow-up of CAR-T cell therapy (every year after the last medication), and survival follow-up (every 3 months after the last medication).
  • a 90-day antibody safety follow-up 90 ⁇ 7 days after the last antibody administration
  • a 2-year short-term safety follow-up of CAR-T cell therapy every 3 months after the last medication until CAR-DNA is not amplified
  • a 15-year long-term safety follow-up of CAR-T cell therapy every year after the last medication
  • survival follow-up every 3 months after the last medication
  • dose groups for dose expansion based on the safety, efficacy signals, cell and antibody PK data obtained in the early stage.
  • Each dose group can be supplemented with 3 to 6 subjects. For example, dosage, administration cycle, administration method, administration process, combination medication sequence, etc.
  • the median age of the 5 subjects enrolled was 64.0 years (range: 60-68 years); most subjects were male (4 subjects, 80%); the median weight of the subjects was 61.0 kg (range: 41-82 kg); the median BMI of the subjects was 22.7 kg/cm 2 (range: 13-29 kg/cm 2 ); the baseline Eastern Cooperative Oncology Group performance status (ECOG) score of all 5 subjects was 1; 60% of the 5 subjects had gastric cancer/esophagogastric junction cancer (3 cases, 60%), and 40% had pancreatic cancer (2 cases, 40%); 40% of the 5 subjects (2 cases) had low expression of CLDN18.2 (the total proportion of IHC staining intensity 1+/2+/3+ positive cells was less than 40% or the IHC staining intensity was only 1+ (any proportion)), 20% (1 case) had medium expression of CLDN18.2 (the total proportion of IHC staining intensity 2+/3+ positive cells was 40%-69%), and 40% (2 cases) had high expression of CLDN18.2 (the total proportion of IHC staining intensity 2
  • lympholysis period 5 subjects received lympholysis treatment.
  • the top three adverse events were decreased lymphocyte count (5 cases, 100%), decreased white blood cell count (4 cases, 80%), and decreased monocyte count (3 cases, 60.0%). All 5 subjects (100%) experienced adverse events during the lymphoproliferative period with CTCAE ⁇ 3, and the most common adverse event during the lymphoproliferative period was a decrease in lymphocyte count (5 cases, 100%). No subject experienced serious adverse events during the lymphoproliferative period.
  • TRAEs treatment-related adverse events
  • SAEs serious adverse events
  • the cell volume of 01003 was in the 50*10 ⁇ 6 dose group, and the other subjects were in the 250*10 ⁇ 6 dose group.
  • Cmax2 and Tmax2 represent the peak concentration and peak time after the second cycle, respectively.
  • PKanalix software (Lixoft, France) was used to calculate the NCA parameters of intensive sampling after the first administration of the antibody. The patient reached peak concentration after the end of the antibody infusion, and then the blood concentration slowly decreased.
  • the geometric mean (geometric coefficient of variation) of the peak concentration (Cmax) was 18.7 ⁇ g/mL (25.9)
  • the geometric mean (geometric coefficient of variation) of the area under the curve (AUClast) was 2417.4 ⁇ g*h/mL (116.7)
  • the geometric mean (geometric coefficient of variation) of the clearance (CL) was 0.315 mL/h/kg (142.2)
  • the geometric mean (geometric coefficient of variation) of the volume of distribution (Vss) was 0.0736 L/kg (34.5)
  • the geometric mean (geometric coefficient of variation) of the half-life was 153.6 h (98.4).
  • the antibody produced approximately 1.43-fold accumulation (trough concentration) after the fourth administration Q3W.
  • the distribution and elimination of antibodies vary greatly from person to person, which may be related to the expansion of CAR-T cells and the expression of target antigens.
  • the blood drug concentration-time curve is shown in Figure 7, and the individual and PK parameters are summarized in
  • R_Cmax and R_Cmin represent the ratios of the peak concentration and trough concentration after the 4th to the 1st administration, respectively.
  • the use of the HB37A6 antibody and CAR-T cells described in this example to treat subjects can partially alleviate the disease of the subjects.
  • Clinical studies have shown that a total of 4 patients with evaluable efficacy were enrolled, of which the best tumor efficacy of 1 subject was partial response (PR), and the best tumor efficacy of 2 subjects was stable disease (SD), with an ORR of 25% and a DCR of 75%.
  • the drug combination therapy (HB37A6 antibody and CAR-T cells) achieved relatively good results in terms of safety (1)
  • CRS As of the data cutoff date, no CRS of cytokine release syndrome occurred in this study;
  • ICANS As of the data cutoff date, no neurological toxicity events were observed in this study.

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Abstract

L'invention concerne un polypeptide récepteur antigénique chimérique régulé par commutateur moléculaire comprenant une séquence scFv de mutation anti-P329G humanisée, une région charnière/région d'espacement, une région transmembranaire, un domaine de signalisation costimulateur et un domaine de signalisation stimulateur ; une cellule effectrice immunitaire modifiée pour exprimer le polypeptide récepteur antigénique chimérique régulé par commutateur moléculaire ; et un procédé de préparation de la cellule effectrice immunitaire. L'invention concerne un anticorps muté P329G se liant de manière spécifique à une molécule CLDN18.2 et un procédé de préparation de l'anticorps muté P329G. Une combinaison pharmaceutique comprenant la cellule effectrice immunitaire et l'anticorps muté P329G peut être utilisée pour traiter une maladie associée à CLDN18.2, telle qu'un cancer exprimant ou surexprimant CLDN18.2.
PCT/CN2024/124141 2023-10-13 2024-10-11 Utilisation d'anticorps cldn18.2 et cellule car-t dans le traitement d'une tumeur solide cldn18.2-positive Pending WO2025077829A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113754780A (zh) * 2020-06-04 2021-12-07 四川科伦博泰生物医药股份有限公司 靶向cldn18.2的嵌合抗原受体、其组合物及用途
US20230030674A1 (en) * 2019-12-06 2023-02-02 Sotio Biotech A.S. Humanized cldn18.2 antibodies
CN115916827A (zh) * 2020-06-19 2023-04-04 豪夫迈·罗氏有限公司 免疫活化Fc结构域结合分子
WO2023093811A1 (fr) * 2021-11-25 2023-06-01 信达细胞制药(苏州)有限公司 Combinaison d'une cellule de récepteur antigénique chimérique de type à régulation de commutation moléculaire et d'un anticorps, et son utilisation
CN116284385A (zh) * 2021-12-07 2023-06-23 信达细胞制药(苏州)有限公司 靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用
CN116648261A (zh) * 2020-12-25 2023-08-25 信达生物制药(苏州)有限公司 Claudin18.2嵌合抗原受体以及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230030674A1 (en) * 2019-12-06 2023-02-02 Sotio Biotech A.S. Humanized cldn18.2 antibodies
CN113754780A (zh) * 2020-06-04 2021-12-07 四川科伦博泰生物医药股份有限公司 靶向cldn18.2的嵌合抗原受体、其组合物及用途
CN115916827A (zh) * 2020-06-19 2023-04-04 豪夫迈·罗氏有限公司 免疫活化Fc结构域结合分子
CN116648261A (zh) * 2020-12-25 2023-08-25 信达生物制药(苏州)有限公司 Claudin18.2嵌合抗原受体以及其用途
WO2023093811A1 (fr) * 2021-11-25 2023-06-01 信达细胞制药(苏州)有限公司 Combinaison d'une cellule de récepteur antigénique chimérique de type à régulation de commutation moléculaire et d'un anticorps, et son utilisation
CN116284385A (zh) * 2021-12-07 2023-06-23 信达细胞制药(苏州)有限公司 靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用

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