WO2021170146A1 - Préparation d'un anticorps anti-cd19 de type nouveau et d'une cellule cd19-car-t, et utilisation associée - Google Patents

Préparation d'un anticorps anti-cd19 de type nouveau et d'une cellule cd19-car-t, et utilisation associée Download PDF

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WO2021170146A1
WO2021170146A1 PCT/CN2021/078544 CN2021078544W WO2021170146A1 WO 2021170146 A1 WO2021170146 A1 WO 2021170146A1 CN 2021078544 W CN2021078544 W CN 2021078544W WO 2021170146 A1 WO2021170146 A1 WO 2021170146A1
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antibody
cells
variable region
present
heavy chain
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PCT/CN2021/078544
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黄纲雄
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福州拓新天成生物科技有限公司
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • A61K39/001112—CD19 or B4
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    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of medicine, and more specifically to the preparation and application of novel anti-CD19 antibodies and CD19-CAR-T cells.
  • B cells include pre-B cells, early-developing B cells and mature B cells. Mature B cells differentiate into plasma cells and malignant B cells through terminal differentiation. Most pre-B acute lymphoblastic leukemia (ALL), non-Hodgkin's malignant lymphoma, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia And some non-acute lymphoblastic leukemias have high expression of CD19. The expression of CD19 on plasma cells further indicates that it can be expressed on different B-cell tumors such as multiple myeloma, plasmacytoma, and Waldenstrom. Therefore, CD19 is considered to be the target of a variety of hematological tumors.
  • the target antigen is often completely or partially missing due to the loss of CD19 antigen fragments, leading to recurrence of leukemia after treatment.
  • the CAR structure when the CAR structure is mistakenly introduced into tumor cells, it can cause the expression of anti-CD19 CAR on the tumor cells.
  • the scFV in the CAR structure can bind to the tumor’s own CD19 molecule. , And then shield the tumor's CD19 molecule, so that tumor cells can no longer be recognized by the scFV of the same structure on CD19-CAR-T cells, resulting in tumor cells that cannot be killed and eventually treatment fails.
  • the purpose of the present invention is to provide CD19 antibodies and CD19-CAR-T cells against recurrent tumors and their preparation and application.
  • the heavy chain variable region includes the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for CD19.
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15.
  • a heavy chain of an antibody having the heavy chain variable region of the first aspect of the present invention.
  • the heavy chain of the antibody also includes a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a light chain variable region of an antibody includes the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for CD19.
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 16.
  • a light chain of an antibody having the light chain variable region of the third aspect of the present invention.
  • the light chain of the antibody also includes a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • an antibody having:
  • the antibody has: the heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.
  • the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
  • the animal is a non-human mammal, preferably a rat, a sheep, or a rabbit.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.
  • the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, and more preferably one.
  • the added, deleted, modified and/or substituted at least one amino acid sequence is an amino acid sequence with at least 80% homology.
  • the derivative sequence after adding, deleting, modifying and/or substituting at least one amino acid has the function of inhibiting cell surface CD19 or recombinant CD19 protein.
  • the antibody is in the form of a drug conjugate.
  • the affinity KD of the derivative sequence for CD19 is 0.01 nM-1 nM, preferably 0.02 nM-0.1 nM.
  • the light chain described in the fourth aspect of the present invention or the antibody described in the fifth aspect of the present invention targets a CD19 molecule with a deletion of the first exon (which does not bind to an epitope corresponding to the first exon of CD19).
  • the sixth aspect of the present invention provides a recombinant protein, the recombinant protein having:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or multimer.
  • the seventh aspect of the present invention provides a CAR construct.
  • the scFV segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to CD19, and the scFv has the first The variable region of the heavy chain described in one aspect and the variable region of the light chain described in the third aspect of the present invention.
  • variable region of the antibody heavy chain and the variable region of the antibody light chain are connected by a connecting peptide.
  • the structure of the single-chain antibody domain is shown in the following formula I or II:
  • V H is an anti-CD19 antibody heavy chain variable region
  • V L of anti-CD19 antibody light chain variable region
  • "-" connecting peptide or a peptide bond
  • the structure of the scFv is shown in Formula II.
  • the heavy chain variable region and the light chain variable region of the scFv are each independently murine, human, rabbit, or human.
  • the scFv is a murine, human, chimeric, or fully humanized single-chain antibody variable region fragment.
  • the structure of the chimeric antigen receptor is shown in the following formula III:
  • L is no or signal peptide sequence
  • scFv is a scFv targeting CD19
  • H is the hinge area
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ .
  • the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CSF2RB, CD4, CD137, IL-2, IFNr, or a combination thereof.
  • the L is a signal peptide derived from CD8.
  • the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, CD80, CD86 or a combination thereof.
  • the H is a hinge region derived from CD8.
  • the TM is a transmembrane region of a protein selected from the group consisting of ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, GD2, CD33, CD37, CD64, CD80 , CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from CD8.
  • the C is a costimulatory signal molecule of a protein selected from the group consisting of: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the C is a costimulatory signal molecule derived from CD28 and/or 4-1BB (CD137).
  • amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 21 and 22.
  • the eighth aspect of the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described in the seventh aspect of the present invention.
  • the cell is a mammalian cell.
  • the immune cells are ex vivo.
  • the immune cells are autologous.
  • the immune cells are non-autologous.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • the immune cells are derived from primates (preferably humans).
  • the immune cell is selected from the following group:
  • CAR-NK cells Chimeric antigen receptor NK cells
  • the immune cells include: NK cells, T cells, NKT cells, ( ⁇ ) T cells, monocytes, or macrophages.
  • the immune cells are CD19-CAR-T cells
  • the ninth aspect of the present invention provides an immunoconjugate, which contains:
  • An antibody portion which is selected from the group consisting of the variable region of the heavy chain as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention
  • the light chain variable region of the fourth aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, or a combination thereof and
  • a coupling portion coupled to the antibody portion The coupling moiety is selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes (etoposides) taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group:
  • Otostatin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • chlortetracycline mettancilol
  • octoxin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • Lastatin doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissus, dihydroxy anthracisin diketone, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, acacia toxin A chain, capsule lotus root toxin A chain, ⁇ -Sarcina, white tree toxin, mitogellin, retstrictocin, phenomycin, enoxomycin
  • the coupling portion is a detectable label.
  • the detectable marker is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or Enzymes capable of producing detectable products, gold nanoparticles/nanorods, viral particles, liposomes, magnetic nanoparticles, nanoparticles of any form, etc.
  • the enzyme includes a prodrug activating enzyme (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)).
  • a prodrug activating enzyme for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • Therapeutic isotope the therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, Or a combination.
  • the immunoconjugate is an antibody drug conjugate.
  • the antibody-drug conjugate is shown in the following molecular formula:
  • D is a drug
  • subscript p is a value selected from 1-10, preferably 1-8.
  • the antibody portion and the coupling portion are coupled through a chemical bond or a linker.
  • the tenth aspect of the present invention provides a use of an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, The light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the first aspect of the present invention.
  • the detection reagent, detection plate or kit is used for:
  • the detection reagent, detection plate or kit is used to diagnose CD19-expressing tumors.
  • the drug is used to treat or prevent CD19-expressing tumors, tumor migration, or tumor resistance.
  • the tumors include solid tumors and blood cancers.
  • the tumor includes a recurrent tumor, preferably a tumor that has recurred after CAR-T cell therapy, such as a recurring blood-borne tumor.
  • the CD19-expressing tumor is selected from the group consisting of bladder cancer, biliary tract cancer, brain cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, neck cancer, kidney cancer, and saliva Cancer, thymic epithelial cancer, thyroid cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma, myeloma, or a combination thereof.
  • the tumor is a drug-resistant tumor.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, which contains:
  • An active ingredient which is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain according to the third aspect of the present invention Variable region, light chain according to the fourth aspect of the present invention, or antibody according to the fifth aspect of the present invention, recombinant protein according to the sixth aspect of the present invention, immune cell according to the eighth aspect of the present invention, The antibody drug conjugate according to the ninth aspect, or a combination thereof; and
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the concentration of the cells is 1 ⁇ 10 3 -1 ⁇ 10 9 cells/ml, preferably 1 ⁇ 10 5 -1 ⁇ 10 8 cells/ml .
  • the pharmaceutical composition also contains other drugs that selectively kill tumor cells (such as nucleic acid drugs, antibody drugs, targeted drugs, other immune cell drugs, other CAR-T drugs, chemotherapy drugs, or Its combination).
  • tumor cells such as nucleic acid drugs, antibody drugs, targeted drugs, other immune cell drugs, other CAR-T drugs, chemotherapy drugs, or Its combination.
  • the pharmaceutical composition is administered by intravenous injection or local injection.
  • the antibody drug includes PD-1 or PD-L1 inhibitor.
  • the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or a combination thereof.
  • the twelfth aspect of the present invention provides a polynucleotide which encodes a polypeptide selected from the following group:
  • nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.: 17 or SEQ ID NO.: 19 in the sequence listing.
  • nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.: 18 or SEQ ID NO.: 20 in the sequence listing.
  • the thirteenth aspect of the present invention provides a vector containing the polynucleotide according to the twelfth aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fourteenth aspect of the present invention provides a genetically engineered host cell containing the vector according to the thirteenth aspect of the present invention or the genome integrated with the polynucleus according to the twelfth aspect of the present invention. Glycidic acid.
  • the fifteenth aspect of the present invention provides a method for detecting CD19 in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps:
  • the sixteenth aspect of the present invention provides a test board, the test board comprising: a substrate (support plate) and a test strip, the test strip containing the antibody according to the fifth aspect of the present invention or the present invention
  • the immunoconjugate of the ninth aspect is not limited to: a substrate (support plate) and a test strip, the test strip containing the antibody according to the fifth aspect of the present invention or the present invention.
  • the seventeenth aspect of the present invention provides a kit including:
  • a first container containing the antibody described in the fifth aspect of the present invention and/or (2) a second container containing the antibody described in the fifth aspect of the present invention
  • the secondary antibody of the antibody
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • the eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • the nineteenth aspect of the present invention provides a method for treating CD19-expressing tumors, the method comprising: administering the antibody according to the fifth aspect of the present invention or the antibody-drug conjugate of the antibody to a subject in need , Or CAR-T cells expressing the antibody, or a combination thereof.
  • the method further includes: administering other drugs or treatment methods to the subject in need for combined therapy.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapeutics, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.
  • a method for preparing a chimeric antibody including the steps:
  • the nucleotide sequence of the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is cloned into an expression vector containing the nucleotide sequence of a human antibody constant region Then, the lentiviral vector is packaged by transfecting engineered cells, and human immune cells are infected with the lentiviral vector to express the human-mouse chimeric antibody.
  • a method for preparing a humanized antibody including the steps:
  • the nucleotide sequence of the CDR region in the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is implanted into a nucleoside containing the FR region of a human antibody After the acid sequence template is cloned into an expression vector containing the constant region of a human antibody, the humanized antibody is expressed by transfecting animal cells.
  • a method for inhibiting tumor cell growth and migration including the steps of: administering the antibody according to the fifth aspect of the present invention and the antibody-drug conjugate of the antibody to a subject in need Or a CAR-T cell expressing the antibody, or a combination thereof.
  • a method for inhibiting tumor growth in a model animal including the steps of: administering the antibody according to the fifth aspect of the present invention and the antibody-drug couple of the antibody to a subject in need. Conjugate, or CAR-T cell expressing the antibody.
  • the drugs can be administered alone or in combination, including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiotherapy.
  • Figure 1 shows the anti-CD19 (hCD19-D-mFc) antibody with the deletion of the first exon in the serum of the mouse after 3 immunizations.
  • Figure 2 shows that HG27 antibody, HG28 antibody and anti-full-length CD19 antibody can all bind to 293FT/hCD19-F cells.
  • Figure 3 shows that the HG27 antibody and the HG28 antibody can bind to the CD19-DhFc fusion protein (the first exon deleted CD19 is fused with a human Fc fragment), while the CD19 antibody (HIB19) cannot bind to the CD19-DhFc fusion protein.
  • Figure 4 shows that there is no competitive binding between HG27 antibody and HG28 antibody and human CD19 antibody (HIB19) ( Figure 4), and the antigen epitopes that they bind to are different. HG27 and HG28 compete with each other for binding to the same epitope ( Figure 4 bottom).
  • Figure 5 shows that CAR-T cells were incubated with three target cells at different effective target ratios. The results showed that G27 CAR-T and HG28 CAR-T cells and CAR-T cells derived from existing CD19 antibody clones (CD19-CAR- T) Have the same effective lethality.
  • Figure 6 shows the use of animal models to evaluate the anti-tumor effects of HG28 CAR-T cells.
  • Figure 6A shows the expression rate of CAR.
  • Figure 6B shows that HG28 CAR-T cells can effectively inhibit the growth of malignant tumors compared with the control group.
  • Figure 6C shows that HG28 CAR-T can be specifically amplified in peripheral blood and maintained for a long time, while the control group TX10 3 CAR-T cannot be amplified.
  • the inventors unexpectedly discovered a new anti-CD19 antibody and a CD19-CAR containing the antibody for the first time.
  • the antibodies of the present invention recognize different epitopes of CD19 and target CD19 molecules with the first exon deleted.
  • the antibody of the present invention can bind to the CD19 antigen with high specificity, and its KD is 9.32 ⁇ 10 -2 nM and 2.09 ⁇ 10 -2 nM, respectively. It has significant anti-tumor activity and can be used for recurrence and resistance. Treatment and detection of drug-induced tumors.
  • conjugate refers to a soluble receptor or a fragment or analog thereof, or an antibody or a fragment or analog thereof capable of binding to a target.
  • CD19 conjugate As used herein, the terms “CD19 conjugate”, “CD19 antibody”, “anti-CD19 antibody”, and “antibody of the present invention” have the same meaning, and refer to antibodies or fragments or analogs thereof that can specifically recognize CD19 and bind to CD19. Things.
  • administering refers to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • administering can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • administering also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including CD19 conjugates and humans or animals, subjects, cells, tissues, physiological regions Chamber or physiological fluid contact.
  • treatment refers to the administration of an internal or external therapeutic agent, including any one of the CD19 conjugates and compositions of the present invention, to a patient who has one or more disease symptoms, and the treatment is known
  • the agent has a therapeutic effect on these symptoms.
  • the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • IgG represents the most important class of immunoglobulins.
  • IgG1, IgG2, IgG3 and IgG4 Due to differences in chemical structure and biological functions, it can be divided into 4 subclasses: IgG1, IgG2, IgG3 and IgG4.
  • the light chain is divided into a kappa or lambda chain by the difference of the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved FR regions (FR). The amino acid sequences of the 4 FRs are relatively conservative and do not directly participate in the binding reaction.
  • Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from amino terminal to carboxyl terminal is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2, and LCDR3;
  • the number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or conform to the numbering rules of Kabat and Chothia (HCDR1 ).
  • the four FR regions in the natural heavy chain and light chain variable regions are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely held together by the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions. Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • the term "antigen-binding fragment” refers to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments that have antigen-binding activity.
  • the Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites.
  • an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • antigenic determinant refers to discrete three-dimensional sites on the antigen that are recognized by the antibody or antigen-binding fragment of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombination techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific and are directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic or phage cloned cell lines.
  • chimeric antibody is a chimeric gene formed by splicing the V region gene of a murine antibody and the C region gene of a human antibody into a chimeric gene, which is then inserted into a vector and transfected into an antibody molecule expressed by a host cell. It not only retains the high specificity and affinity of the parent mouse antibody, but also enables the human Fc segment to effectively mediate the biological effect function.
  • humanized antibody is a modified form of the variable region of the murine antibody of the present invention, which has CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody) , And FR regions and constant regions that are basically derived from human antibody sequences; that is, the CDR region sequences of murine antibodies are grafted onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, expression vectors can be constructed to express recombinant antibodies that mimic the properties of specific naturally-occurring antibodies.
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably At most 3 or 1 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • enhancing ADCC effector function can mean enhanced potency or enhanced efficacy.
  • concentration of the antibody half maximum effective concentration
  • efficacy used in the experimental context means the maximum possible effector function of the antibody at the saturation level.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • Fc ⁇ R antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • complement protein components recognize the antibody bound on the target cell, which then results in the lysis of the target cell.
  • effector function includes biochemical events caused by the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include Fc ⁇ R-mediated effector functions, such as ADCC and ADCP, and complement-mediated effector functions, such as CDC.
  • chimeric antigen receptor or "CAR” as used herein refers to a polypeptide that includes an extracellular domain capable of binding antigen, a transmembrane domain, and a domain that transmits cytoplasmic signals to the structure (ie, intracellular signal domain).
  • Signal domain refers to a protein that transmits information into the cell to regulate cell activity by generating a second messenger through a certain signal transduction pathway, or a protein that functions as an effector by corresponding to such a messenger, including the primary signal domain, and It may include functional signaling domains derived from the stimulatory molecules defined below (ie, costimulatory signal domains).
  • the intracellular signal domain generates signals that can promote the immune effector functions of CAR cells (such as CAR T cells). Examples of immune effector functions, such as in CART cells, include cytolytic activity and auxiliary activity, including cytokine secretion.
  • the term "primary signal domain” modulates the initial activation of the TCR complex in a stimulating manner.
  • the primary signal domain is triggered by, for example, the binding of the TCR/CD3 complex and peptide-loaded MHC molecules, thereby mediating T cell responses (including but not limited to proliferation, activation, differentiation, etc.).
  • the primary signaling domain that acts in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or the signaling motif of ITAM.
  • ITAM-containing primary signal domains examples include, but are not limited to, those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS" ) And the sequence of CD66d.
  • the intracellular signaling domain in any one or more of the CARs of the present invention includes an intracellular signaling sequence, such as the primary signaling domain of CD3 ⁇ .
  • costimulatory signal domain refers to a "costimulatory molecule", which is a related binding partner on T cells, which specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, for example, but not limited to ,proliferation.
  • Co-stimulatory molecules are non-antigen receptor cell surface molecules or their ligands required for effective immune response.
  • Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and CD19 (CD137).
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain containing derived from Stimulate the functional signaling domain of the molecule.
  • the CAR comprises a chimeric fusion protein, the protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain containing a function derived from a costimulatory molecule Sexual signaling domains and functional signaling domains derived from stimulating molecules.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular conduction domain, and the intracellular signaling domain comprises one or more costimulatory molecules derived from At least two functional signaling domains and a functional signaling domain derived from a stimulating molecule.
  • the CAR contains an optional leader sequence at the amino acid (ND end) of the CAR fusion protein.
  • the CAR also contains a leader sequence at the N-terminus of the extracellular antigen recognition domain, where the leader sequence is optionally cut from the antigen recognition domain (such as scFv) during the cellular processing and localization of the CAR to the cell membrane.
  • CD3 ⁇ herein is defined as the protein provided by GenBan accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes and the like.
  • CD3 ⁇ domain is defined as the amino acid residues from the cytoplasmic domain of the ⁇ chain, which are sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ includes residues 52 to 164 of GenBan accession number BAG36664.1, and its functional orthologs-from non-human species such as mice, rodents, monkeys, apes, etc. Valence residues.
  • CD19 herein refers to a member of the TNFR superfamily, which has the amino acid sequence of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.; "CD19 costimulatory domain” is defined as the amino acid sequence 214-255 of GenBank ACC. No. AAA62478.2, or equivalent residues from non-classified species such as mice, rodents, monkeys, apes, etc.
  • CD19 includes, but is not limited to, variants, isotypes, and species homologs of human CD19.
  • the humanized antibody of the present invention can cross-react with CD19 of a species other than human.
  • antibodies can be completely specific for one or more human CD19 proteins and can exhibit species or other types of non-human cross-reactivity.
  • the complete amino acid sequence of an exemplary human CD19 has SwissPort accession number P15391.
  • CD19 is also known as B cell surface antigen B4, B cell antigen CD19, CD19 antigen or Leu-12.
  • Human CD19 is called Gene ID: 930 by Entrez Gene, and HGNC: 1633 by HGNC.
  • CD19 can be coded by a gene called CD19.
  • the use of "human CD19” herein covers all known or undiscovered alleles and polymorphic forms of human CD19.
  • the CD19 molecule used as an immune antigen is a CD19 molecule with the first exon deleted, and the anti-CD19 antibody of the present invention does not bind to the epitope corresponding to the first exon of the CD19 molecule.
  • the target antigen is often completely or partially missing due to the loss of CD19 antigen fragments, leading to recurrence of leukemia after treatment.
  • the present invention provides new CD19 antibodies, designed to target new epitopes of CD19 molecules, not only against the original CD19 molecules, but also against existing CD19 antibodies and CD19-CAR-T treatments The latter part of the antigen fragment lacks the CD19 molecule. Therefore, the CAR-T cell therapy of the present invention can be used to treat the original ALL/lymphoma patients, and can also be used to treat the patients who have relapsed after the above-mentioned existing treatments;
  • the CAR structure when the CAR structure is mistakenly introduced into tumor cells, it can cause the expression of anti-CD19 CAR on the tumor cells.
  • the scFV in the CAR structure is combined with the tumor’s own CD19 molecules, and then Shielding the tumor's CD19 molecule makes the tumor cells no longer be able to be recognized by the scFV of the same structure on the CD19-CAR-T cells, causing the tumor cells to be unable to be killed and ultimately the treatment fails.
  • the scFV of the antibody of the present invention can recognize different epitopes of CD19.
  • the antibody derived from it or the scFV of CAR-T cells can recognize the above-mentioned shielded tumor cells and can be used to treat the above-mentioned reasons that lead to the failure of existing CAR-T cell therapy. patient.
  • the anti-CD19 antibody or its scFV of the present invention can also be used to detect the tumor cells of the above-mentioned treatment failure patients or patients who relapsed due to antigen deletion or modulation after treatment, and these tumor cells cannot be identified and detected by the existing CD19 antibody itself.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, human-animal chimeric antibodies, preferably humanized antibodies, and more preferably fully humanized antibodies.
  • the antibody derivatives of the present invention may be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other antibody derivatives known in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the animal is preferably a mammal, such as a mouse.
  • the present invention provides murine antibodies HG27 and HG28.
  • the antibody HG27 includes the heavy chain variable region CDRs shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6 shown in the light chain variable region CDR
  • antibody HG28 includes SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 shown in the heavy chain variable region CDR, and the light chain variable region CDR shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the antibody of the present invention targets the CD19 molecule with the first exon deleted, that is, does not bind to the epitope corresponding to the first exon of CD19.
  • amino acid sequences involved in the antibodies of the present invention are shown in Table 1.
  • the invention provides variants of antibodies or fragments thereof that bind to human CD19. Therefore, the present invention provides antibodies or fragments thereof, which have a heavy chain and/or light chain variable region that is at least 80% identical to the variable region sequence of the heavy chain or light chain.
  • the amino acid sequence identity of the variable region of the heavy chain and/or light chain is at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98% , Most particularly 99%, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequence identity.
  • This article refers to the identity of the amino acid sequence or the percentage of amino acid residues in the sequence that are identical to the humanized antibody or fragment thereof that binds to human CD19.
  • sequence identity can be determined by standard methods commonly used to compare the similarity of the amino acid positions of two polypeptides. Using a computer program such as BLAST or FASTA, the best matches of the respective amino acids of the two polypeptides (along the full length of one or two sequences or along predetermined portions of one or both sequences) are aligned. The program provides default open penalties and default gap penalties. Score matrices such as PAM250 (standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, Volume 5, Third Supplement (1978)) can be compared with Used in conjunction with computer programs. For example, percent identity can be calculated as: the total number of identical matches multiplied by 100, then divided by the total length of the longer sequence in the matching span and the number of gaps poured into the longer sequence to align the two sequences.
  • the present invention also provides humanized antibody fragments that bind to human CD19, the fragments being selected from the group consisting of Fab, Fab', Fab'-SH, Fd, dAb, F(ab')2, scFv, bispecific single-chain Fv two Polymers, diabodies, tri-chain antibodies, and scFv genetically fused to the same or different antibodies.
  • Preferred fragments are scFv, bispecific single chain Fv dimers and diabodies.
  • the present invention also provides a full-length humanized antibody that binds to human CD19.
  • the present invention also provides isolated nucleic acids and vectors encoding the antibodies and fragments thereof of the present invention, and host cells containing the nucleic acids or vectors.
  • the nucleic acid can be located in intact cells, in cell lysates, or in a partially purified or substantially purified form.
  • Standard molecular biology techniques can be used to obtain the nucleic acid of the present invention.
  • standard PCR amplification or cDNA cloning techniques can be used to obtain the light chain and heavy chain encoding the antibody or the cDNA encoding the VH and VL segments.
  • antibodies obtained from an immunoglobulin gene library for example, using phage display technology
  • one or more nucleic acids encoding the antibody can be recovered from the library.
  • the method of introducing exogenous nucleic acid into a host cell is generally known in the art and can vary with the host cell used.
  • the nucleic acid encoding the antibody of the present invention can be integrated into an expression vector.
  • a variety of expression vectors can be used for protein expression.
  • Expression vectors can include self-replicating extrachromosomal vectors, or vectors integrated into the host genome.
  • Expression vectors used in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As known in the art, a variety of expression vectors are commercially available or otherwise. It can be used in the present invention to express antibodies.
  • the nucleic acid encoding the heavy chain variable region is as shown in the sequence listing SEQ ID NO.: 17 or the sequence listing SEQ ID NO.: 19; and/or, encoding the light chain variable region
  • the nucleic acid is shown in the sequence list SEQ ID NO.: 18 or the sequence list SEQ ID NO.: 20.
  • nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO.: 17 in the sequence listing, and the nucleic acid encoding the variable region of the light chain is shown in SEQ ID NO.: 18 in the sequence listing.
  • the nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO.: 19 in the sequence listing, and the nucleic acid encoding the variable region of the light chain is shown in SEQ ID NO.: 20 in the sequence listing.
  • the method for preparing the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein through gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein through a method of artificial full-sequence synthesis .
  • the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately substituted, deleted, altered, inserted or added to provide a polynucleotide homolog.
  • the polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
  • any method suitable for producing monoclonal antibodies can be used to produce the anti-CD19 antibodies of the present invention.
  • animals can be immunized with linked or naturally occurring CD19 homodimers or fragments thereof.
  • Appropriate immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more approaches can be used.
  • the fusion protein of the human CD19 extramembrane protein with the first exon deleted and the murine Fc fragment is used as the immune antigen to produce CD19-specific antibodies, and the biological activity of the antibodies is screened.
  • An exemplary method of producing the anti-CD19 antibody of the present invention is described in Example 1.
  • Fully humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and the IgG4 subtype is used. It is easy to achieve the optimization of the necessary constant domain sequence to produce the desired biological activity by screening antibodies with the biological assays described in the examples below.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof can be used in the compounds and methods of the present invention.
  • Example 3 An exemplary method of humanizing the anti-CD19 antibody of the present invention is described in Example 3.
  • sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can also be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell is a variety of conventional host cells in the field, as long as the above-mentioned recombinant expression vector can stably replicate itself and the nucleic acid carried by it can be effectively expressed.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • Preferred host cells include E. coli TG1 or BL21 cells (expressing single-chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full-length IgG antibodies)
  • the step of transforming host cells with recombinant DNA described in the present invention can be performed by techniques well known in the art.
  • the obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. According to the host cell used, it is cultured in a conventional medium under suitable conditions.
  • the transformed host cell is cultured under conditions suitable for expression of the antibody of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibody of the present invention is purified by conventional separation and purification means well-known to the person.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the present invention provides multiple chimeric antigen receptors (CAR), which contain antibodies or antibody fragments engineered to enhance binding to CD19 protein.
  • CAR chimeric antigen receptors
  • the present invention provides engineered cells (such as T cells) that express CAR, wherein the CAR T cells ("CART") exhibit anti-tumor properties.
  • cells are transformed with CAR, and CAR is expressed on the cell surface.
  • a viral vector encoding CAR is used to transduce cells (such as T cells).
  • the viral vector is a lentiviral vector.
  • the cell can stably express CAR.
  • the anti-CD19 protein binding portion of the CAR is a scFv antibody fragment.
  • the antibody fragment is functional, so that compared with the IgG antibody from which it is derived, it maintains equivalent affinity binding, for example, it binds to the same antigen with comparable efficacy.
  • the antibody fragment is functional, so that it provides a biochemical reaction. The reaction may include, but is not limited to, activating an immune response, inhibiting the initiation of signal transduction from its target antigen, inhibiting kinase activity, etc. .
  • the anti-CD19 antigen-binding domain of the CAR is a humanized scFv antibody fragment relative to the murine sequence scFv from which it is derived.
  • the CAR of the present invention combines the antigen-binding domain of a specific antibody and an intracellular signal transduction molecule.
  • intracellular signaling molecules include, but are not limited to, CD3 ⁇ chains, 4-1BB and CD28 signaling modules, and combinations thereof.
  • the present invention provides cells (such as T cells) engineered to express chimeric antigen receptors (CAR), wherein the CAR T cells ("CART") exhibit anti-tumor properties.
  • the antigen binding domain of the CAR comprises a humanized anti-CD19 antibody fragment, which comprises scFV. Therefore, the present invention provides a CD19-CAR containing a humanized anti-CD19 binding domain that is engineered into T cells, and a method of using it for adoptive immunotherapy.
  • CD19-CAR contains at least one intracellular signaling domain, which selects CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 ⁇ signaling domain, and any combination thereof. In another preferred example, CD19-CAR contains at least one intracellular signaling domain derived from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.
  • the CAR of the present invention combines the antigen-binding domain of a specific antibody and intracellular signal transduction molecules while adding IFN ⁇ expression elements.
  • intracellular signaling molecules include, but are not limited to, CD3 ⁇ chains, 4-1BB and CD28 signaling modules, and combinations thereof.
  • the present invention provides cells (such as T cells) engineered to express chimeric antigen receptors (CAR), wherein the CAR T cells ("CART") exhibit anti-tumor properties.
  • the antigen binding domain of the CAR comprises a humanized anti-CD19 antibody fragment, which comprises scFV.
  • the present invention provides the use of the antibody of the present invention, for example, for the preparation of diagnostic preparations, or preparation of drugs for the prevention and/or treatment of CD19-related diseases.
  • the CD19-related diseases include cancer, autoimmune diseases, viral infections, graft-versus-host disease, inflammatory diseases, immune diseases, or combinations thereof.
  • the cancer includes solid tumors and blood cancers
  • the solid tumors are selected from the following group: bladder cancer, biliary tract cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head cancer , Leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, saliva cancer, thymic epithelial cancer and thyroid cancer, or a combination thereof;
  • the autoimmune diseases include : Systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, or a combination thereof.
  • the present invention includes therapeutic applications with cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention.
  • the transduced T cells can target CD19, a marker of tumor cells, and coordinately activate T cells, causing T cell immune responses, thereby significantly improving its killing efficiency on tumor cells.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, which comprises the following steps: administering the CAR-T cell of the present invention to the mammal.
  • the present invention includes a type of cell therapy in which the patient's autologous T cells (or heterologous donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by T cells in a non-MHC-restricted manner.
  • one CAR-T can treat all cancers that express the antigen.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • anti-CD19 CAR-T cells cause a specific immune response against CD19-positive cells.
  • the present invention should be construed to include the Any number of changes in one.
  • Cancers that can be treated include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors.
  • the cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors.
  • the types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma, and melanoma. It also includes adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is cancer of the blood or bone marrow.
  • leukemias include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin's lymphoma (painless and high-grade form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as acute lymphoblastic leukemia, acute myeloid leuk
  • a solid tumor is an abnormal mass of tissue that does not usually contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, and so on.
  • the treatable cancer is a CD19-positive tumor, such as leukemia, lymphoma, and the like.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunity and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from mammals (preferably humans) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits.
  • the mammalian recipient can be a human, and the CAR-modified cell can be autologous relative to the recipient.
  • the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method for treating tumors, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include the target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelate Mixtures such as EDTA or glutathione
  • adjuvants for example, aluminum hydroxide
  • preservatives for example, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the number and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.
  • the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be 104 to 109 doses cells / kg body weight, preferably 105 to 106 cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses.
  • the cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation.
  • the compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • the treatment modality includes, but is not limited to, treatment with the following agents: the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies Or other immunotherapeutics.
  • the cell composition of the present invention is administered to bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, simultaneously, or after). patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (for example, before, simultaneously, or after).
  • the subject may undergo the standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to the practice accepted in the art.
  • 1 ⁇ 10 6 to 1 ⁇ 10 10 modified T cells (for example, CAR-T cells) of the present invention can be administered to the patient by, for example, intravenous infusion, per treatment or per course of treatment. .
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein or ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention can also be used for cell therapy by expressing the nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used to bind CD19 protein molecules, and therefore can be used to prevent and treat CD19-related diseases.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 100 mg/kg body weight per day.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, which is more Preferably, the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the antibodies of the present invention can be used in detection applications, for example, to detect samples, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or a fragment thereof) of the present invention.
  • the kit further includes a container, instructions for use, buffer, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the present invention also provides a method for detecting cells overexpressing CD19 protein, which includes the following steps: the above-mentioned protein is contacted with the sample to be tested in vitro, and the binding of the above-mentioned protein to the sample to be tested is detected.
  • overexpression is conventional in the art, and refers to the overexpression of the RNA or protein of the CD19 protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and changes in protein degradation), and due to changes in protein Changes in transport mode (increased nuclear localization) result in local overexpression and increased functional activity (as in the case of increased enzymatic hydrolysis of the substrate).
  • the detection method of the binding is a conventional detection method in the art, preferably FACS detection.
  • the present invention provides a composition for detecting cells overexpressing CD19 protein, which includes the above-mentioned protein as an active ingredient. Preferably, it also includes a compound composed of functional fragments of the above-mentioned protein as an active ingredient.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the CD19 antibody of the present invention can target existing CD19 antibodies and CD19 molecules with partial antigen fragments missing after CD19-CAR-T cell therapy, and can be used for the treatment of relapsed leukemia.
  • the scFV of the present invention can identify tumor cells partially shielded from CD19 molecules after the existing CD19-CAR-T cell therapy, and can be used to treat patients whose CAR-T cell therapy fails due to the above-mentioned reasons.
  • the CD19 antibody or its scFV of the present invention can also be used to detect tumor cells in patients who have failed the above-mentioned treatment or who have relapsed due to antigen deletion or modulation after treatment.
  • mice Female, 8-week-old Balb/c mice were selected, and the fusion protein (hCD19-D-mFc) of human-derived CD19 extra-membrane protein with the deletion of the first exon expressed in E. coli and mouse Fc fragment (hCD19-D-mFc) was used as Immune antigen.
  • hCD19-D-mFc human-derived CD19 extra-membrane protein with the deletion of the first exon expressed in E. coli and mouse Fc fragment
  • hCD19-D-mFc mouse Fc fragment
  • the immunized mouse spleen was taken to separate the cells, and the SP2/0 cells were fused with PEG1500.
  • the fused cells were suspended in 20% FCS IMDM culture medium and seeded in 96-well culture plates, and HAT was added for screening.
  • the supernatant was screened by ELISA and FACS to obtain two positive clones, HG27 antibody and HG28 antibody.
  • the selected anti-CD19 antibodies (HG27 antibody and HG28 antibody) were incubated with 293FT cells expressing human CD19 extra-membrane full-length protein (hCD19-F), and the binding activity was detected by flow cytometry.
  • HG27, HG28 and anti-CD19 antibodies can all bind to 293FT/hCD19-F cells.
  • the fusion protein (hCD19-D-hFc) of CD19 with the first exon deletion and the human Fc fragment was used as a substrate for ELISA experiments to identify whether the screened antibodies could specifically bind to the CD19 protein with the first exon deletion.
  • the known CD19-positive leukemia cell CA46 is used as the target cell to detect antibody binding/competition.
  • Human lymphoma cells (CA46) were used as target cells, incubated with 10ug excess HG27 and HG28 antibodies at 4°C for 30 minutes, washed with 1% calf serum in PBS, and 0.1ul anti-human CD19 antibody (anti-hCD19- FITC, eBioscience) or 0.1ug streptavidin (streptavidin) labeled homemade antibodies HG27-biotin and HG28-biotin, incubated at 4°C for 20 minutes, washed and analyzed by flow cytometry.
  • CA46 Human lymphoma cells
  • RNA is extracted from hybridoma cells (HG27 and HG28), and DNA strands are synthesized by reverse transcription. Connect to the T vector, select clones, sequence analysis, and determine the amino acid sequence and CDR regions of the antibody's light and heavy chains.
  • the single-chain antibody fragments (scFv) of HG27 and HG28 were combined with the hinge region and transmembrane region of CD8, the intracellular signaling domain of CD28 and/or 4-1BB, and the activation functional domain of CD3 ⁇ , respectively.
  • the lentiviral vector cell culture supernatant was collected, filtered with a 0.45 ⁇ m filter, and concentrated by centrifugation at 4°C and 3000 rpm for 5 minutes to obtain the lentiviral vector stock solution. It can also be purified by chromatography or ion exchange.
  • the lentiviral vector was quickly frozen in liquid nitrogen after titer identification, and then stored at minus 80°C for later use.
  • PBMC peripheral blood mononuclear cells
  • the activated T lymphocytes were collected and resuspended in RPMI1640 medium. Infect 1 ⁇ 10 6 activated T lymphocytes with HG27 and HG28 lentivirus respectively, add the cell suspension to a 6-well plate, and incubate overnight in a 37°C, 5% CO2 incubator. The next day, centrifuge again and replace with fresh medium, add fresh medium every 2 days, and continue to expand the culture. At the 8th day of culture, CAR-T cells (HG27 CAR-T, HG28 CAR-T) were collected by centrifugation, resuspended in a suitable freezing solution, and frozen in liquid nitrogen for later use.
  • chimeric antigen receptor T cells HG27 CAR-T, HG28 CAR-T, TX10 3 CAR-T (non-CD19 targeting), CD19 CAR-T, T), and target cells (chronic myeloid leukemia cells) K562, leukemia cell line Nalm6, human Burkitt's lymphoma cells CA46 and Raji), the tumor cells were labeled with CFSE, and the labeled tumor cells were seeded into 96-well plates at a density of 5 ⁇ 10 4 /100ul/well ml.
  • Different CARs -T cells were added to a 96-well plate for co-cultivation of tumors according to 100ul/well and an effective target ratio of 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1. After 18 hours of co-cultivation, aspirate the cells from each well into a flow tube, and add DAPI to label dead cells before going on the machine.
  • the killing ratio of tumor cells CFSE+DAPI+cells/CFSE+cells, the killing efficiency of CAR-T cells to tumors is calculated by the killing ratio of tumor cells.
  • HG27 CAR-T or HG28 CAR-T cells did not kill the negative control cell K562, which shows that HG27 and HG28 can specifically kill CD19+ tumor cells.
  • T cells CD19-CAR-T have the same or stronger killing ability.
  • the clone number of the existing CD19 antibody is FMC63(B19) (FITC), and the CD19-CAR-T is self-made.
  • the TX10 3 CAR-T and HG28 CAR-T cells were prepared, and the CAR expression rate is shown in Figure 6A, which were used in animal models to evaluate anti-tumor effects.
  • the Nalm6 leukemia tumor cell line (Nalm6-Luc) carrying firefly luciferase was injected into mice through the tail vein, a total of 8 mice, 5x10 ⁇ 4 cells per mouse, and the injection dose was 100ul. Three days after tumor inoculation, mice were injected with D-Luciferase in the abdomen to perform live imaging.
  • mice were randomly divided into 2 groups, 4 mice in each group, for treatment, one group used HG28CAR-T cells, and the other group used TX10 3 -CAR- T cells (non-CD19 targeting), the therapeutic dose is 5x10 ⁇ 6 CAR+ cells, and the treatment day is recorded as day0. Imaging was performed on day3, day8, and day15 after treatment to observe the therapeutic effect.
  • HG28 CAR-T cells can effectively inhibit the growth of malignant tumors compared with the control group.
  • Peripheral blood was taken on day5, day9, day12, and day16 to detect the expression level of CAR-T cells.
  • HG28 CAR-T can be specifically amplified in peripheral blood and maintained for a long time, while the control group TX10 3 CAR-T cannot be amplified.

Abstract

L'invention concerne la préparation d'un anticorps anti-CD19 de type nouveau et d'une cellule CD19-CAR-T, ainsi qu'une utilisation associée. L'anticorps anti-CD19 cible une molécule CD19, qui supprime un premier exon, et peut être utilisé pour traiter et détecter des tumeurs récurrentes et résistantes aux médicaments.
PCT/CN2021/078544 2020-02-27 2021-03-01 Préparation d'un anticorps anti-cd19 de type nouveau et d'une cellule cd19-car-t, et utilisation associée WO2021170146A1 (fr)

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