WO2023186100A1 - 抗ror1的抗体及其用途 - Google Patents

抗ror1的抗体及其用途 Download PDF

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WO2023186100A1
WO2023186100A1 PCT/CN2023/085493 CN2023085493W WO2023186100A1 WO 2023186100 A1 WO2023186100 A1 WO 2023186100A1 CN 2023085493 W CN2023085493 W CN 2023085493W WO 2023186100 A1 WO2023186100 A1 WO 2023186100A1
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antibody
antigen
seq
cancer
cells
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PCT/CN2023/085493
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English (en)
French (fr)
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廖敬礼
付雅媛
曹卓晓
刘雷
唐任宏
任晋生
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山东先声生物制药有限公司
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Publication of WO2023186100A1 publication Critical patent/WO2023186100A1/zh

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    • 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
    • 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

Definitions

  • the present invention relates to the fields of bioengineering and biomedicine, and mainly relates to an anti-human ROR1 antibody or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cells, preparation methods, pharmaceutical compositions, and their uses for treating diseases. , such as the treatment of tumors.
  • ROR1 is a transmembrane receptor tyrosine kinase protein and a member of the receptor tyrosine kinases (RTKs) family.
  • the ROR1 encoding gene has a total length of 2814bp and is located on chromosome 1p31.3.
  • ROR1 membrane protein consists of 937 amino acids and has a molecular weight of approximately 105kDa.
  • the structure of ROR1 is highly conserved among biological species. For example, the amino acid sequence homology of ROR1 between humans and mice reaches 97%.
  • the human ROR1 molecule consists of three parts, including an extracellular region, a transmembrane region, and an intracellular region.
  • the extracellular region includes an immunoglobulin-like domain (Ig) and two cysteine-rich coiled domains ( FZD) and a juxtamembrane Kringle domain (KZD).
  • the intracellular region contains a tyrosine kinase domain (TKD), two serine/threonine-rich domains (S/TRD) and a proline-rich domain. set domain (PRD).
  • ROR1 has low or no expression in normal human tissues, but is highly expressed in a variety of malignant tumors or tissues, such as chronic lymphocytic leukemia (CLL), breast cancer, ovarian cancer, melanoma, lung adenocarcinoma, etc.
  • CLL chronic lymphocytic leukemia
  • breast cancer breast cancer
  • ovarian cancer ovarian cancer
  • melanoma melanoma
  • lung adenocarcinoma etc.
  • the characteristics of high expression in tumors and low expression in healthy cells greatly enhance the clinical value of ROR1.
  • a large number of studies have shown that ROR1 plays an important role in promoting tumor growth and metastasis, inducing drug resistance in tumor cells, and inhibiting apoptosis.
  • ROR1 can mediate the non-canonical Wnt signaling pathway, especially Wnt5a, participate in the phosphorylation of NF- ⁇ B subunit p65, activate the NF- ⁇ B pathway in tumor cells, and promote cell migration and Invasion. Therefore, ROR1, as a highly identifiable tumor-specific marker, has become an attractive target in tumor treatment.
  • ROR1 is highly expressed in a variety of malignant tumors or tissues and is lowly expressed in healthy cells, suggesting that ROR1 can be used as a highly identifiable tumor-specific marker and can be used in drugs to treat tumors. in preparation.
  • the present disclosure provides an antibody or antigen-binding fragment that specifically binds ROR1, the antibody or antigen-binding fragment comprising a combination of heavy chain CDRs and a combination of light chain CDRs:
  • the heavy chain CDRs combination includes: CDR1-VH, CDR2-VH and CDR3-VH; the CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or are combined with the sequence Compared to sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • the light chain CDRs combination includes: CDR1-VL, CDR2-VL and CDR3-VL.
  • the CDR1-VL, CDR2-VL and CDR3-VL have any sequence combination selected from the following or are consistent with the sequence combination. Compare sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • Each CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is encoded according to the popular analysis method of KABAT, IMGT or Chothia; preferably, the substitution is a substitution of a conservative amino acid.
  • the antibody or antigen-binding fragment comprises a combination of heavy chain CDRs and light chain CDRs selected from: VH1+VL1, VH2+VL2, or VH3+VL3, and combinations with the heavy chain and
  • the sequence of the light chain CDRs combination is compared to a CDRs combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions; preferably, the substitutions are conservative amino acid substitutions.
  • the heavy chain variable region and the framework region of the light chain variable region of the antibody or antigen-binding fragment of the invention are derived from a human germline heavy chain template and a human germline light chain template, wherein:
  • the framework region sequence is derived from the combined sequence of human germline heavy chain IGHV4-38-2*01 and IGHJ6*01; it contains FR1, FR2, and FR3 of IGHV4-38-2*01 shown in SEQ ID NO: 24 Area and SEQ ID NO: FR4 area of IGHJ6*01 shown in 25;
  • the framework region sequence is derived from the combined sequence of human germline light chain IGKV4-1*01 and IGKJ4*01; it contains the FR1, FR2, FR3 regions and SEQ ID of IGKV4-1*01 shown in SEQ ID NO: 21 NO: FR4 area of IGKJ4*01 shown in 23;
  • the framework region sequence is derived from the combined sequence of human germline light chain IGKV3-11*01 and IGKJ4*01; it contains the FR1, FR2, FR3 regions and SEQ ID of IGKV3-11*01 shown in SEQ ID NO: 22 NO: FR4 area of IGKJ4*01 shown in 23.
  • the antibody or antigen-binding fragment of the invention further includes one or more mutations selected from the group consisting of:
  • the framework region of the heavy chain variable region includes: Q1D, Q1E, V2L, S30T, P41F, K44N, G45K, V72R or S80F; preferably includes Q1E, S30T and V72R; or preferably includes Q1E, S30T, V72R and S80F; Or preferably include Q1E, S30T, P41F and V72R; Or preferably include Q1D, V2L, S30T and V72R; Or preferably include Q1E, S30T, K44N, G45K and V72R;
  • the framework region of the light chain variable region includes: A47P, V62I or G72R; preferably includes G72R; or preferably includes A47P and G72R; or preferably includes V62I and G72R.
  • the antibody or antigen-binding fragment of the invention comprises:
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 9, 16, 17, 18, 19 or 20;
  • the light chain variable region has the sequence shown in SEQ ID NO: 10, 13, 14 or 15;
  • the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment of the invention are selected from the group consisting of:
  • the antibody or antigen-binding fragment of the present invention is (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; (3) a fully human antibody or a fragment thereof; preferably , the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), monovalent antibodies, multivalent antibodies, fully Long antibody, antibody fragment, naked antibody, conjugated antibody, humanized antibody, fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody or single domain antibody.
  • the antibody of the present invention comprises the sequence of any one of the constant regions of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably it contains human or murine antibodies IgG1, IgG2 , IgG3 or IgG4 constant region sequence.
  • the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, Nanobodies and minimal recognition units of antibodies.
  • the antibody or antigen-binding fragment of the present invention is further coupled with a therapeutic agent or tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutics or immunomodulators, and the tracer Selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.
  • the therapeutic agent is selected from radioactive isotopes, chemotherapeutics or immunomodulators
  • the tracer Selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.
  • the present invention also provides a multispecific antigen-binding molecule, the multispecific antigen-binding molecule comprising a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprising the above-mentioned third antigen-binding module.
  • the second antigen-binding module specifically binds to other antigens other than ROR1 or binds to a different ROR1 antigen epitope than the first antigen-binding module; preferably, the other antigens are selected from CD3, CD7, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66(a-d), CD74 , CD80, CD126, CD138, BCMA, HLA-DR, HER2, VEGF, P1GF, HER3/ERBB3, HER4/ERBB4, IL-2, IL-6, PD-1, PD-L1, TRAIL-R1 or TRAIL-R2 ;
  • the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific
  • the present invention also provides a chimeric antigen receptor (CAR), which at least includes an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, so The extracellular antigen-binding domain includes the ROR1 antibody or antigen-binding fragment described in the first aspect.
  • CAR chimeric antigen receptor
  • the present invention also provides an immune effector cell, said immune effector cell comprising the chimeric antigen receptor described in the above fourth aspect or a nucleic acid comprising the chimeric antigen receptor described in the above fourth aspect.
  • the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells; preferably, the immune effector cells are allogeneic immune Effector cells or autologous immune cells.
  • the present invention also provides an isolated nucleic acid molecule encoding the antibody, antigen-binding fragment, or any combination of the above-mentioned first aspect, the multi-specific antigen binding of the third aspect Molecule or fourth aspect of chimeric antigen receptor.
  • the present invention also provides an expression vector comprising the nucleic acid molecule described in the sixth aspect.
  • the present invention provides the nucleic acid molecule described in the sixth aspect, or the isolated host cell of the expression vector described in the seventh aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell cells; more preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cells are selected from Expi293 or CHO cells.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment described in the first aspect, or the multispecific antigen-binding molecule described in the third aspect, under appropriate conditions.
  • the host cell of the eighth aspect and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.
  • the present invention provides a method for preparing the immune effector cells described in the fifth aspect, the method comprising introducing the CAR encoding the fourth aspect into the immune effector cells, optionally, The method further includes activating the immune effector cells to express the CAR described in the fourth aspect.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment described in the first aspect, the immune effector cell described in the fifth aspect, the sixth aspect
  • the pharmaceutical composition also contains a pharmaceutically acceptable carrier, diluent or auxiliary agent; preferably, the pharmaceutical composition also contains an additional anti-tumor agent.
  • the present invention provides the antibody or antigen-binding fragment described in the first aspect, the multispecific antigen-binding molecule described in the third aspect, the chimeric antigen receptor described in the fourth aspect. body, the immune effector cell described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the expression vector described in the seventh aspect, the cell described in the eighth aspect, the ninth or tenth aspect
  • the tumor diseases are preferably melanoma, clear cell sarcoma, chronic lymphocytic Leukemia, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer cancer.
  • the present invention provides a method for preventing and/or treating tumor diseases, comprising administering to a patient in need an effective amount of the antibody or antigen-binding fragment described in the first aspect, a third The multispecific antigen-binding molecule described in the first aspect, the chimeric antigen receptor described in the fourth aspect, the immune effector cell described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the seventh aspect
  • the tumor disease is preferably melanoma, clear cell sarcoma, chronic lymphocytic leukemia, head and neck cancer, and bladder cancer.
  • the present invention provides the antibody or antigen-binding fragment described in the first aspect, the multispecific antigen-binding molecule described in the third aspect, the chimeric antigen receptor described in the fourth aspect. body, the immune effector cell described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the expression vector described in the seventh aspect, the cell described in the eighth aspect, the ninth or tenth aspect
  • the product prepared by the method described in the aspect, or the pharmaceutical composition described in the eleventh aspect is used for and/or treating tumor diseases; the tumor diseases are preferably melanoma, clear cell sarcoma, chronic lymphocytic leukemia, head and neck uterine cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, salivary gland cancer, lung cancer, liver cancer, skin cancer, or brain cancer.
  • the present invention provides a kit comprising the antibody or antigen-binding fragment described in the first aspect, the multispecific antigen-binding molecule described in the third aspect, the antibody or antigen-binding fragment described in the fourth aspect, The chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the fifth aspect, the isolated nucleic acid molecule described in the sixth aspect, the expression vector described in the seventh aspect, the cell described in the eighth aspect, The product prepared by the method described in the ninth or tenth aspect, or the pharmaceutical composition described in the eleventh aspect, and instructions for use.
  • antibody refers to an immunoglobulin molecule that specifically binds or is immunoreactive to a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugated antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies, antibody conjugates) and Antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab')2 fragments those capable of specifically Intact antibody molecules that sexually bind the target protein as well as incomplete antibody fragments such as Fab and F(ab')2 fragments, which lack the Fc fragment of the intact antibody (cleared more quickly from the animal circulation) and therefore lack Fc-mediated Effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the content of which is incorporated herein by reference).
  • the "antibodies” herein can be derived from any animal, including but not limited to humans and non-human animals.
  • the non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • the term "monospecific” herein refers to having one or more binding sites, where each binding site binds the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen-binding sites, one of the at least two antigen-binding sites Each antigen-binding site binds to a different epitope of the same antigen or to a different epitope of a different antigen.
  • terms such as “bispecific,””trispecific,””tetraspecific,” etc. refer to the number of different epitopes to which the antibody/antigen-binding molecule can bind.
  • Fully-length antibody “intact antibody” and “intact antibody” are used herein interchangeably and mean that they have a structure that is substantially similar to that of a native antibody.
  • the term "antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of the full-length antibody.
  • the antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, tribody, affibody, Nanobody, aptamer or domain antibody.
  • binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains of one arm of the antibody Fv fragments consisting of; (V) dAbs containing VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) dAbs consisting of VH or VL A dAb consisting of a domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally
  • the two domains of the Fv fragment, VL and VH are encoded by separate genes, the two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • ROR1 is a transmembrane protein in the receptor tyrosine kinase (RTK) family.
  • the ROR1 gene encodes two well-defined isoforms: a short 393 aa intracellular protein (isoform 2) and a long 937 aa type I transmembrane protein (isoform I).
  • the long cell surface isoform is expressed on primary human B chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, a subset of B acute lymphoblastic leukemia, and on many tumors, including those associated with a metastatic phenotype.
  • ROR1 is mainly expressed during embryonic development, but its expression is attenuated during fetal development.
  • ROR1 is used by certain B-cell malignancies (such as, but not limited to, lymphoma, CLL, and B-ALL) as well as many solid tumor malignancies (such as, but not limited to, adrenal cancer, bladder cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, cancer, prostate cancer, ovarian cancer, skin cancer, testicular cancer, uterine cancer and neuroblastoma) are abnormally expressed.
  • B-cell malignancies such as, but not limited to, lymphoma, CLL, and B-ALL
  • solid tumor malignancies such as, but not limited to, adrenal cancer, bladder cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, cancer, prostate cancer, ovarian cancer, skin cancer, testicular cancer, uterine cancer and neuroblastoma
  • the term "bispecific antibody” refers to an antibody, typically a human or humanized antibody, with monoclonal binding specificity for at least two different antigens.
  • one of the binding specificities can be detected against an epitope of ROR1, and the other can be detected against another epitope of ROR1 or any other antigen besides ROR1, for example, against a cell surface protein, a receptor, Receptor subunits, tissue-specific antigens, viral-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins are detected.
  • chimeric antibody refers to an antibody that has variable sequences of immunoglobulins derived from one source organism (e.g., rat or mouse) and derived from a different organism (e.g., human). The constant region of an immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; The above is incorporated herein by reference.
  • CDR complementarity determining region
  • FR framework region
  • the amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT) but not considered to be within a different set of criteria (such as KABAT or IMGT). One or more of these locations may also be found in extended hypervariable zones.
  • the invention includes antibodies containing modifications in these hybrid hypervariable positions.
  • variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect the sheet structure , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held closely together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, MD. 1987; which is incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, second CDR and third CDR of the heavy chain variable region (VH) respectively.
  • These three CDRs constitute the heavy chain variable region (VH).
  • the CDR combination of the light chain (or its variable region) (VHCDR combination);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR of the light chain variable region (VL) respectively.
  • Three CDRs, these three CDRs constitute the CDR combination of the light chain (or its variable region) (VLCDR combination).
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method of production of the antibody.
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody (including the heavy chain of a Fv, scFv, or Fab).
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).
  • heavy chain constant region herein refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, that are relative to the antibody's Variable domains have more conserved amino acid sequences.
  • "Heavy chain constant region” includes at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain When an antibody is used, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxyl-terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen.
  • the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxyl-terminal portion of the antibody resulting from papain hydrolysis of the intact antibody, which typically contains the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain.
  • the Fc region can include Or excluding Lys447.
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance immune response, etc.
  • the term "fully human antibody” herein refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • naked antibody herein refers to an antibody that is not linked, fused, or conjugated to another agent or molecule (eg, a label or drug), peptide or polypeptide.
  • naked antibodies expressed from mammalian host cells can be glycosylated by the host cell's glycosylation machinery (eg, glycosylases).
  • naked antibodies are not glycosylated when expressed by a host cell that does not possess its own glycosylation machinery (eg, glycosylase).
  • the naked antibody is an intact antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of an intact antibody, such as a Fab antibody.
  • conjugated antibody refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • diabody herein refers to a bivalent bispecific antibody that binds to different epitopes on the same or different antigens.
  • percent (%) sequence identity refers to the alignment of sequences and the introduction of gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, the candidate and reference After introducing gaps in one or both of the sequences, and non-homologous sequences can be ignored for comparison purposes), the amino acid (or nucleotide) residues of the candidate sequence are compared with the amino acid (or nucleotide) residues of the reference sequence. ) percentage of residues that are identical. For the purpose of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity.
  • the length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.
  • amino acids herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to conservative amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:
  • Acidic amino acids Asp(D) and Glu(E);
  • Non-polar uncharged amino acids Cys(C), Met(M) and Pro(P);
  • Aromatic amino acids Phe(F), Tyr(Y) and Trp(W).
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, e.g., by an antibody or its antigen Specific recognition of binding fragments.
  • An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • a variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate.
  • solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • antibody conjugate refers to a conjugate/conjugate in which an antibody molecule is chemically bonded to another molecule, either directly or through a linker.
  • An example is an antibody-drug conjugate (ADC), where the drug molecule is the other molecule.
  • ADC antibody-drug conjugate
  • chimeric antigen receptor herein refers to a recombinant protein containing at least (1) an extracellular antigen-binding domain, such as a variable heavy or light chain of an antibody, and (2) anchoring the CAR into transmembrane domain of immune effector cells, and (3) intracellular signaling domain.
  • the extracellular antigen binding domain of the CAR comprises a scFv.
  • a scFv can be derived from a fusion of the variable heavy and light domains of an antibody.
  • the scFv may be derived from Fab's (rather than antibodies, e.g. obtained from a Fab library).
  • the scFv is fused to a transmembrane domain and then to an intracellular signaling domain.
  • nucleic acid herein includes any compound and/or substance that contains a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA containing two A polymer that is a mixture of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/ or expression of the encoded molecule such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
  • vector includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors).
  • DNA vectors eg, plasmids
  • RNA vectors eg. RNA vectors
  • viruses or other suitable replicons eg, viral vectors.
  • a variety of vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • Expression vectors of the present invention contain polynucleotide sequences as well as additional sequence elements for, for example, expression of proteins and/or integration of these polynucleotide sequences into the genome of mammalian cells.
  • Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences that direct gene transcription, such as promoter and enhancer regions.
  • kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites in order to direct efficient transcription of the gene carried on the expression vector.
  • Expression vectors of the invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
  • host cell herein refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cells are included herein.
  • composition refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain unacceptable toxicity to the subject administered the pharmaceutical composition of additional ingredients.
  • the terms "subject,” “subject,” and “patient” refer to an organism undergoing treatment for a particular disease or condition (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), sheep and horses, etc.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject, such as cell proliferative disorders (such as cancer) or infectious disease) progression.
  • Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • an effective amount herein refers to an amount of a therapeutic agent that is effective to prevent or alleviate the symptoms of a disease or the progression of a disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • the therapeutically effective agent Amount refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • appropriate conditions refers to conditions suitable for culturing various host cells, including eukaryotic cells and prokaryotic cells.
  • cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and tumor as used herein are not mutually exclusive.
  • anti-tumor agent refers to anti-tumor drugs, which are a class of drugs used to treat tumor diseases, such as chemotherapy drugs, biological agents, etc.
  • Figure 1A shows the binding reaction between human ROR1-his protein and positive antibodies detected by ELISA.
  • Figure 1B shows the binding reaction between human ROR1-Ig-his protein and positive antibodies detected by ELISA.
  • Figure 1C shows the binding reaction between human ROR1-KZD-his protein and positive antibodies detected by ELISA.
  • Antibody binding reaction Figure 1D shows the binding reaction of mouse ROR1-his protein and positive antibody detected by ELISA.
  • Figure 2 shows the binding reaction between endogenous cell MDA-MB-231 and positive antibodies detected by FACS.
  • Figure 3A shows the FACS detection results of the Flp-in CHO stably transduced cell line expressing human ROR1 protein.
  • Figure 3B shows the FACS detection result of the Flp-in CHO stably transduced cell line expressing the mouse ROR1 protein.
  • Figure 4A shows the binding reaction between the FACS detection control antibody and MDA-MB-231 tumor cells.
  • Figure 4B shows the binding reaction between the FACS detection control antibody and Flp-in CHO-hROR1 recombinant cells.
  • Figure 4C shows the FACS detection control antibody and Flp-in Binding reaction of CHO-mROR1 recombinant cells.
  • Figure 5A is an ELISA for detecting the binding potency of mouse serum to human ROR1-his protein after immunization.
  • Figure 5B is an ELISA for detecting the binding potency of mouse serum to mouse ROR1-his protein after immunization.
  • Figure 5C is an ELISA for detecting the binding potency of mouse serum to human ROR1-his protein after immunization. The binding potency of mouse serum to human ROR1-Ig-his protein.
  • Figure 5D shows the ELISA detection of the binding potency of mouse serum to human ROR1-KZD-his protein after immunization.
  • Figure 6A shows the ELISA detection of the binding reaction between humanized antibodies and hROR1-his protein.
  • Figure 6B shows the ELISA detection of the binding reaction between humanized antibodies and hROR1-Ig-his protein.
  • Figure 6C shows the ELISA detection of the binding reaction between humanized antibodies and hROR1- The binding reaction of KZD-his protein,
  • Figure 6D is the ELISA detection of the binding reaction of humanized antibodies and mouse ROR1-his protein,
  • Figure 6E is the ELISA detection of the binding reaction of humanized antibodies and Flp-in CHO cells.
  • Figure 7 shows the FACS detection of the binding reaction between humanized antibodies and MDA-MB-231 cells.
  • the UC961 sequence comes from patent WO2020198531A2, and the I2A27 sequence comes from patent WO2018237335A1 (see Table 1).
  • UC961 binds to the ROR1 extracellular Ig domain, and I2A27 binds to the ROR1 extracellular KZD domain.
  • the VH and VL sequences of the clones that recognize the human ROR1 epitope were recombined.
  • the protein in the cell culture supernatant was purified using a Protein A column (GE, Cat. No. 17549802).
  • the Protein A column is first equilibrated with 3 to 5 column volumes of equilibrium buffer (PBS phosphate buffer, pH7.4) (20X PBS buffer 500ml, Sangon, Cat. No. B548117-0500), and then the clarified culture supernatant is Load the sample onto the Protein A column and control the flow rate at 10 mL/min. After loading the sample, wash the Protein A column with equilibrium buffer. The volume of the equilibrium buffer is 3 to 5 times the bed volume of the Protein A column. The protein bound to the protein A column was eluted with eluent (0.02M citrate buffer, pH 3.5).
  • the obtained protein was diluted to 1 ⁇ g/ml, 50 ⁇ l per well was added to a 96-well plate (coning, 9018), incubated at 4°C overnight, washed twice with PBST, and then added with 0.5% skim milk (raw milk). (Worker, A600669-0250) for 2 hours at room temperature. Pour off the blocking solution and wash the plate twice with PBST. Add 50 ⁇ l/well of 100 nM control antibody as a starting concentration and 10 times gradient dilution of the control antibody. After incubation at room temperature for 1 hour, wash the plate with PBST.
  • the detection results of different proteins and positive antibodies are shown in Table 2 and Figure 1A-1D, indicating that the extracellular peptide produced by Baiying Biotechnology Human ROR1-his and human ROR1-Ig-his can be combined with UC961. Extracellular full-length human ROR1-his, human ROR1-KZD-his and extracellular full-length mouse ROR1-his can bind to I2A27.
  • the negative control antibody hIgG1 is an antibody against chicken egg lysozyme anti-hel-hIgG1 (purchased from Baiying, B117901). The data in the figure is the OD 450nm value.
  • the endogenous cells MDA-MB-231 (from the Cell Bank of the Chinese Academy of Sciences in Shanghai) expressing human ROR1 protein were expanded and cultured in T-75 cell culture flasks to the logarithmic growth phase. The culture supernatant was discarded by centrifugation, and the cell pellet was washed with PBS. Wash 2 times, use primary antibody: 100nM UC961-hIgG1 and I2A27-hIgG1, secondary antibody: Alexa 647 AffiniPure Goat Anti-Human IgG (H+L) (purchased from Jackson Immuno, Cat. No.: 109-605-088), detected and analyzed by FACS (FACS Canto TM , purchased from BD Company). The results are shown in Table 3 and Figure 2. Endogenous cell MDA-MB-231 has binding activity to both UC961-hIgG1 and I2A27-hIgG1, indicating that MDA-MB-231 expresses human ROR1 protein.
  • the nucleotide sequence encoding the human ROR1 amino acid sequence was cloned into the PcDNA5/FRT vector (Ubao Biotechnology, VT8066). After transfection of the Flp-inCHO cell line, the nucleotide sequence was incubated with 600 ⁇ g/ml hygromycete. Selectively cultivate for 2 weeks in F12K Medium (Gibco, Cat. No. 21127030) medium containing 10% (w/w) fetal bovine serum (ExCell Bio, Cat. No. FND500) (ThermoFisher, Cat. No.
  • mice 10687010 and use mouse anti-human ROR1 Antibody (UC961, self-produced) was used as the primary antibody, goat anti-mouse IgG (H+L) antibody (Jackson, Cat. No.: 115605006) was used as the secondary antibody, and detection was carried out in the flow cytometer FACS CantoII (purchased from BD Biosciences). Cells with high expression levels and single peak shapes were amplified, and the amplified cells were retested by flow cytometry analysis. The results are shown in Table 4 and Figure 3A. Select cells with better growth, higher fluorescence intensity, and uniformity. The positive cell groups with better sex were further expanded and cultured and frozen in liquid nitrogen.
  • the nucleotide sequence encoding the mouse ROR1 amino acid sequence was cloned into the PcDNA5/FRT vector (Ubao Biotech, VT8066). After transfection of the Flp-in CHO cell line, F12K Medium (Gibco , Catalog No. 21127030) culture medium for 2 weeks, using mouse anti-mouse ROR1 antibody (I2A-27, self-produced) as the primary antibody, goat anti-mouse IgG (H+L) antibody (Jackson, Catalog No.: 115605006) as the primary antibody The secondary antibody is detected in the flow cytometer FACS CantoII (purchased from BD Biosciences).
  • FACS detects the binding activity of control antibodies to cells expressing human ROR1 and mouse ROR1.
  • the IgG subtype control is human IgG1.
  • UC961-hIgG1 and I2A27-hIgG1 have good binding activity to MDA-MB-231 tumor cells expressing human ROR1 protein.
  • the binding abilities of the two positive antibodies are basically the same; IgG
  • the isotype control is mouse IgG2a.
  • UC961-mIgG2a has good binding activity to Flp-inCHO-hROR1 recombinant cells expressing human ROR1 protein, but weak binding activity to Flp-inCHO-mROR1 recombinant cells expressing mouse ROR1 protein.
  • I2A27-mIgG2a has good binding activity to recombinant cells expressing Flp-inCHO-hROR1 expressing human ROR1 protein and Flp-inCHO-mROR1 expressing mouse ROR1 protein.
  • Anti-human ROR1 monoclonal antibodies were generated by immunizing mice.
  • Five 6- to 8-week-old female SJL mice purchased from Shanghai Slack Experimental Animal Co., Ltd.) were used in the experiment. They were numbered: 2428, 2429, 2430, 2431, and 2432. The mice were raised under SPF conditions. After purchase, the mice were kept in a laboratory environment for 1 week, with a 12/12 hour light/dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. Mice that have adapted to the environment are immunized according to the following protocol. During the initial immunization, human ROR1 protein fused to human Fc (hROR1ECD-hFc, ACRO, Cat.
  • RO1-H5250 RO1-H5250
  • adjuvant Titer max purchased from sigma, Cat. T2684
  • oligonucleotide CPG ODN 1826, Synthesized from Shanghai Sangon Biotechnology
  • human ROR1 was mixed with the adjuvant Imject Alum (purchased from Thermo Fisher Scientific, Cat. 77161) and CPG and injected into the abdominal cavity, and each mouse was injected with 50 ⁇ g of antigen; No.
  • hROR1ECD-hFc Imject Alum and CPG, mix well and then inject into the back and foot pads.
  • booster immunization human ROR1, titer max and CPG are mixed and emulsified and then injected into the back. Subsequent booster immunizations were carried out alternately as the first and second booster immunizations, with a total of 5 immunizations. Each mouse was injected with 25 ⁇ g of antigen each time, with an interval of 7 days between each immunization. Carry out blood collection from mice on the 5th day after the second and fourth boosting immunization respectively, separate the serum, and use the enzyme-linked immunosorbent (ELISA) method (refer to Example 1.2) to measure the titer of specific antibodies in the serum. .
  • ELISA enzyme-linked immunosorbent
  • hROR1 ECD-hFc immunogen (ACRO, Cat. RO1-H5250) was injected into the abdominal cavity, sole and back of each selected mouse. The mice were sacrificed 3 days later, and spleen cells and lymphocytes were collected. After centrifugation at 1500 rpm/min, the supernatant was discarded, and ACK lysis solution (purchased from Gibco, Cat. A1049201) was added to the cells to lyse the red blood cells doped in the cells to obtain a cell suspension. The cells were washed three times with DMEM basic medium (purchased from Gibco, Cat. No.
  • mice myeloma cells SP2/0 purchased from ATCC, Cat. CRL-1581
  • the fused cells were diluted to contain 20% (w/w) fetal calf serum (purchased from ExCell Bio, Cat. FND500), 1xHAT (purchased from sigma, Cat. H0262-10VL), bovine insulin (purchased from Yeason, Cat.
  • qualified positive clones were selected and subcloned using semi-solid medium (purchased from stemcell, Cat.03810). After 7 days, the grown clones were picked one by one into a 96-well culture plate and cultured in a medium containing 10% ( w/w) fetal calf serum, 1xHT (purchased from sigma, Cat.H0137-10VL) for expanded culture in DMEM medium, and ELISA was used for preliminary screening one day later to select monoclonal amplifications with binding activity to human ROR1 protein. Continue culturing in a 24-well plate.
  • the culture supernatant was further tested to evaluate its binding activity to human ROR1 overexpressing cells and endogenous cells MDA-MB-231.
  • the optimal clones were selected and placed in a The optimal clone is expanded and cultured in DMEM culture medium with 10% (w/w) FBS under the conditions of 37°C and 5% (v/v) CO2 , and then frozen in liquid nitrogen to obtain the hybridoma cells of the present invention. , and can be used for subsequent antibody production and purification.
  • mROR1-1 see Table 10 for the amino acid sequence of the heavy and light chain variable regions, and the CDRs sequences of the three division methods are shown in Table 11).
  • IMGT http://imgt.cines.fr
  • human antibody heavy and light chain variable region germline gene database we selected heavy chain and light chain variable region germline genes with high homology to mouse antibodies.
  • the CDRs of the mouse antibody are transplanted into the corresponding human template respectively to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • humanized monoclonal antibodies are obtained by performing reverse mutations on embedded residues, residues that directly interact with the CDR region, and residues in the framework region that have an important impact on the conformation of VL and VH. .
  • the CDR amino acid residues of the antibody are determined and annotated by the kabat numbering system.
  • the humanized light chain templates of the positive clone mROR1-1 are IGKV4-1*01, IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV4-38-2*01 and IGHJ6*01.
  • the positive clone mROR1-1 will be The CDRs of the heavy and light chains of cloned mROR1-1 (divided according to Kabat) were transplanted into its human template respectively, to obtain the corresponding humanized version of hROR1-1.
  • the key amino acids in the FR region sequence of the humanized antibody of hROR1-1 are back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity (the antibody has sites prone to chemical modification, we point mutations at the site to eliminate the risk of modification).
  • the specific reverse mutation design is shown in Table 12.
  • Graft means implanting the mouse antibody CDR into the human germline template FR region sequence; L47M means mutating L at position 47 of Graft to M, and so on.
  • the numbering of the reverse mutation amino acids is the natural sequence numbering.
  • variable region after backmutation of the hROR1-1 humanized antibody is as follows:
  • the hROR1-1.VL1 amino acid sequence is shown in SEQ ID NO: 13:
  • the hROR1-1.VL2 amino acid sequence is shown in SEQ ID NO: 14:
  • the hROR1-1.VL3 amino acid sequence is shown in SEQ ID NO: 15:
  • the hROR1-1.VH1 amino acid sequence is shown in SEQ ID NO: 16:
  • the hROR1-1.VH2 amino acid sequence is shown in SEQ ID NO: 17:
  • the hROR1-1.VH3 amino acid sequence is shown in SEQ ID NO: 18:
  • the hROR1-1.VH4 amino acid sequence is shown in SEQ ID NO: 19:
  • the hROR1-1.VH5 amino acid sequence is shown in SEQ ID NO: 20:
  • amino acid sequence of humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 21:
  • amino acid sequence of humanized light chain template IGKV3-11*01 is shown in SEQ ID NO: 22:
  • amino acid sequence of humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 23:
  • amino acid sequence of humanized heavy chain template IGHV4-38-2*01 is shown in SEQ ID NO: 24:
  • amino acid sequence of humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 25:
  • the present invention selects different light chain and heavy chain sequences from the reverse mutation design of the humanized antibody light chain and heavy chain variable regions of hROR1-1 for cross-combination, and finally obtains a variety of humanized hROR1-1 Antibodies, the corresponding amino acid sequences of the heavy and light chain variable regions of each antibody are detailed in Table 13 below:
  • Enzyme-linked immunosorbent assay is used to detect the binding of humanized antibodies to human ROR1 protein.
  • Example 1.2 In order to detect the binding activity of the ROR1 humanized antibody to the human ROR1 full-length protein and different epitope proteins, the same detection method as the enzyme-linked immunosorbent assay (ELISA) in Example 1.2 was used to detect the binding of the humanized antibody to the human ROR1 protein.
  • the test results are shown in Tables 14 to 18 and Figures 6A to 6E, indicating that the purified and produced humanized antibodies bind to human ROR1-his, human ROR1-KZD-his and mouse ROR1-his to varying degrees at the ELISA level. , does not bind to human ROR1-Ig-his and Flp-in CHO empty cells at the ELISA level.
  • the data in the table are OD 450nm values.
  • Anti-test antibodies were captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer were HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C and the sample block was set to 16°C, both preconditioned with running buffer.
  • the protein A chip is first used to capture the antibody to be tested, then a single concentration of ROR1 antigen protein is injected, the binding and dissociation process of the antibody and antigen protein is recorded, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration.
  • Binding was measured by injecting different concentrations of human ROR1-his in solution for 240 seconds with a flow rate of 30 ⁇ L/min, starting from 200 nM (see detailed results for actual concentrations tested), diluted 1:1, for a total of 5 concentrations.
  • the dissociation phase is monitored for up to 600 s and triggered by switching from sample solution to running buffer.
  • 10mM glycine solution pH 1.5
  • Wash at a flow rate of 30 ⁇ L/min for 30 seconds to regenerate the surface.
  • the Langmuir 1:1 model was used.

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Abstract

一种特异性结合ROR1(酪氨酸激酶样孤儿受体1)的抗体及其应用，具体公开了结合ROR1的鼠源和人源化抗体及其制备方法和应用，并且与ROR1蛋白具有较好的亲和力，因此能够运用于治疗肿瘤等药物的制备中。

Description

抗ROR1的抗体及其用途
本申请要求于2022年4月2日提交中国专利局、申请号为202210343431.2、发明名称为“抗ROR1的抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本发明涉及生物工程、生物医药领域，主要涉及一种抗人ROR1的抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于治疗疾病的用途，例如治疗肿瘤的用途。
背景技术
ROR1是一种跨膜受体酪氨酸激酶蛋白，是受体酪氨酸激酶(RTKs)家族成员之一。ROR1编码基因共2814bp，位于染色体1p31.3。ROR1膜蛋白由937个氨基酸组成，分子量约105kDa。ROR1结构在生物种间高度保守，如人与鼠的ROR1氨基酸序列同源性可达97％。人源ROR1分子由三部分组成，包括胞外区、跨膜区和胞内区，胞外区包括一个免疫球蛋白样结构域(Ig)、两个富含半胱氨酸卷曲的结构域(FZD)和一个近膜Kringle结构域(KZD)，胞内区含有一个酪氨酸激酶结构域(TKD)、两个丝/苏氨酸富集结构域(S/TRD)和一个脯氨酸富集结构域(PRD)。
ROR1在人正常组织中低表达或不表达，但在多种恶性肿瘤或组织中高度表达，如慢性淋巴细胞白血病(CLL)、乳腺癌、卵巢癌、黑色素瘤、肺腺癌等，这种在肿瘤高表达、在健康细胞低表达的特点使ROR1的临床价值大幅提升。大量研究表明，ROR1在促进肿瘤的生长和转移、诱导肿瘤细胞耐药和抑制细胞凋亡等方面发挥着重要作用。而关于ROR1的作用机制，主流的说法是ROR1可介导非经典的Wnt信号通路，尤其是Wnt5a，参与NF-κB亚基p65的磷酸化，激活肿瘤细胞中NF-κB通路，促进细胞迁移和侵袭。因此，ROR1作为一种具有高度识别性的肿瘤特异性标志物，已成为肿瘤治疗中一个极具吸引力的靶点。
发明内容
发明人发现ROR1具有在多种恶性肿瘤或组织中高度表达、而在健康细胞中低表达的特点，提示ROR1可以作为一种具有高度识别性的肿瘤特异性标志物，能够运用于治疗肿瘤等药物的制备中。
在第一个方面，本发明公开提供了一种特异性结合ROR1的抗体或抗原结合片段，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
(1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：

和，
(2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、IMGT或Chothia的通行分析方法编码；优选地，所述替换为保守氨基酸的替换。
在一个具体的实施方案中，所述的抗体或抗原结合片段包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、或VH3+VL3，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合；优选地，所述替换为保守氨基酸的替换。
在另一个具体的实施方案中，本发明的抗体或抗原结合片段的重链可变区和轻链可变区的框架区来源于人种系重链模板和人种系轻链模板，其中：
(1)框架区序列来源于人种系重链IGHV4-38-2*01和IGHJ6*01的组合序列；其包含SEQ ID NO：24所示IGHV4-38-2*01的FR1、FR2、FR3区和SEQ ID NO：25所示IGHJ6*01的FR4区；
(2)框架区序列来源于人种系轻链IGKV4-1*01和IGKJ4*01的组合序列；其包含SEQ ID NO：21所示IGKV4-1*01的FR1、FR2、FR3区和SEQ ID NO：23所示IGKJ4*01的FR4区；
(3)框架区序列来源于人种系轻链IGKV3-11*01和IGKJ4*01的组合序列；其包含SEQ ID NO：22所示IGKV3-11*01的FR1、FR2、FR3区和SEQ ID NO：23所示IGKJ4*01的FR4区。
在另一个具体实施方案中，本发明的抗体或抗原结合片段，根据Kabat编号系统编号，还包括选自下组的一种或多种突变，其中：
(1)重链可变区的框架区包括：Q1D、Q1E、V2L、S30T、P41F、K44N、G45K、V72R或S80F；优选包括Q1E、S30T和V72R；或优选包括Q1E、S30T、V72R和S80F；或优选包括Q1E、S30T、P41F和V72R；或优选包括Q1D、V2L、S30T和V72R；或优选包括Q1E、S30T、K44N、G45K和V72R；
(2)轻链可变区的框架区包括：A47P、V62I或G72R；优选包括G72R；或优选包括A47P和G72R；或优选包括V62I和G72R。
在一个具体的实施方案中，本发明的抗体或抗原结合片段包含：
(1)重链可变区具有SEQ ID NO:9、16、17、18、19或20所示序列；
(2)轻链可变区具有SEQ ID NO:10、13、14或15所示序列；
(3)与上述(1)～(2)中任一所述序列相比具有至少90％同一性的氨基酸序列，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
在另一个具体的实施方案中，本发明的抗体或抗原结合片段的重链可变区和轻链可变区选自如下组：
(1)具有SEQ ID NO.9所示的VH和SEQ ID NO.10所示的VL；
(2)具有SEQ ID NO.16所示的VH和SEQ ID NO.13所示的VL；
(3)具有SEQ ID NO.16所示的VH和SEQ ID NO.14所示的VL；
(4)具有SEQ ID NO.16所示的VH和SEQ ID NO.15所示的VL；
(5)具有SEQ ID NO.17所示的VH和SEQ ID NO.13所示的VL；
(6)具有SEQ ID NO.17所示的VH和SEQ ID NO.14所示的VL；
(7)具有SEQ ID NO.17所示的VH和SEQ ID NO.15所示的VL；
(8)具有SEQ ID NO.18所示的VH和SEQ ID NO.13所示的VL；
(9)具有SEQ ID NO.18所示的VH和SEQ ID NO.14所示的VL；
(10)具有SEQ ID NO.18所示的VH和SEQ ID NO.15所示的VL；
(11)具有SEQ ID NO.19所示的VH和SEQ ID NO.13所示的VL；
(12)具有SEQ ID NO.19所示的VH和SEQ ID NO.14所示的VL；
(13)具有SEQ ID NO.19所示的VH和SEQ ID NO.15所示的VL；
(14)具有SEQ ID NO.20所示的VH和SEQ ID NO.13所示的VL；
(15)具有SEQ ID NO.20所示的VH和SEQ ID NO.14所示的VL；
(16)具有SEQ ID NO.20所示的VH和SEQ ID NO.15所示的VL；
(17)具有与上述(1)～(16)任一序列组合相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合。
在一个优选实施方案中，本发明的抗体或抗原结合片段与人ROR1结合的解离常数(KD) 不大于4×10-7M。
在一个优选实施方案中，本发明的抗体或抗原结合片段为(1)嵌合抗体或其片段；(2)人源化抗体或其片段；(3)全人源抗体或其片段；优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在另一个优选实施方案中，本发明的抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在另一个优选实施方案中，本发明的抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
在第二个方面，本发明的抗体或抗原结合片段进一步还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
在第三个方面，本发明还提供一种多特异性抗原结合分子，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含上述第一方面所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合ROR1以外的其他抗原或结合与第一抗原结合模块不同的ROR1抗原表位；优选地，所述其他抗原选自CD3、CD7、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、BCMA、HLA-DR、HER2、VEGF、P1GF、HER3/ERBB3,HER4/ERBB4、IL-2、IL-6、PD-1、PD-L1、TRAIL-R1或TRAIL-R2；优选地，所述多特异性抗原结合分子为双特异性抗体、三特异性抗体或四特异性抗体。
在第四个方面，本发明还提供一种嵌合抗原受体(CAR)，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述第一个方面所述ROR1抗体或抗原结合片段。
在第五个方面，本发明还提供一种免疫效应细胞，所述免疫效应细胞包含上述第四个方面所述嵌合抗原受体或包含上述第四个方面所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自，炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；优选地，所述免疫效应细胞为同种异体免疫 效应细胞或自体免疫细胞。
在第六个方面，本发明还提供一种分离的核酸分子，所述核酸分子编码上述第一个方面所述的抗体、抗原结合片段、或任意组合，第三个方面的多特异性抗原结合分子或第四个方面的嵌合抗原受体。
在第七个方面，本发明还提供了一种包含第六个方面所述的核酸分子的表达载体。
在第八个方面，本发明提供了上述第六个方面所述的核酸分子，或第七个方面所述的表达载体的分离的宿主细胞；优选地，所述宿主细胞是真核细胞或原核细胞；更优选地，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选地，所述宿主细胞选自Expi293或CHO细胞。
在第九个方面，本发明提供了一种制备上述第一个方面所述的抗体或抗原结合片段、或第三个方面所述的多特异性抗原结合分子的方法，在适当的条件下上述第八个方面所述的宿主细胞，并分离抗体或抗原结合片段或多特异性抗原结合分子。
在第十个方面，本发明提供了一种制备上述第五个方面所述的免疫效应细胞的方法，所述方法包括将编码第四个方面所述的CAR导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达第四个方面所述的CAR。
在第十一个方面，本发明提供了一种药物组合物，所述组合物包含上述第一个方面所述的抗体或抗原结合片段、第五个方面所述的免疫效应细胞、第六个方面所述的分离的核酸分子、第七个方面所述的表达载体、第八个方面所述的细胞、或第九个或第十个方面所述方法制备的产品；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；优选地，所述药物组合物还包含额外的抗肿瘤剂。
在第十二个方面，本发明提供了上述第一个方面所述的抗体或抗原结合片段、第三个方面所述的多特异性抗原结合分子、第四个方面所述的嵌合抗原受体、第五个方面所述的免疫效应细胞、第六个方面所述的分离的核酸分子、第七个方面所述的表达载体、第八个方面所述的细胞、第九或第十个方面所述方法制备的产品、或第十一个方面所述的药物组合物在制备预防和/或治疗肿瘤疾病的药物中的用途；所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
在第十三个方面，本发明提供了一种预防和/或治疗肿瘤疾病的方法，包含向有此需要的患者施用有效量的上述第一个方面所述的抗体或抗原结合片段、第三个方面所述的多特异性抗原结合分子、第四个方面所述的嵌合抗原受体、第五个方面所述的免疫效应细胞、第六个方面所述的分离的核酸分子、第七个方面所述的表达载体、第八个方面所述的细胞、第九或 第十个方面所述方法制备的产品、或第十一个方面所述的药物组合物；所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
在第十四个方面，本发明提供了上述第一个方面所述的抗体或抗原结合片段、第三个方面所述的多特异性抗原结合分子、第四个方面所述的嵌合抗原受体、第五个方面所述的免疫效应细胞、第六个方面所述的分离的核酸分子、第七个方面所述的表达载体、第八个方面所述的细胞、第九或第十个方面所述方法制备的产品、或第十一个方面所述的药物组合物用于和/或治疗肿瘤疾病；所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
在第十五个方面，本发明提供了一种试剂盒，包含上述第一个方面所述的抗体或抗原结合片段、第三个方面所述的多特异性抗原结合分子、第四个方面所述的嵌合抗原受体、第五个方面所述的免疫效应细胞、第六个方面所述的分离的核酸分子、第七个方面所述的表达载体、第八个方面所述的细胞、第九或第十个方面所述方法制备的产品、或第十一个方面所述的药物组合物，以及使用说明。
有益效果：发明人基于杂交瘤技术，开发并制备了上述针对ROR1的新型鼠源及人源化抗体，其可以：(1)特异性的结合人ROR1；(2)由于ROR1在人的肿瘤细胞系上高表达，此抗体也可介导对ROR1高表达肿瘤细胞的杀伤作用。
术语定义和说明
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“单特异性”是指具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的 每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文“全长抗体”、“完好抗体”和“完整抗体”可互换使用，是指其具有基本上与天然抗体结构相似的结构。
如本文所用，术语“抗原结合片段”是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“ROR1”是受体酪氨酸激酶(RTK)家族中的跨膜蛋白。ROR1基因编码两个明确定义的同种型：短的393个氨基酸(aa)的细胞内蛋白质(同种型2)和长的937aa的I型跨膜蛋白质(同种型I)。长细胞表面同种型在原发性人B慢性淋巴细胞白血病(B-CLL)和套细胞淋巴瘤、B急性淋巴细胞白血病的一部分以及许多肿瘤(包括与转移表型相关的肿瘤)上表达。ROR1主要在胚胎发育过程中表达，但其表达在胎儿发育过程中减弱。然而，ROR1被某些B细胞恶性肿瘤(例如但不限于淋巴瘤、CLL和B-ALL)以及许多实体瘤恶性肿瘤(例如但不限于肾上腺癌、膀胱癌、乳腺癌、结肠癌、肺癌、胰腺癌、前列腺癌、卵巢癌、皮肤癌、睾丸癌、子宫癌和神经母细胞瘤)异常表达。
如本文所用，术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对ROR1的抗原表位而被检测，另一个可以针对ROR1的另一个抗原表位或除ROR1外的任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“嵌合”抗体是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985 J Immunol Methods 125:191-202； 以上通过援引加入并入本文。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含以下之一：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据EU编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不意图包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“裸抗体”是指不与另一种作用剂或分子(例如标记或药物)、肽或多肽连接、融合或缀合的抗体。在具体的实施方案中，由哺乳动物宿主细胞表达的裸抗体可被宿主细胞的糖基化机器(例如糖基化酶)糖基化。在某些实施方案，当通过不具有其自身糖基化机器(例如糖基化酶)的宿主细胞表达时，裸抗体不被糖基化。在某些实施方案中，裸抗体是完整抗体，而在其它实施方案中，裸抗体是完整抗体的抗原结合片段，例如Fab抗体。
本文术语“缀合抗体”是指可与药学上可接受的载体或稀释剂缔合的抗体，其可为单克隆抗体、嵌合抗体、人源化抗体或人抗体。
本文术语“双抗体”是指二价的双特异性抗体，可以与相同或不同抗原上的不同表位结合。
如本文所用，术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸彼此属于保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)酸性氨基酸：Asp(D)和Glu(E)；
(2)碱性氨基酸：Lys(K)、Arg(R)和His(H)；
(3)亲水性不带电荷氨基酸：Ser(S)、Thr(T)、Asn(N)和Gln(Q)；
(4)脂肪族不带电荷氨基酸：Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)；
(5)非极性不带电荷的氨基酸：Cys(C)、Met(M)和Pro(P)；
(6)芳香族氨基酸：Phe(F)、Tyr(Y)和Trp(W)。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
本文术语“嵌合抗原受体(CAR)”是指这样的重组蛋白，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。在某些实施方式中，CAR的细胞外抗原结合结构域包含scFv。scFv可以源自融合抗体的可变重和轻区。替代地或另外，scFv可以衍生自Fab’s(而不是抗体，例如获自Fab文库)。在某些实施方式中，将scFv融合至跨膜结构域，然后融合至细胞内信号传导结构域。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/ 或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823 B1)。
如本文所用，术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、绵羊和马等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂 量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“适当的条件”指适合培养各种宿主细胞的条件，其中宿主细胞包括真核细胞和原核细胞。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。
本文术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“抗肿瘤剂”指抗肿瘤药物，其为治疗肿瘤疾病的一类药物，例如化疗药物、生物制剂等。
附图说明
图1A为ELISA检测人ROR1-his蛋白与阳性抗体的结合反应、图1B为ELISA检测人ROR1-Ig-his蛋白与阳性抗体的结合反应、图1C为ELISA检测人ROR1-KZD-his蛋白与阳性抗体的结合反应、图1D为ELISA检测鼠ROR1-his蛋白与阳性抗体的结合反应。
图2为FACS检测内源细胞MDA-MB-231与阳性抗体的结合反应。
图3A为表达人ROR1蛋白的Flp-in CHO稳转细胞系FACS检测结果、图3B为表达鼠ROR1蛋白的Flp-in CHO稳转细胞系FACS检测结果。
图4A为FACS检测对照抗体与MDA-MB-231肿瘤细胞的结合反应、图4B为FACS检测对照抗体与Flp-in CHO-hROR1重组细胞的结合反应、图4C为FACS检测对照抗体与Flp-in CHO-mROR1重组细胞的结合反应。
图5A为ELISA检测免疫后小鼠血清对人ROR1-his蛋白结合的效价、图5B为ELISA检测免疫后小鼠血清对鼠ROR1-his蛋白结合的效价、图5C为ELISA检测免疫后小鼠血清对人ROR1-Ig-his蛋白结合的效价、图5D为ELISA检测免疫后小鼠血清对人ROR1-KZD-his蛋白结合的效价。
图6A为ELISA检测人源化抗体与hROR1-his蛋白的结合反应、图6B为ELISA检测人源化抗体与hROR1-Ig-his蛋白的结合反应、图6C为ELISA检测人源化抗体与hROR1-KZD-his蛋白的结合反应、图6D为ELISA检测人源化抗体与鼠ROR1-his蛋白的结合反应、图6E为ELISA检测人源化抗体与Flp-in CHO细胞的结合反应。
图7为FACS检测人源化抗体与MDA-MB-231细胞的结合反应。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1对照抗体制备、内源细胞鉴定和过表达细胞株的制备
1.1对照抗体的制备
UC961序列来自专利WO2020198531A2，I2A27序列来自专利WO2018237335A1(见表1)，UC961结合在ROR1胞外Ig domain，I2A27结合在ROR1胞外KZD domain，将上述识别人ROR1表位的克隆的VH和VL序列重组到人IgG1/鼠IgG2a的CH和CL表达载体，然后再通过3个GGGGS连接子连接后重组到人IgG1Fc/鼠IgG2a Fc的表达载体中，分别获得重组质粒UC961-hIgG1、I2A27-hIgG1、UC961-mIgG2a和I2A27-mIgG2a。
将质粒和转染试剂PEI(Polysciences，24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(Thermofisher，A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天，加入OPM-293ProFeed(上海奥浦迈，F081918-001)和6g/L葡萄糖(Sigma，G7528)。转染第六天，收集细胞上清。
用Protein A柱(GE，货号17549802)纯化细胞培养上清液中的蛋白。Protein A柱先用3～5倍柱体积的平衡缓冲液(PBS磷酸缓冲液，pH7.4)(20X PBS缓冲剂500ml，生工，货号B548117-0500)平衡，然后将澄清的培养上清液上样到Protein A柱，控制流速在10mL/分钟。上样完毕后用平衡缓冲液清洗Protein A柱，平衡缓冲液的体积为Protein A柱柱床体积的3～5倍。用洗脱液(0.02M柠檬酸缓冲液，pH3.5)洗脱结合在蛋白A柱上的蛋白。收集洗脱的蛋白，加入Tris缓冲液(国药，30188336)至pH中性。样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE，28990946)进一步纯化，去除聚集体，收集单体的峰，然后用0.22μm的滤器(Millipore，SLGVR13SL)进行无菌过滤，SEC-HPLC纯度检测合格后，使用Nano drop 8000(Thermo，Thermo NanoDrop 8000)进行浓度测定分装备用。
表1.对照抗体序列信息
1.2抗原蛋白的制备
委托通用泰州市百英生物科技有限公司生产胞外全长人ROR1-his蛋白，人ROR1-Ig-his蛋白，人ROR1-KZD-his蛋白和胞外全长鼠ROR1-his蛋白，用于杂交瘤筛选和人源化抗体鉴定。
胞外全长人ROR1-his氨基酸序列(SEQ ID NO：5)：

人ROR1-Ig-his氨基酸序列(SEQ ID NO：6)：
人ROR1-KZD-his氨基酸序列(SEQ ID NO：7)：
胞外全长鼠ROR1-his氨基酸序列(SEQ ID NO：8)：
为了检测蛋白的结合活性，将获得的蛋白稀释到1μg/ml，每孔50μl加入到96孔板(coning，9018)中，4℃过夜孵育，PBST洗涤2次，再加入0.5％脱脂牛奶(生工，A600669-0250)室温封闭2小时，倒掉封闭液，用PBST洗板2次，以50μl/孔加入100nM为起始浓度，10倍梯度稀释的对照抗体，室温孵育1小时后，用PBST洗板3次，加入HRP(辣根过氧化物酶)标记的二抗(购自Jackson，109-035-088)，室温孵育1小时后，用PBST洗板5次。加入TMB底物50μl/孔，室温孵育5-10分钟后，加入终止液(1.0N HCl)50μl/孔。用ELISA读板机(Multimode Plate Reader，EnSight，购自Perkin Elmer)读取OD450nm数值，不同蛋白与阳性抗体的检测结果如表2和图1A-1D所示，说明百英生物生产的胞外全长人ROR1-his，人ROR1-Ig-his可与UC961结合。胞外全长人ROR1-his，人ROR1-KZD-his和胞外全长鼠ROR1-his可与I2A27结合。其中阴性对照抗体hIgG1为针对鸡卵溶菌酶的抗体anti-hel-hIgG1(购自百英，B117901)，图中的数据为OD450nm值。
表2.ELISA检测ROR1蛋白与阳性对照抗体的结合反应

1.3内源性表达人ROR1蛋白的MDA-MB-231细胞株鉴定
将表达人ROR1蛋白的内源性细胞MDA-MB-231(来自上海中科院细胞库)在T-75细胞培养瓶中扩大培养至对数生长期，离心弃去培养基上清，细胞沉淀用PBS洗涤2次，用一抗：100nM UC961-hIgG1和I2A27-hIgG1，二抗：Alexa647 AffiniPure Goat Anti-Human IgG(H+L)(购自Jackson Immuno，货号：109-605-088)，经FACS(FACS CantoTM，购自BD公司)检测和分析。结果如表3和图2所示，内源细胞MDA-MB-231与UC961-hIgG1和I2A27-hIgG1均有结合活性，说明MDA-MB-231表达人ROR1蛋白。
表3.FACS检测内源细胞MDA-MB-231与阳性对照抗体的结合反应
1.4表达人ROR1和鼠ROR1蛋白的Flp-inCHO重组细胞株制备
编码人ROR1氨基酸序列(SEQ ID NO：5)的核苷酸序列被克隆到PcDNA5/FRT载体(优宝生物，VT8066)，对Flp-inCHO细胞系进行转染后，在含600μg/ml潮霉素(ThermoFisher,货号10687010)的含10％(w/w)胎牛血清(ExCell Bio，货号FND500)的F12K Medium(Gibco,货号21127030)培养基中选择性培养2周，用鼠抗人源ROR1抗体(UC961，自产)作为一抗，山羊抗鼠IgG(H+L)抗体(Jackson，货号：115605006)作为二抗，在流式细胞仪FACS CantoII(购自BD Biosciences)中进行检测，对表达量高且峰形单一的细胞进行扩增，对扩增后的细胞经流式细胞分析法进行复测，结果如表4和图3A所示，选择长势较好、荧光强度较高、均一性较好的阳性细胞群继续扩大培养并液氮冻存。
表4.表达人ROR1蛋白的Flp-inCHO稳转细胞系的FACS检测结果
编码鼠ROR1氨基酸序列(SEQ ID NO：8)的核苷酸序列被克隆到PcDNA5/FRT载体(优宝生物，VT8066)。对Flp-in CHO细胞系进行转染后，在含600μg/ml潮霉素(ThermoFisher,货号10687010)的含10％(w/w)胎牛血清(ExCell Bio，货号FND500)的F12K Medium(Gibco,货号21127030)培养基中选择性培养2周，用鼠抗鼠源ROR1抗体(I2A-27，自产)作为一抗，山羊抗鼠IgG(H+L)抗体(Jackson，货号：115605006)作为二抗，在流式细胞仪FACS CantoII(购自BD Biosciences)中进行检测，对表达量高且峰形单一的细胞进行扩增，对扩增后的细胞经流式细胞分析法进行复测，结果如表5和图3B所示，选择长势较好、荧光强度较高、均一性较好的阳性细胞群继续扩大培养并液氮冻存。
表5.表达鼠ROR1蛋白的Flp-inCHO稳转细胞系FACS检测结果
1.5细胞系与对照抗体的结合实验
FACS检测对照抗体与表达人ROR1和鼠ROR1的细胞的结合活性。如表6-表8和图4A- 图4C所示，IgG亚型对照为人IgG1，UC961-hIgG1和I2A27-hIgG1与表达人ROR1蛋白的MDA-MB-231肿瘤细胞有很好的结合活性，两个阳性抗体的结合能力基本一致；IgG亚型对照为鼠IgG2a，UC961-mIgG2a与表达人ROR1蛋白的Flp-inCHO-hROR1重组细胞有很好的结合活性，与表达鼠ROR1蛋白的Flp-inCHO-mROR1重组细胞结合活性弱。I2A27-mIgG2a与表达人ROR1蛋白的Flp-inCHO-hROR1和表达鼠ROR1蛋白的Flp-inCHO-mROR1的重组细胞都有很好的结合活性。
表6.FACS检测对照抗体与MDA-MB-231肿瘤细胞的结合反应
表7.FACS检测对照抗体与Flp-in CHO-hROR1重组细胞的结合反应
表8.FACS检测对照抗体与Flp-in CHO-mROR1重组细胞的结合反应
实施例2抗人ROR1杂交瘤单克隆抗体的制备
2.1动物免疫
抗人ROR1单克隆抗体通过免疫小鼠产生。实验用6～8周龄的雌性SJL小鼠5只(购自上海斯莱克实验动物有限公司)，分别编号为:2428，2429，2430，2431，2432，小鼠在SPF条件下饲养。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。初次免疫时，使用融合了人Fc的人ROR1蛋白(hROR1ECD-hFc,ACRO，Cat.RO1-H5250)、佐剂Titer max(购自sigma,Cat.T2684)和寡核苷酸CPG(ODN 1826,合成自上海生工生物)混合乳化后注射足垫和背部，人ROR1与佐剂Imject Alum(购自Thermo fisher scientific,Cat.77161)和CPG混合后注射腹腔，每只小鼠注射50μg抗原；第一次加强免疫时，使用hROR1ECD-hFc、Imject Alum和CPG混匀后注射背部和足垫。第二次加强免疫时使用人ROR1、titer max和CPG混合乳化后注射背部。后续的加强免疫按照第一次和第二次加强免疫的方式交替进行，共免疫5次，每次每只小鼠注射25μg抗原，每次免疫之间均间隔7天。分别在第二次和第四次加强免疫后的第5天进行小鼠的采血操作，分离血清，使用酶联免疫吸附(ELISA)方法(参考实施列1.2)测定血清中特异性抗体的滴度。
实验结果如表9和图5A-图5D所示，经以上蛋白免疫的小鼠的第四次免疫后的血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中空白对照为1％(w/w)BSA，表中的数据为OD450nm值。
表9.ELISA检测第四次免疫后小鼠血清对人ROR1-his、人ROR1-Ig-his、人ROR1-KZD-his和鼠ROR1-his蛋白结合的效价

2.2脾细胞融合和杂交瘤筛选
向每只选出的小鼠腹腔、足底和背部共注射50μg hROR1 ECD-hFc免疫原(ACRO，Cat.RO1-H5250)，3天后处死小鼠，收集脾细胞和淋巴细胞。1500rpm/min离心后，弃掉上清，在细胞中加入ACK裂解液(购自Gibco,Cat.A1049201)，裂解细胞中掺杂的红细胞，获得细胞悬液。用DMEM基础培养基(购自Gibco，货号10569044)1500rpm/min清洗细胞3次，然后将活细胞与小鼠骨髓瘤细胞SP2/0(购自ATCC，Cat.CRL-1581)按数目2:1混合，采用电融合方法(仪器为BTX 2001+)进行细胞融合。融合后的细胞稀释到含20％(w/w)胎牛血清(购自ExCell Bio，Cat.FND500)、1xHAT(购自sigma，Cat.H0262-10VL)、牛胰岛素(购自Yeason，Cat.40107ES25)、NEAA(Gibco，Cat.11140050)的DMEM培养基中，然后按5x104个/200μL每孔加入96孔细胞培养板中，放入5％(v/v)CO2、37℃培养箱中培养。7天后用ELISA方法筛选融合板上清以确认对人ROR1蛋白的结合活性；对于筛选后获得的阳性克隆上清，FACS确认与内源性细胞MDA-MB-231的结合活性。
根据筛选结果，挑选符合条件的阳性克隆，用半固体培养基(购自stemcell，Cat.03810)进行亚克隆，7天后将长出的克隆逐个挑入96孔培养板中，在含10％(w/w)胎牛血清、1xHT(购自sigma，Cat.H0137-10VL)的DMEM培养基中扩大培养，1天后用ELISA进行初步筛选，挑选出与人ROR1蛋白有结合活性的单克隆扩增到24孔板继续培养。3天后对培养上清进行进一步检测，评估其与人ROR1过表达细胞和内源性细胞MDA-MB-231的结合活性，根据24孔板样品检测结果，挑选出最优的克隆，置于含10％(w/w)FBS的DMEM培养基中，在37℃、5％(v/v)CO2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体生产和纯化。
实施例3杂交瘤阳性克隆轻重链可变区氨基酸序列测定
收集处于对数生长期的杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)充分裂解细胞后于-80℃保存待测。委托苏州金唯智生物科技有限公司完成杂交瘤阳性克隆轻重链可变区氨基酸序列的测定，对测序结果使用MOE软件进行分析，根据可变区编码蛋白氨基酸序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列后，从中筛选获得1个克隆：mROR1-1(重轻链可变区氨基酸序列详见表10，三种划分方式的CDRs序列见表11)。
表10.杂交瘤阳性克隆mROR1-1轻重链可变区氨基酸序列信息
注：人IgG1Fc序列中加粗为hinge区序列，其包含C220S突变，用下划线表示。
表11.mROR1-1抗体根据三种方式划分的重轻链CDR序列
委托通用生物系统(安徽)有限公司合成编码上述克隆的重链及轻链可变区的核苷酸序列，并通过序列为GGGGSGGGGSGGGGS(SEQ ID NO：12)的linker连接，然后克隆到包含信号肽MGWSWILLFLLSVTAGVHS(SEQ ID NO:32)和人源抗体Fc(SEQ ID NO：11)的表达载体pTT5-huFc(C220S)上，最终获得人鼠嵌合抗体cROR1-1，表达纯化步骤参照实施例1.1。
实施例4 ROR1鼠源抗体的人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。基于抗体的三维结构，对包埋残基、与CDR区有直接相互作用的残基以及对VL和VH的构象有重要影响的构架区的残基进行回复突变，即得到人源化单克隆抗体。其中抗体的CDR氨基酸残基由kabat编号系统确定并注释。
阳性克隆mROR1-1的人源化轻链模板为IGKV4-1*01、IGKV3-11*01和IGKJ4*01，人源化重链模板为IGHV4-38-2*01和IGHJ6*01，将阳性克隆mROR1-1重轻链的CDR(根据Kabat划分)分别移植到其人源模板中，即获得对应的人源化版本hROR1-1。根据需要，将hROR1-1的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力(抗体存在易发生化学修饰的位点，我们对这些位点进行点突变以消除修饰风险)。具体回复突变设计见表12。
表12：hROR1-1的人源化抗体回复突变设计
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；L47M表示将Graft第47位L突变成M，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
hROR1-1人源化抗体回复突变后的可变区具体序列如下：
hROR1-1.VL1氨基酸序列如SEQ ID NO：13所示：
hROR1-1.VL2氨基酸序列如SEQ ID NO：14所示：
hROR1-1.VL3氨基酸序列如SEQ ID NO：15所示：
hROR1-1.VH1氨基酸序列如SEQ ID NO：16所示：
hROR1-1.VH2氨基酸序列如SEQ ID NO：17所示：
hROR1-1.VH3氨基酸序列如SEQ ID NO：18所示：
hROR1-1.VH4氨基酸序列如SEQ ID NO：19所示：
hROR1-1.VH5氨基酸序列如SEQ ID NO：20所示：
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：21所示：
人源化轻链模板IGKV3-11*01氨基酸序列如SEQ ID NO：22所示：
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：23所示：
人源化重链模板IGHV4-38-2*01氨基酸序列如SEQ ID NO：24所示：
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：25所示：
本发明分别从上述hROR1-1的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种hROR1-1人源化抗体，各抗体的重轻链可变区氨基酸序列对应情况详见下表13：
表13.hROR1-1抗体重轻链可变区氨基酸序列对应情况
委托通用生物系统(安徽)有限公司合成编码(表13)的重链及轻链可变区的核苷酸序列，并通过序列为GGGGSGGGGSGGGGS(SEQ ID NO：12)的linker连接，然后克隆到包含信号肽MGWSWILLFLLSVTAGVHS(SEQ ID NO:32)和人源抗体Fc(SEQ ID NO:11)的表达载体pTT5-huFc(C220S)上，最终获得不同的人源化抗体，表达纯化步骤参照实施例1.1。
实施例5 ROR1人源化抗体的鉴定
5.1酶联免疫吸附实验(ELISA)检测人源化抗体与人ROR1蛋白的结合。
为了检测ROR1人源化抗体与人ROR1全长蛋白和不同表位蛋白的结合活性，采用实施例1.2酶联免疫吸附实验(ELISA)同样的检测方法检测人源化抗体与人ROR1蛋白的结合，检测结果如表14-表18和图6A-图6E，说明纯化生产后的人源化抗体与人ROR1-his，人ROR1-KZD-his和鼠ROR1-his在ELISA水平上有不同程度的结合，与人ROR1-Ig-his和Flp-in CHO空细胞在ELISA水平上均不结合，表中的数据为OD450nm值。
表14.ELISA检测hROR1-1人源化抗体与hROR1-his的结合反应
表15.ELISA检测hROR1-1人源化抗体与hROR1-Ig-his的结合反应
表16.ELISA检测hROR1-1人源化抗体与hROR1-KZD-his的结合反应

表17.ELISA检测hROR1-1人源化抗体与mROR1-his的结合反应
表18.Cell-based ELISA检测hROR1-1人源化抗体与Flp-in CHO的结合反应

5.2流式细胞实验(FACS)检测人源化抗体与内源细胞MDA-MB-231结合活性
为了检测ROR1人源化抗体与人ROR1全长蛋白的结合活性，采用实施例1.5中FACS同样的检测方法检测人源化抗体与人ROR1全长蛋白的结合，检测结果如表19和图7，说明人源化抗体与MDA-MB-231细胞均有不同程度的特异性结合，表中的数据为MFI值。
表19.FACS检测hROR1-1人源化抗体与MDA-MB-231细胞的结合反应
5.3 ROR1人源化抗体的亲和力检测
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗待测抗体。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃，样品块设置为16℃，两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的ROR1抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的人ROR1-his持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1:1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5) 以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异，也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1:1模型。选择结合能力好的VL1组合检测与人ROR1蛋白的结合速率(Ka)、解离速率(Kd)及结合亲和力(KD)。如表20所示，其中UC961-hIgG1和I2A27-hIgG1抗体作为对照，人源化抗体与人ROR1蛋白的KD值与阳性对照抗体和嵌合抗体相当，具有较好的结合活性。
表20人源化抗体与人ROR1蛋白的结合亲和力

Claims (24)

  1. 一种特异性结合ROR1的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
    (1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    和，
    (2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、IMGT或Chothia的通行分析方法编码；
    优选地，所述替换为保守氨基酸的替换。
  2. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、或VH3+VL3，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合；
    优选地，所述替换为保守氨基酸的替换。
  3. 根据权利要求2所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段的重链可变区和轻链可变区的框架区来源于人种系重链模板和人种系轻链模板，其中：
    (1)框架区序列来源于人种系重链IGHV4-38-2*01和IGHJ6*01的组合序列；其包含SEQ ID NO：24所示IGHV4-38-2*01的FR1、FR2、FR3区和SEQ ID NO：25所示IGHJ6*01的 FR4区；
    (2)框架区序列来源于人种系轻链IGKV4-1*01和IGKJ4*01的组合序列；其包含SEQ ID NO：21所示IGKV4-1*01的FR1、FR2、FR3区和SEQ ID NO：23所示IGKJ4*01的FR4区；
    (3)框架区序列来源于人种系轻链IGKV3-11*01和IGKJ4*01的组合序列；其包含SEQ ID NO：22所示IGKV3-11*01的FR1、FR2、FR3区和SEQ ID NO：23所示IGKJ4*01的FR4区。
  4. 根据权利要求3所述的抗体或抗原结合片段，其特征在于，根据Kabat编号系统编号，所述抗体或抗原结合片段的重链可变区和轻链可变区的框架区还包括选自下组的一种或多种突变，其中：
    (1)重链可变区的框架区包括：Q1D、Q1E、V2L、S30T、P41F、K44N、G45K、V72R或S80F；优选包括Q1E、S30T和V72R；或优选包括Q1E、S30T、V72R和S80F；或优选包括Q1E、S30T、P41F和V72R；或优选包括Q1D、V2L、S30T和V72R；或优选包括Q1E、S30T、K44N、G45K和V72R；
    (2)轻链可变区的框架区包括：A47P、V62I或G72R；优选包括G72R；或优选包括A47P和G72R；或优选包括V62I和G72R。
  5. 根据权利要求1-4任一项所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链可变区具有SEQ ID NO:9、16、17、18、19或20所示序列；
    (2)轻链可变区具有SEQ ID NO:10、13、14或15所示序列；
    (3)与上述(1)～(2)中任一所述序列相比具有至少90％同一性的氨基酸序列，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
  6. 根据权利要求5所述的抗体或抗原结合片段，其特征在于，所述的重链可变区和轻链可变区选自如下组：
    (1)具有SEQ ID NO.9所示的VH和SEQ ID NO.10所示的VL；
    (2)具有SEQ ID NO.16所示的VH和SEQ ID NO.13所示的VL；
    (3)具有SEQ ID NO.16所示的VH和SEQ ID NO.14所示的VL；
    (4)具有SEQ ID NO.16所示的VH和SEQ ID NO.15所示的VL；
    (5)具有SEQ ID NO.17所示的VH和SEQ ID NO.13所示的VL；
    (6)具有SEQ ID NO.17所示的VH和SEQ ID NO.14所示的VL；
    (7)具有SEQ ID NO.17所示的VH和SEQ ID NO.15所示的VL；
    (8)具有SEQ ID NO.18所示的VH和SEQ ID NO.13所示的VL；
    (9)具有SEQ ID NO.18所示的VH和SEQ ID NO.14所示的VL；
    (10)具有SEQ ID NO.18所示的VH和SEQ ID NO.15所示的VL；
    (11)具有SEQ ID NO.19所示的VH和SEQ ID NO.13所示的VL；
    (12)具有SEQ ID NO.19所示的VH和SEQ ID NO.14所示的VL；
    (13)具有SEQ ID NO.19所示的VH和SEQ ID NO.15所示的VL；
    (14)具有SEQ ID NO.20所示的VH和SEQ ID NO.13所示的VL；
    (15)具有SEQ ID NO.20所示的VH和SEQ ID NO.14所示的VL；
    (16)具有SEQ ID NO.20所示的VH和SEQ ID NO.15所示的VL；
    (17)具有与上述(1)～(16)任一序列组合相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合。
  7. 根据权利要求1-6任一项所述的抗体或抗原结合片段，其特征在于，其与人ROR1结合的解离常数(KD)不大于4×10-7M。
  8. 根据权利要求1-7任一项所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段为：
    (1)嵌合抗体或其片段；
    (2)人源化抗体或其片段；
    (3)全人源抗体或其片段；
    优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  9. 根据权利要求1-8任一项的抗体或抗原结合片段，其特征在于，所述抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  10. 根据权利要求1-9任一项的抗体或抗原结合片段，其特征在于，所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
  11. 根据权利要求1-10任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段进一步还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
  12. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含第一抗 原结合模块和第二抗原结合模块，所述第一抗原结合模块包含权利要求1-11任一项所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合ROR1以外的其他抗原或结合与第一抗原结合模块不同的ROR1抗原表位；
    优选地，所述其他抗原选自CD3、CD7、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、BCMA、HLA-DR、HER2、VEGF、P1GF、HER3/ERBB3,HER4/ERBB4、IL-2、IL-6、PD-1、PD-L1、TRAIL-R1或TRAIL-R2；
    优选地，所述多特异性抗原结合分子为双特异性抗体、三特异性抗体或四特异性抗体。
  13. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1-11任一项所述ROR1抗体或抗原结合片段。
  14. 一种免疫效应细胞，其特征在于，所述免疫效应细胞包含权利要求13所述嵌合抗原受体或包含编码权利要求13所述嵌合抗原受体的核酸片段；
    优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自，炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
    优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
  15. 一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1-11任一项所述的抗体、抗原结合片段、或其任意组合，权利要求12所述的多特异性抗原结合分子或权利要求13所述的嵌合抗原受体。
  16. 包含权利要求15所述分离的核酸分子的表达载体。
  17. 包含权利要求15所述的分离的核酸分子、或权利要求16所述的表达载体的分离的宿主细胞；优选地，所述宿主细胞是真核细胞或原核细胞；更优选地，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选地，所述宿主细胞选自Expi293或CHO细胞。
  18. 一种制备权利要求1-11任一项所述抗体或抗原结合片段或权利要求12所述多特异性抗原结合分子的方法，其特征在于，在适当的条件下培养权利要求17所述的宿主细胞，并分离抗体或抗原结合片段或多特异性抗原结合分子。
  19. 一种制备权利要求14所述免疫效应细胞的方法，其特征在于，所述方法包括将编码权利要求13所述CAR的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免 疫效应细胞表达权利要求13所述CAR。
  20. 一种药物组合物，其特征在于，所述组合物包含权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12所述的多特异性抗原结合分子、权利要求13所述的嵌合抗原受体、权利要求14所述的免疫效应细胞、权利要求15的分离的核酸分子、权利要求16的表达载体、权利要求17的细胞，或权利要求18或19所述方法制备的产品；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；优选地，所述药物组合物还包含额外的抗肿瘤剂。
  21. 权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12所述的多特异性抗原结合分子、权利要求13所述嵌合抗原受体、权利要求14所述的免疫效应细胞、权利要求15的分离的核酸分子、权利要求16的表达载体、权利要求17的细胞，权利要求18或19所述方法制备的产品、或权利要求20所述的药物组合物在制备预防和/或治疗肿瘤疾病的药物中的用途；
    优选地，所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
  22. 一种预防和/或治疗肿瘤疾病的方法，包含向有此需要的患者施用有效量的权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12所述的多特异性抗原结合分子、权利要求13所述嵌合抗原受体、权利要求14所述的免疫效应细胞、权利要求15的分离的核酸分子、权利要求16的表达载体、权利要求18或19所述方法制备的产品、或权利要求20所述的药物组合物；
    优选地，所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
  23. 权利要求1-11任一项所述的抗体或抗原结合片段、权利要求12所述的多特异性抗原结合分子、权利要求13所述嵌合抗原受体、利要求14所述的免疫效应细胞、权利要求15的分离的核酸分子、权利要求16的表达载体、权利要求17的细胞、权利要求18或19所述方法制备的产品、或权利要求20所述的药物组合物，其特征在于，用于和/或治疗肿瘤疾病；
    优选地，所述肿瘤疾病优选黑色素瘤、透明细胞肉瘤、慢性淋巴细胞白血病、头部和颈部癌症、膀胱癌、乳腺癌、结肠癌、卵巢癌、子宫内膜癌、胃癌、胰腺癌、肾癌、前列腺癌、涎腺癌、肺癌、肝癌、皮肤癌、或脑癌。
  24. 一种试剂盒，其包含权利要求1-11任一项的抗体或其抗原结合片段、权利要求12 所述的多特异性抗原结合分子、权利要求13所述嵌合抗原受体、权利要求14所述的免疫效应细胞、权利要求15的分离的核酸分子、权利要求16的表达载体、权利要求17的细胞、权利要求18或19所述方法制备的产品、或权利要求20所述的药物组合物；任选地，还包含使用说明。
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