WO2021143914A1 - Anticorps anti-ox40, son procédé de production et son application - Google Patents

Anticorps anti-ox40, son procédé de production et son application Download PDF

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WO2021143914A1
WO2021143914A1 PCT/CN2021/072586 CN2021072586W WO2021143914A1 WO 2021143914 A1 WO2021143914 A1 WO 2021143914A1 CN 2021072586 W CN2021072586 W CN 2021072586W WO 2021143914 A1 WO2021143914 A1 WO 2021143914A1
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antibody
amino acid
acid sequence
seq
heavy chain
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王双
曾大地
王荣娟
焦莎莎
张畅
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迈威(上海)生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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

Definitions

  • the present invention belongs to the field of antibody engineering, and specifically relates to an anti-OX40 antibody, its production method and application, in particular to an anti-human OX40 humanized antibody, its recombinant expression method and its application in the treatment of solid tumors.
  • OX40 also known as CD134, ACT45, and TNFRSF4, belongs to the tumor necrosis factor receptor (TNFR) superfamily, and is an activating receptor expressed on the surface of activated CD4+T and CD8+T cells.
  • OX40 signal can activate the downstream NF- ⁇ B, PI3K and PKB pathways. The continuous activation of these pathways can ultimately prolong the survival time of T cells, expand T cell memory, and promote the cell killing ability of T cells.
  • OX40 can also inhibit The differentiation and activity of regulatory T cells (Treg) improve the immunosuppressive effect in the tumor microenvironment and further enhance the function of effector T cells.
  • Treg regulatory T cells
  • the OX40 gene is located on human chromosome 1 (mouse chromosome 4) and encodes a 50kD transmembrane glycoprotein.
  • the extracellular region has 191 amino acids and contains three complete and one shorter cysteine-rich domains (CRDs). It is mainly expressed on activated effector T cells (Teffs) and regulatory T cells (Tregs), but also on NKT cells, NK cells and neutrophils.
  • OX40 combines with the ligand OX40L (CD252, TNFSF4) to deliver costimulatory signals.
  • the OX40L gene is located on chromosome 1 of humans and mice, and encodes a 34kD type 2 transmembrane glycoprotein.
  • OX40L can be expressed on antigen presenting cells (APC), such as B cells, dendritic cells, and macrophages; in addition, it can also be induced on other cell types such as Langerhans cells, endothelial cells, smooth muscle cells, mast cells, and NK cells Express.
  • APC antigen presenting cells
  • OX40 and OX40L participates in a variety of physiological reactions between T cells and lymphocytes and non-lymphocytes.
  • the interaction of OX40 and OX40L can recruit TNFR-related (TRAFs) molecules in the intracellular region of OX40 to form a signaling complex containing IKK ⁇ and IKK ⁇ as well as PI3k and PKB (Akt);
  • TRFs TNFR-related
  • IKK ⁇ and IKK ⁇ PI3k and PKB
  • Akt PI3k and PKB
  • Anti-OX40 activating antibody can play a similar function to OX40L to activate antigen-dependent T effector cells, and can play an anti-tumor effect by eliminating the inhibitory function of Treg cells.
  • OX40 fully human anti-tumor necrosis factor receptor superfamily member 4
  • OX40 monoclonal antibody of Cinda Biopharmaceuticals the recombinant fully human anti-OX40 monoclonal antibody of Livzon Pharmaceutical Group Clone antibody injection.
  • Most of the OX40 monoclonal antibodies in the prior art obtain anti-OX40 antibodies through hybridoma technology.
  • the target antibody can also be obtained through methods such as transgenic mouse technology, phage antibody library technology, and B cell sorting technology.
  • the evaluation and analysis methods are the same as or similar to the hybridoma antibody preparation scheme.
  • the present invention obtains murine antibodies through hybridoma technology, obtains candidate activated anti-OX40 murine antibodies through analysis of antibody binding activity, activation activity, and blocking activity; After designing, the obtained humanized antibody is analyzed again through antibody activity analysis (binding activity, activation activity, blocking activity) and pharmacodynamic analysis in mice to finally determine the activated anti-OX40 humanized antibody.
  • antibody activity analysis binding activity, activation activity, blocking activity
  • the present invention provides an antibody or fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
  • VH CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 40, 52, 64,
  • VH CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 41, 53, 65,
  • VH CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 42, 54, 66;
  • the light chain variable region contains:
  • VL CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 46, 58, 70,
  • VL CDR2 is selected from the amino acid sequence shown in SEQ ID NO: 47, 59, 71,
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 2, 6, 10, 14, 22, 24, or 30.
  • the heavy chain variable region of the antibody or fragment thereof of the present invention includes the amino acid sequence shown in SEQ ID NO: 4, 8, 12, 16, 18, 20, 26, 28, and 32.
  • the antibody or fragment thereof of the present invention is characterized in that:
  • the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 2, 14, 22, and 30, and the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 4, 16, 18, and 20;
  • the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 6, 24, and the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 8, 26, 28, 32;
  • the antibody or fragment thereof of the present invention is characterized in that:
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 8;
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 10
  • the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12;
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16, 18, or 20;
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16, 18 or 20;
  • the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 26 or 28;
  • the present invention provides an immunoconjugate comprising
  • the coupling part includes a detectable label, a cytotoxic molecule, a biologically active molecule, and the like.
  • the cytotoxic molecules include diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, leuculin and the like.
  • the present invention provides a multispecific antibody or derivative thereof, which is characterized by comprising at least one antigen binding domain of the antibody or fragment thereof according to the first aspect of the present invention.
  • the multispecific antibody or derivative thereof of the present invention wherein the other targets include PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, CD47, 4-1BB, CD73, ROR1, HER2, HER3, EGFR, etc.
  • the heavy chain antibody of the present invention does not have an Fc region.
  • composition of the present invention further includes other active ingredients for treating tumors.
  • the present invention provides a kit comprising the antibody or fragment thereof described in the first aspect of the present invention, for qualitative or quantitative detection of OX40.
  • the present invention provides a nucleic acid that encodes the antibody or fragment thereof of the first aspect of the present invention, or encodes the multispecific antibody or derivative thereof of the third aspect of the present invention.
  • the present invention provides a vector comprising the nucleic acid according to the seventh aspect of the present invention.
  • the present invention provides the antibody or fragment thereof of the first aspect of the present invention, the antibody conjugate of the second aspect of the present invention, the multispecific antibody or derivative thereof of the third aspect of the present invention, and the first aspect of the present invention.
  • molecular adjuvants used to enhance specific immune responses are used in combination with adjuvants such as CpG for tumor vaccines and other purposes.
  • the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in preparing drugs for inducing OX40+ cells to produce IL-8 and for initiating NF ⁇ B gene transcription.
  • the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in the preparation of drugs that stimulate PBMC to produce IL-2 and IFN- ⁇ .
  • the present invention provides the antibody or fragment thereof according to the first aspect of the present invention, the antibody conjugate according to the second aspect of the present invention, the multispecific antibody or derivative thereof according to the third aspect of the present invention, and the present invention Use of the heavy chain antibody of the fourth aspect and the composition of the fifth aspect of the present invention in the preparation of drugs for inhibiting the growth and metastasis of solid tumors.
  • the use of the present invention is characterized in that the digestive system tumors include liver cancer, pancreatic cancer, gastric cancer, duodenal cancer, colorectal cancer, and esophageal cancer.
  • the use of the present invention is characterized in that the respiratory system tumors include small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma, laryngeal carcinoma, mesothelioma and the like.
  • the use of the present invention is characterized in that the urogenital system tumors include breast cancer, ovarian cancer and the like.
  • the present invention provides a method for producing antibodies, including:
  • the present invention provides a method for preventing and/or treating diseases mediated by OX40, the method comprising administering the antibody or fragments thereof, antibody conjugates, Multispecific antibodies or derivatives thereof, heavy chain antibodies, compositions, nucleic acids, recombinant vectors or recombinant host cells.
  • the method for preventing and/or treating a disease mediated by OX40 of the present invention wherein the respiratory tumor of the subject includes small cell lung cancer, non-small cell lung cancer, nasopharyngeal carcinoma , Laryngeal cancer, mesothelioma, etc.
  • the method for preventing and/or treating diseases mediated by OX40 according to the present invention wherein the urogenital system tumors suffered by the subject include breast cancer, ovarian cancer and the like.
  • human OX40 refers to an OX40 protein having a human amino acid sequence, such as the amino acid sequence of Genbank Accession No. NP_003318.
  • monkey OX40 and mouse OX40 refer to the OX40 sequences of monkey and mouse respectively, for example, sequences with Genbank accession number NP_001090870 and Genbank accession number NP_035789, respectively.
  • antibody herein is meant to include full-length antibodies and any antigen-binding fragments (ie, antigen-binding portions) or single chains thereof.
  • Full-length antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains, the heavy and light chains are connected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH for short) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the constant region of the light chain consists of a domain CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDR), which are separated by more conservative framework regions (FR) regions.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, FR3, FR4 from the amino terminal to the carboxy terminal.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (for example, effector cells) and the first component (C1q) of the traditional complement system.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigen specificities.
  • an isolated antibody that specifically binds to OX40 protein does not substantially contain antibodies that specifically bind to antigens other than OX40 protein.
  • isolated antibodies that specifically bind to human OX40 protein may have cross-binding properties to other antigens, such as OX40 proteins of other species.
  • the isolated antibody contains substantially no other cellular materials and/or chemical substances.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to an antibody molecule product of single molecular composition.
  • the monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.
  • the DNA sequences CTGACT and CAGGTT share 50% homology (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum homology.
  • Such comparison can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol., 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl.
  • Biosci., 4:11-17, 1988 that have been integrated into the ALIGN program (version 2.0), and use the PAM120 weight residue table (weightresiduetable)
  • a gap length penalty of 12 and a gap penalty of 4 are used to determine the percent homology between two amino acid sequences.
  • the Needleman and Wunsch (J.MoI.Biol., 48:444-453, 1970) algorithms in the GAP program integrated into the GCG software package (available on www.gcg.com) can be used, and Blossum 62 Matrix or PAM250 matrix and gap weight of 16, 14, 12, 10, 8, 6 or 4 and length weight of 1, 2, 3, 4, 5 or 6 to determine the percentage difference between two amino acid sequences. Source.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, OX40 protein). It has been confirmed that the antigen-binding function of antibodies can be implemented by fragments of full-length antibodies.
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by recombination through a synthetic linker that makes the two into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called Single-chain Fc (scFv); see, for example, Bird et al., (1988) Science242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
  • scFv Single-chain Fc
  • These single chain antibodies are also intended to be included in the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.
  • the antigen-binding fragments of the present invention include those capable of specifically binding to OX40.
  • antibody binding fragments include, for example, but not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single chain Fv (scFv) fragments, and single domain fragments.
  • the Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • the difference between Fab' fragments and Fab fragments is the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody.
  • Fab' fragments are generated by cleaving the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • the "Fc” region is a crystallizable constant region of an antibody fragment that does not contain an antigen-specific binding region.
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody.
  • IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.
  • the "Fv” fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site.
  • This region is composed of a dimer (VH-VL dimer) of one heavy chain and one light chain variable domain in tight non-covalent bonding.
  • VH-VL dimer dimer
  • the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer.
  • the six CDRs confer target binding specificity to the antibody.
  • even a single variable domain (or half of an Fv containing only three CDRs specific for the target) may have the ability to recognize and bind to the target, although its affinity is lower than the entire binding site.
  • Single chain Fv or “scFv” antibody binding fragments comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form a structure that facilitates target binding.
  • a "single domain fragment” consists of a single VH or VL domain that shows sufficient affinity for OX40.
  • the single domain fragments are camelized (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).
  • the anti-OX40 antibodies of the present invention include derivatized antibodies.
  • derivatized antibodies are usually glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, and linked to cell ligands or other proteins.
  • protecting/blocking groups proteolytic cleavage, and linked to cell ligands or other proteins.
  • Any of numerous chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the derivative may contain one or more unnatural amino acids, for example, using ambrx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).
  • the anti-OX40 antibody may be an antibody whose sequence has been modified to change at least one constant region-mediated biological effector function.
  • an anti-OX40 antibody can be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, for example, with one or more Fc receptors (Fc ⁇ R) such as The binding of FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA and/or FcyRIIIB is reduced.
  • Fc ⁇ R binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at specific regions necessary for Fc ⁇ R interaction (see, for example, Canfield and Morrison, 1991, J. Exp. Med.
  • the reduction in the Fc ⁇ R binding capacity of an antibody can also reduce other effector functions that depend on Fc ⁇ R interactions, such as opsonization, phagocytosis, and antigen-dependent cytotoxicity ("ADCC").
  • ADCC antigen-dependent cytotoxicity
  • a variant CH2 domain with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region can show The affinity for Fc ⁇ RIIB is reduced.
  • a variant CH2 domain with V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutions in the CH2 domain of the Fc region can show Greater affinity for Fc ⁇ RIIIA.
  • the anti-OX40 antibodies of the present invention may have alterations in biological activity that result in increased or decreased opsonization, phagocytosis, or ADCC.
  • Such changes are known in the art.
  • US Patent No. 5,834,597 describes modifications in antibodies that reduce ADCC activity.
  • An exemplary ADCC lowering variant corresponds to "Mutant 3" (also referred to as "M3", shown in Figure 4 of US Patent No. 5,834,597), in which residues 234 and 237 (using EU numbering) are substituted with alanine.
  • Mutant 3 (also called “M3”) variants can be used in many antibody isotypes, such as human IgG2M3.
  • Additional substitutions that can modify the Fc ⁇ R binding and/or ADCC effector function of the anti-OX40 antibody include K322A substitutions or L234A and L235A dual substitutions in the Fc region, such as human IgG1 with L234A/L235A dual substitutions. See, for example, Hezareh et al. J. Virol., 75(24): 12161-12168 (2001).
  • the anti-OX40 antibody has low levels of fucose or lacks fucose.
  • Antibodies lacking fucose have been associated with enhanced ADCC activity, especially at low doses of antibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73.
  • the method of making antibodies with less fucose involves growth in rat myeloma YB2/0 cells (ATCC CRL 1662).
  • YB2/0 cells express low levels of FUT8 mRNA, which encodes ⁇ -1,6-fucosyltransferase, an enzyme necessary for the fucosylation of polypeptides.
  • the anti-OX40 antibody may comprise a modified (or variant) CH2 domain or the entire Fc domain that includes amino acid substitutions that increase the binding to Fc ⁇ RIIB and/or the binding of the corresponding wild-type CH2 or Fc region. Or the combination of technology and Fc ⁇ RIIIA.
  • the variant CH2 or variant Fc domain has been described in US Patent Application No. 2014/0377253.
  • the variant CH2 or variant Fc domain usually includes one or more substitutions at position 263, position 266, position 273, and position 305, where the number of residues in the Fc domain is the same as the EU index in Kabat .
  • the anti-OX40 antibody comprises one or more substitutions selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W relative to the wild-type CH2 domain.
  • one or more substitutions of the CH2 domain are selected from V263L, V273E, V273F, V273M, V273S, and V273Y.
  • one or more substitutions of the IgG1 CH2 domain can be V273E.
  • the anti-OX40 antibody of the invention comprises a variant IgG1 CH2 domain, which comprises the amino acid substitution V263L.
  • variant CH2 or variant Fc domains that can provide increased binding to Fc ⁇ RIIB and/or reduced binding to Fc ⁇ RIIIA compared to the binding of the corresponding wild-type CH2 or Fc region are included in Vonderheide et al. Clin. Cancer Those found in Res., 19(5), 1035-1043 (2013), such as S267E or S267E/L328F in human IgG1.
  • the anti-OX40 antibody includes modifications that increase or decrease its binding affinity to the fetal Fc receptor FcRn, for example, by mutating immunoglobulin constant region segments at specific regions involved in FcRn interaction (see, e.g., WO 2005/123780).
  • the anti-OX40 antibody of the IgG class is mutated so that at least one of the amino acid residues 250, 314 and 428 of the heavy chain constant region is substituted alone or in any combination thereof, for example at positions 250 and 428 , Or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428, where positions 250 and 428 are a specific combination.
  • the substituted amino acid residue can be any amino acid residue except threonine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine acid.
  • threonine including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine acid.
  • the substituted amino acid residue can be any amino acid residue except leucine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine acid.
  • leucine including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, Histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine acid.
  • the substituted amino acid residue can be any amino acid residue except methionine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine , Histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine acid.
  • An exemplary substitution known to modify Fc effector function is the Fc substitution M428L, which can occur in combination with the Fc substitution T250Q. Additional specific combinations of suitable amino acid substitutions are identified in Table 1 of US Patent No. 7,217,797. Such mutations increase binding to FcRn, which protect the antibody from degradation and increase its half-life.
  • mouse-derived antibody refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences.
  • the murine antibody of the present invention may comprise amino acid residues not encoded by the mouse germline immunoglobulin sequence, for example, mutations introduced by random or point mutations in vitro or somatic mutations in vivo. However, the term “murine antibody” does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse framework sequence.
  • chimeric antibody refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally speaking, a chimeric antibody refers to an antibody that combines the genetic material of one species with the genetic material of another species.
  • humanized form of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulins.
  • a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all of the FR regions Or substantially all of those of human immunoglobulin sequences.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • Human antibodies include antibodies having the amino acid sequence of human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or animals that are transgenic for one or more human immunoglobulins and are not Express endogenous immunoglobulins. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741.
  • transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes to produce human antibodies. See, for example, PCT Publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598.
  • companies such as LakePharma, Inc.
  • antibody that recognizes an antigen and "antibody specific for an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” herein.
  • an antibody that "specifically binds to human OX40” refers to an antibody that binds to human OX40 (or OX40 of other non-human species) but does not substantially bind to non-OX40 proteins.
  • the antibody "high affinity" binding human OX40 protein i.e., a KD value of 5.0x10 -8 M or less, preferably 1.0x10 -8 M or less, more preferably 5.0x10 -9 M or less.
  • high affinity for an IgG antibody means that the KD for the antigen is 1.0x 10 -6 M or less, preferably 5.0x 10 -8 M or less, more preferably 1.0x 10 -8 M or less, 5.0x 10 -9 M or less, more preferably 1.0 x 10 -9 M or less.
  • "high affinity" binding may vary.
  • “high affinity” binding of IgM subtypes means that the KD is 10 -6 M or less, preferably 10 -7 M or less, and more preferably 10 -8 M or less.
  • Kassoc or “Ka” refers to the rate of association of a specific antibody-antigen interaction
  • Kdis or “Kd” refers to the rate of dissociation of a specific antibody-antigen interaction
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (Kd/Ka) and is expressed in molar concentration (M).
  • M molar concentration
  • the KD value of the antibody can be determined by a method known in the art.
  • a preferred way to determine the KD of an antibody is to use a surface plasmon resonance (SPR) measurement, preferably to use a biosensing system such as the BiacoreTM system.
  • SPR surface plasmon resonance
  • EC50 also called half-maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximum effect.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, Examples include non-human primates, sheep, dogs, cats, cows, and horses.
  • therapeutically effective amount refers to an amount of the antibody of the present invention that is sufficient to prevent or alleviate the symptoms associated with a disease or condition (e.g., cancer) and/or reduce the severity of the disease.
  • the therapeutically effective amount is related to the disease to be treated, and those skilled in the art can easily distinguish the actual effective amount.
  • OX40 antibody has binding specificity to human OX40 and other beneficial functional characteristics.
  • the antibody of the present invention specifically binds to human OX40 with high affinity, for example, a KD value of 1 ⁇ 10 -8 M or less.
  • the antibody also has cross-reactivity with monkey OX40 and does not bind to mouse OX40.
  • the antibody of the present invention has a good anti-tumor effect in vivo. After the antibody administration is stopped, the tumor will not grow, and even the tumor will be completely eliminated, and immune memory can be produced.
  • the antibodies of the present invention may be polyclonal, monoclonal, genetically engineered, and/or substantially modified in other ways, including but not limited to chimeric antibodies, humanized antibodies, and human antibodies.
  • the constant region is an isotype selected from: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM.
  • the anti-OX40 antibodies described herein comprise IgG1.
  • the anti-OX40 antibody comprises IgG2 or IgG4.
  • the "constant region" of an antibody includes natural constant regions, allotypes or natural variants, such as D356E and L358M in human IgG1, or A431G. See, for example, Jefferis and Lefranc, MAbs, 1(4): 332-338 (July to August 2009).
  • a preferred antibody of the invention is a monoclonal antibody.
  • the antibody may be, for example, a murine, chimeric or humanized monoclonal antibody.
  • the light chain constant region of an anti-OX40 antibody can be a C kappa ( ⁇ ) region or a C lambda ( ⁇ ) region.
  • the lambda region can be any of the known subtypes, such as lambda 1, lambda 2, lambda 3, or lambda 4.
  • the anti-OX40 antibody comprises a C kappa ( ⁇ ) region.
  • the antibody binds to human OX40 with an affinity ranging from about 1 pM to about 100 nM, or an affinity ranging between any of the foregoing values, such as but not limited to about 0.001 to 10 nM, 0.001 to 5 nM, 0.01 to 100 nM, 0.01 to 50 nM, 0.01 to 10 nM, 0.01 to 5 nM, or 0.01 to 1 nM.
  • Anti-OX40 antibodies generally include a heavy chain comprising a variable region (VH) with three complementarity determining regions ("CDR"), which are referred to herein as (in N ⁇ C order) VH CDR# 1.
  • VH CDR#2 and VH CDR#3 and a light chain comprising a variable region (VL) with three complementarity determining regions, which are referred to herein as (in N ⁇ C order) VL CDR#1, VL CDR#2 and VL CDR#3.
  • VH CDR#2 and VH CDR#3 a light chain comprising a variable region (VL) with three complementarity determining regions
  • VL CDR#1 variable region
  • VL CDR#2 and VL CDR#3 are referred to herein as (in N ⁇ C order) VL CDR#1, VL CDR#2 and VL CDR#3.
  • amino acid sequences of exemplary CDRs as well as the amino acid sequences of the VH and VL regions of the
  • the present invention provides a new humanized anti-OX40 activating antibody with a clear structure of amino acid sequence, high affinity to OX40, definite effect of activating the OX40 signaling pathway, low heterogeneity and high clinical Application potential.
  • hz25A7m8 can make the tumor completely regress, and the activity is better than PC2 and PC3 (the variable region sequence comes from Xinda patent: WO2018177220A1; heavy chain: SEQ NO ID: 111, light chain: SEQ NO ID: 130; also abbreviated in the present invention : XD; the same below) and other control antibodies.
  • Figure 1 ELISA results of the binding activity of murine monoclonal antibodies to OX40.
  • Figure 2 Detection result of IL8 expression in HT1080-hOX40 cells activated by anti-OX40 murine monoclonal antibody. The antibody concentration of each antibody gradually decreases from left to right.
  • Figure 3 The result of binding of anti-OX40 murine monoclonal antibody to HT1080-hOX40 cells.
  • Figure 4 Anti-OX40 chimeric antibodies ch25A7, ch27G12, and ch11F7 Fortebio determined affinity results.
  • Figure 5B NF ⁇ B pathway activation-fluorescein detection method to identify the activation activity of the OX40 chimeric antibody.
  • the antibody concentration of each antibody gradually decreases from left to right.
  • FIG. 6 Chimeric antibodies ch25A7 and ch27G12 block the activity of OX40L in binding to HT1080-huOX40 cells.
  • Figure 8 The result of the species-specific identification of the anti-OX40 antibody hz25A7m8.
  • Figure 9 The result of species-specific identification of the anti-OX40 antibody hz27G12H1L2.
  • FIG. 10A Immunological function (IFN- ⁇ ) of OX40 antibody hz25A7 and hz27G12, Von is PC1, BMS is PC2, and XD is PC3.
  • Figure 10C In vitro testing of the immunological function (cell proliferation) of OX40 antibodies hz25A7 and hz27G12, Von is PC1, BMS is PC2, and XD is PC3.
  • Figure 11B hz25A7m8, hz27G12H1L2 effectively inhibited tumor growth in the MC38 model of OX40 humanized mouse colorectal cancer (average data), BMS is PC2, XD is PC3.
  • Figure 12A The results of immunological memory analysis of subcutaneous tumor formation in mice after anti-OX40 antibody treatment successively (MC38), BMS is PC2; hz25A7m8, hz27G12H1L2, BMS, and isotype control groups are treated with 0h, 10mg/kg, ip , Biw ⁇ 4.
  • FIG. 13 FACS method to detect immune memory T cell grouping results, BMS is PC2.
  • the present invention provides antibodies or fragments thereof, including heavy chain variable regions and light chain variable regions, wherein
  • variable region of the heavy chain contains:
  • VH CDR3 is selected from the amino acid sequence shown in SEQ ID NO: 42, 54, 66;
  • VL CDR1 is selected from the amino acid sequence shown in SEQ ID NO: 46, 58, 70,
  • the heavy chain variable region is selected from SEQ ID NO: 6, the amino acid sequence shown in 24 has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% homology .
  • the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 8, 26, 28, 32 with 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% identical Origin
  • the heavy chain variable region is SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 10 has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology.
  • the light chain The variable region is SEQ ID NO: The amino acid sequence shown in 12 has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology.
  • the present invention also provides an immunoconjugate, which comprises
  • the present invention also provides a multispecific antibody or derivative thereof, which comprises at least one antigen binding domain of the antibody or fragment thereof according to the present invention.
  • the present invention also provides a heavy chain antibody, which is a dimeric heavy chain antibody obtained on the basis of the antibody of the present invention.
  • the present invention also provides a composition comprising
  • the present invention also provides a recombinant vector or recombinant host cell, which includes the encoding nucleic acid of the present invention.
  • the present invention also provides the antibody or its fragment, the antibody conjugate, the multispecific antibody or its derivative, the heavy chain antibody, the composition, the nucleic acid, the recombinant vector or the recombinant Uses of host cells include:
  • the present invention also provides a method for producing antibodies, including:
  • mice were immunized with human OX40/mFc recombinant protein (serial number: P43489-1, 29aa-216aa), and serum titer was detected by ELISA with a 96-well ELISA plate coated with recombinant human OX40-his protein ; Mice whose serum titers meet the fusion requirements are used for the next cell fusion.
  • human OX40/mFc recombinant protein serial number: P43489-1, 29aa-216aa
  • Example 2 Screening of anti-human OX40 antibody-positive hybridoma cell lines.
  • test hole A492 value is greater than 2.1 times the negative control hole A492 value and it is judged as positive. In order to determine the reliability of positive clones, a second round of screening was carried out every other day after the first screening change.
  • Anti-OX40 murine monoclonal antibody activates HT1080-hOX40 cells to express IL8 activity screening.
  • Digest HT1080-hOX40 cells (Kangyuan Bochuang: Cat.No: KC-0140) the day before the experiment, resuspend the cells in complete medium (1640 medium + 10% FBS + 0.5 ⁇ g/mL), and adjust the cell density to 10 5 cells/mL, add 200 ⁇ L of cell suspension to each well of a 96-well cell culture plate. Then the cell culture plate was placed in a 37°C 5% CO2 incubator and cultured overnight. The next day, the antibody to be tested was diluted to an appropriate concentration, and added to the cell culture plate, and incubated in a 37°C 5% CO2 incubator for 6 hours.
  • PC1 namely Vonlerolizumab, the variable region sequence is derived from WHO Drug information, Vol. 31, NO. 3, 2017, also referred to as Von in the present invention, the same below.
  • the cell culture supernatant was aspirated, and the IL-8 ELISA quantitative kit (product number: batch number) was used to detect the content of IL-8 in the supernatant according to the instructions. The results are shown in Figure 2. Most of the clones can activate HT1080-hOX40 cells well and increase the level of IL8 expression.
  • Example 3 Sequence determination of murine anti-human OX40 antibody.
  • the m25A7, m27G12, and m11F7 hybridoma cells were extracted according to the TRIzol kit (Cat:15596026, Invitrogen) instructions to extract total cellular RNA; M-MuLV reverse transcriptase (Cat:M0253S, NEB) was used to reverse the total RNA of hybridoma cells Record into cDNA; use degenerate primers and Phusion kit (Cat: E0553L, NEB) to amplify antibody light chain variable region IgVL ( ⁇ ) and heavy chain variable region V H sequence; use gel recovery kit (Cat: AP -GX-250, Axygen) Purify the PCR amplification product; according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biological), connect the amplified PCR product to the T vector and transform E.
  • M-MuLV reverse transcriptase (Cat:M0253S, NEB) was used to reverse the total RNA of hybridom
  • sequence 3 The nucleotide sequence of the murine antibody m25A7 heavy chain variable region DNA is shown in sequence 1, and the amino acid sequence of the murine antibody m25A7 heavy chain variable region inferred from the DNA sequence is shown in sequence 2.
  • sequence 3 The nucleotide sequence of the light chain variable region DNA of the mouse antibody m25A7 is shown in sequence 3.
  • the amino acid sequence of the light chain variable region of the mouse antibody m25A7 is inferred in sequence 4; the nucleoside of the mouse antibody m27G12 heavy chain variable region DNA The acid sequence is shown in sequence 5, and the amino acid sequence of the variable region of the murine antibody m27G12 heavy chain is inferred from the DNA sequence in sequence 6.
  • the nucleotide sequence of the light chain variable region DNA of the murine antibody m27G12 is shown in Sequence 7. From the DNA sequence, the amino acid sequence of the light chain variable region of the murine antibody m27G12 is shown in Sequence 8.
  • the nucleotide sequence of the variable region DNA of the murine antibody m11F7 heavy chain is shown in Sequence 9.
  • the amino acid sequence of the variable region of the murine antibody m11F7 heavy chain is inferred in Sequence 10.
  • the nucleotide sequence of the light chain variable region DNA of the mouse antibody m11F7 is shown in Sequence 11, and the amino acid sequence of the light chain variable region of the mouse antibody m11F7 is inferred from this DNA sequence.
  • Example 4 Preparation of anti-human OX40 chimeric antibody.
  • the human-mouse chimeric light chain (pKN019-Ch25A7L) and human-mouse chimeric heavy chain (pKN041-Ch25A7H) expression plasmids of m25A7 were obtained, and the human-mouse chimeric light chain of m27G12 (pKN019-Ch27G12L) And human-mouse chimeric heavy chain (pKN041-Ch27G12H) expression plasmids, and m11F7 human-mouse chimeric light chain (pKN019-Ch11F7L) and human-mouse chimeric heavy chain (pKN041-Ch11F7H) expression plasmids.
  • a large number of plasmids containing human-mouse chimeric antibody light chain and heavy chain were isolated and amplified by transferring into E. coli. According to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the light and heavy chain plasmids of the chimeric antibodies Ch 25A7, Ch 27G12 and Ch11F7 were paired and transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column to obtain the m25A7, m27G12, and m11F7 chimeric antibodies.
  • Example 5 Activity analysis of anti-human OX40 chimeric antibody.
  • the anti-human antibody Fc segment capture antibody (AHC) biological probe is used to capture the Fc segment of the antibody to determine the affinity of the antibody.
  • AHC anti-human antibody Fc segment capture antibody
  • the anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and the control antibody PC1 were diluted to 4ug/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat:18-0015, PALL) for 120s.
  • the reaction curves of anti-human OX40 chimeric antibodies ch25A7, ch27G12, ch11F7 and human OX40 recombinant protein are shown in Figure 4 (Von is the positive control PC1). The curves are fitted and the affinity is calculated. The results show that the chimeric antibodies ch25A7, ch27G12, The affinities (KD, Table 1) of ch11F7 are: 1.36E-08, 4.34E-08, 6.61E-09. The results show that the chimeric antibodies ch25A7, ch27G12, and ch11F7 have high affinity with human OX40 recombinant protein, which is lower than the control antibody PC1.
  • the NF ⁇ B reporter gene plasmid (pGL4.32[luc2P/NF- ⁇ B-RE/Hygro]) was transfected into HT1080-hOX40 cells by liposome method. Change the medium 24 hours after transfection and continue to culture the cells for 24 hours to ensure that the cells recover for 48 hours after transfection.
  • the antibody to be tested is then diluted to an appropriate concentration and added to the cell culture plate, and incubated in a 37°C 5% CO2 incubator for 18 hours. Remove the cell treatment medium, wash the cells thoroughly with PBS, and finally dry the cells thoroughly.
  • Example 6 ELISA to detect the inhibitory effect of anti-human OX40 chimeric antibody on the binding of human OX40/OX40L.
  • the human OX40 was diluted to 1 ⁇ g/mL, coated overnight at 4°C, and sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes.
  • the ch25A7, ch27G12 and the control antibody PC1, and the isotype control NC-hIgG1 (initial concentration 20 ⁇ g/mL, 3-fold serial dilution, 8 gradients) were reacted in a 37°C constant temperature incubator for 60 minutes, and then 10 ⁇ g/mL OX40L- mFc (OX40L serial number: NP_003317, 51-Leu 183, mFc Tag) was incubated with the antibody, and reacted in a constant temperature incubator at 37°C for 60 minutes.
  • Example 7 Humanization and recombinant expression of anti-human OX40 monoclonal antibodies 25A7 and 27G12.
  • a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then according to the homology comparison result, in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) to find the most similar human antibody template, select VH3( 3-21) as the basic template, combined with the full sequence blast results, considering the frequency of rearranged antibodies in specific FR region amino acids, and the HCDR3 sequence, CDR grafting, to achieve the m25A7 heavy chain variable region (VH) Highly humanized in the Framework area.
  • VH heavy chain variable region
  • the nucleotide sequence of the variable region of the humanized light chain 1 is shown in Sequence 15 and the amino acid sequence. See sequence 16; the nucleotide sequence of the variable region of humanized light chain 2 is shown in sequence 17, and the amino acid sequence is shown in sequence 18; the nucleotide sequence of the variable region of humanized light chain 3 is shown in sequence 19, and the amino acid sequence is shown in sequence 20. Subsequently, according to the characteristics of the murine 25A7 sequence, the CDR-transplanted humanized heavy chain variable region sequence was designed for back mutation, and the back mutation site is shown in Table 2.
  • the final selection of the heavy chain variable sequence The region nucleotide sequence is shown in Sequence 21, the amino acid sequence is shown in Sequence 22, the light chain variable region nucleotide sequence is shown in Sequence 15, and the amino acid sequence is shown in Sequence 16.
  • the 25A7 humanized light chain hz25A7_L1 and humanized heavy chain mutants (parental hz25A7_H1, mutant hz25A7_H1m1-hz25A7_H1m8) were paired (Table 3) and transferred into HEK293 cells for recombinant expression.
  • This table shows the sequences obtained from various combinations of humanized 25A7 light and heavy chains.
  • 25A7-1 the antibody is composed of 25A7 humanized light chain hz25A7_L1 and humanized heavy chain hz25A7_H1, and others are analogized.
  • CDR antigen complementarity determinant
  • the nucleotide sequence of the humanized heavy chain variable region of m27G12 antibody CDR Grafted is shown in Sequence 23, and the amino acid sequence is shown in Sequence 24; the nucleotide sequence of the humanized light chain 1 variable region is shown in Sequence 25, and the amino acid sequence See sequence 26; the humanized light chain 2 variable region nucleotide sequence is shown in sequence 27, and the amino acid sequence is shown in sequence 28.
  • the 27G12 humanized heavy chain hz27G12H1 and the humanized light chain hz27G12L1 and hz27G12L2 were paired and transferred into HEK293 cells for recombinant expression.
  • Example 8 Activity analysis of anti-human OX40 humanized antibody.
  • Example 9 The species cross of hz25A7-mut8 and hz27G12-H1L2.
  • Human OX40 (serial number: P43489-1, 29aa-216aa), cynomolgus OX40 (Cat: 90846-C08H, Beijing Yiqiao Shenzhou), and mouse OX40 (Cat: 50808-MCCH, Beijing Yiqiao Shenzhou) cells
  • the outer region recombinant protein was diluted to 1 ⁇ g/mL and coated overnight at 4°C; after washing the plate 3 times with PBS, add 5% BSA PBS, block at 37°C for 60 min, wash the plate 3 times with PBST; add h25A7mut8, h27G12H1L2, 37 Incubate at °C for 60min, wash the plate with PBST 4 times; add 1:5000 diluted HRP-goat anti-human IgG antibody (Cat:115-035-071, Jackson ImmunoResearch), incubate at 37°C for 30min, wash the plate with PBST 4 times; add TMB The substrate develops color.
  • Example 10 In vitro testing of the immunological functions of OX40 antibodies hz25A7 and hz27G12.
  • peripheral blood mononuclear cell PBMC
  • PBMC peripheral blood mononuclear cell
  • OX40 antibody or control antibody PBMC
  • cell proliferation and cytokine (IL-2 , IFN- ⁇ ) secretion level to evaluate the functional activity of OX40 antibody.
  • the anti-human CD3 antibody and the anti-human CD28 antibody are individually or together coated on the enzyme-linked plate as a control well; the anti-human OX40 antibody is coated on the enzyme-linked plate as the well to be tested.
  • the experimental results show that, like the control antibody, hz25A7m8 (labeled KNAb-1 in this example), hz27G12H1L2 (labeled KNAb-2 in this example) can significantly increase the cytokine IL-2 and IL-2 in the culture medium in a dose-dependent manner.
  • the concentration of IFN- ⁇ suggests that it can activate T lymphocytes and enhance their function of secreting cytokines.
  • the cell proliferation test results showed ( Figure 10C) that both hz25A7m8 and hz27G12H1L2 can effectively promote cell proliferation, and the ability to promote cell proliferation is equivalent to that of the control antibodies PC2 and PC3.
  • Example 11 In vivo activity analysis of anti-OX40 antibodies hz25A7m8 and hz27G12H1L2.
  • MC38 mouse colorectal cancer cells are cultured in DMEM medium containing 10% inactivated fetal bovine serum, 100U/ml penicillin and 100 ⁇ g/ml streptomycin in a 37°C, 5% CO2 incubator, every 3 to After 4 days when the cells are overgrown, the cells are divided into flasks and passaged.
  • the tumor cells in logarithmic growth phase are harvested, resuspended in PBS and inoculated subcutaneously on the right flank of human OX40 transgenic mice, and grouped when the average tumor volume reaches about 60-100mm3 Administration, 6 animals in each group, according to the dose of 10mg/kg, twice a week, a total of 4 administrations.
  • the tumor volume was measured with vernier calipers three times a week, and the long and short diameters of the tumor were measured.
  • the mice were weighed.
  • the relationship between the change of mouse body weight and the time of administration was recorded.
  • the mice were euthanized, the tumors were stripped and weighed, and the tumors stripped from the control group and the test group were placed neatly and photographed.
  • MC38 mouse colorectal cancer tumor cells and Hepa1-6 mouse liver cancer tumor cells use DMEM medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin, 100 ⁇ g/ml streptomycin and 2mM glutamine Cultivate in a 37°C, 5% CO 2 incubator, and sub-flask the cells every 3 to 4 days after the cells are fully grown, harvest the tumor cells in the logarithmic growth phase, and inoculate them in the scheme 1 of this embodiment through hz25A7m8, hz27G12H1L2 , HuOX40 mice whose MC38 tumors completely disappeared after PC2 treatment, and C57BL/6J mice that have not been inoculated with tumors, the tumor cells resuspended in PBS were inoculated subcutaneously on the left and right flanks of experimental animals.
  • the tumor volume is measured 2-3 times with a vernier caliper every week, and the long and short diameters of the tumor are measured.
  • the tumor-bearing mice were dissected in a sterile environment to obtain the spleen; the 70 ⁇ M cell screen was placed in a sterile dish, the spleen was transferred to the cell screen, and the spleen was ground into discrete single cells with a grinding rod; the cells were collected Transfer the suspension to a 50 mL centrifuge tube, centrifuge at 200 ⁇ g for 10 min; discard the supernatant, lyse the red blood cells, wash twice, and process the cell suspension through a 40 ⁇ M cell screen to obtain a spleen single cell suspension; suspend the spleen single cell Transfer the solution to a flow cytometer, add the corresponding antibody (anti-mouse CD3/CD4/CD44/CD62L) according to the experimental design, and incubate for 30 minutes in the dark; add PBS to wash once, resuspend the cells and perform flow cytometry.
  • the 70 ⁇ M cell screen was placed in a sterile dish, the spleen was transferred to

Abstract

La présente invention concerne un anticorps ou un fragment de celui-ci contre un récepteur activé (OX40) exprimé sur les surfaces de lymphocytes T CD4+ et T CD8+, et l'utilisation de l'anticorps ou du fragment de celui-ci dans la prévention ou le traitement de maladies. L'anticorps présente une affinité élevée pour OX40 et présente un effet évident d'activation de la voie de signalisation OX40, et a un spectre relativement large d'effets de stimulation immunitaire, peut amplifier la réponse de lymphocytes T à la mémoire immunitaire, obtenir de meilleurs effets d'inhibition et de destruction tumorale dans des essais in vivo sur des modèles animaux, et possède donc de bonnes perspectives d'application clinique.
PCT/CN2021/072586 2020-01-19 2021-01-19 Anticorps anti-ox40, son procédé de production et son application WO2021143914A1 (fr)

Applications Claiming Priority (2)

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CN202010063141.3A CN113135994A (zh) 2020-01-19 2020-01-19 一种激活型抗ox40抗体、生产方法及应用
CN202010063141.3 2020-01-19

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WO2023109901A1 (fr) * 2021-12-17 2023-06-22 Shanghai Henlius Biotech, Inc. Anticorps anti-ox40 et procédés d'utilisation
CN114591988A (zh) * 2022-03-30 2022-06-07 北京贝来生物科技有限公司 一种激活肿瘤免疫的基因修饰干细胞制备方法

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