WO2019218944A2 - 靶向axl的抗体及抗体-药物偶联物及其制备方法和用途 - Google Patents

靶向axl的抗体及抗体-药物偶联物及其制备方法和用途 Download PDF

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WO2019218944A2
WO2019218944A2 PCT/CN2019/086475 CN2019086475W WO2019218944A2 WO 2019218944 A2 WO2019218944 A2 WO 2019218944A2 CN 2019086475 W CN2019086475 W CN 2019086475W WO 2019218944 A2 WO2019218944 A2 WO 2019218944A2
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antibody
seq
axl
variable region
heavy chain
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PCT/CN2019/086475
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English (en)
French (fr)
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WO2019218944A3 (zh
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余科
沈竞康
孟韬
裴金鹏
马兰萍
王昕�
金锐
杜志彦
陈驎
于霆
张永良
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复旦大学
中国科学院上海药物研究所
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Priority to CN201980002582.8A priority Critical patent/CN110770256B/zh
Priority to CA3100260A priority patent/CA3100260A1/en
Priority to EP19803867.1A priority patent/EP3808773A4/en
Priority to JP2020564443A priority patent/JP2021530436A/ja
Priority to US17/055,499 priority patent/US11939386B2/en
Publication of WO2019218944A2 publication Critical patent/WO2019218944A2/zh
Publication of WO2019218944A3 publication Critical patent/WO2019218944A3/zh

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    • 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
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6877—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the antibody being an immunoglobulin containing regions, domains or residues from different species
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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Definitions

  • the present invention relates to the field of medicines, and more particularly to antibodies and antibody drug conjugates (ADCs) targeted to AXL, methods for their preparation, and uses.
  • ADCs antibody drug conjugates
  • AXL is a member of the TAM family of receptor tyrosine kinase subfamilies.
  • the TAM family includes Tyro-3, Axl and Mer, and their ligands are protein molecules encoded by growth arrest specific gene 6 (Gas6).
  • AXL is activated by binding to Gas6, which activates downstream signal transduction pathways such as PI3K/AKT, RAS/ERK and ⁇ -Catenin/TCF, thereby regulating cell proliferation, apoptosis, chemotaxis, adhesion and recognition.
  • Physiological function is activated by binding to Gas6, which activates downstream signal transduction pathways such as PI3K/AKT, RAS/ERK and ⁇ -Catenin/TCF, thereby regulating cell proliferation, apoptosis, chemotaxis, adhesion and recognition.
  • AXL is activated in a variety of cancers, such as lung cancer, breast cancer, prostate cancer, thyroid cancer, endometrial cancer, ovarian cancer and kidney cancer, and is involved in tumor cell epithelial-mesenchymal transition.
  • Multiple mechanisms such as angiogenesis, apoptosis, and immune regulation, and with poor prognosis (Cancer Cell 2015, 27: 533-46) and resistance in many cases (Oncotarget 2015, 6: 15321-31; Cancer Res. 2013, 19:279-90) related, including EGFR inhibitor refractory lung cancer (Nat. Genet.
  • AXL is associated with acquired resistance to traditional chemotherapy and radiation therapy (Theranostics 2016, 6: 1205-19). Increased sensitivity of drug-resistant cells to cytotoxic drugs and targeted inhibitors after inhibition of AXL (Nat. Commun. 2016, 7:13898).
  • AXL is abnormally activated in tumor tissues compared with normal tissues, especially in high-invasive, high-metastatic basal-like and/or triple-negative breast cancer, metastatic lung cancer, pancreatic cancer, and the like, and Compared to other targets, antibodies targeting AXL can be rapidly and massively internalized, and AXL may be a more preferred antibody-drug conjugate (ADC) development target.
  • ADC antibody-drug conjugate
  • Antibody drug conjugates generally consist of three components: an antibody or antibody ligand, a small molecule drug, and a linker that couples the ligand to the drug.
  • highly active cytotoxic drugs are usually lysine residues attached to the surface of the ligand via a linker, or cysteine residues in the hinge region of the antibody (by the chain)
  • the partial drug-ligand ratio (DAR) is 2-4 on the partial disulfide bond reduction.
  • the large amount of lysine residues (more than 80) on the surface of the antibody and the non-selectivity of the coupling reaction lead to uncertainty in the number and location of the coupling, which in turn leads to heterogeneity of the resulting antibody drug conjugate.
  • Genmabu reported a class of antibody conjugates targeting AXL (CN201580045131.4), which are also antibody drug conjugates based on traditional coupling techniques.
  • antibody drug conjugates may seem simple, but whether an antibody drug conjugate can be a safe and effective drug is very complex and unpredictable, depending on many factors, such as:
  • Characteristics of the target Whether the target can be effectively endocytosed, the expression level of the target, whether the target has sufficient expression level difference in cancer cells and normal cells, whether the target will be secreted or exfoliated to the extracellular part And into the blood.
  • Characteristics of mAb Whether the specificity of the mAb is good enough for the target (no cross-reactivity with other proteins), the stability of the mAb and the rate and extent of endocytosis after binding to the target.
  • the present invention provides an antibody targeting human AXL which has the biological activity of blocking AXL, inhibits tumor growth and metastasis activity, and can reduce the emergence of anti-tumor drug resistance.
  • the invention also provides antibody drug conjugates that target AXL, which have significant anti-tumor effects on tumor cells with high expression of AXL.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining AXL binding affinity.
  • the heavy chain variable region comprises the following complementarity determining regions:
  • SEQ ID NO.: 1 SEQ ID NO.: 2, SEQ ID NO.: 3 heavy chain complementarity determining region HCDR1, HCDR2, HCDR3;
  • SEQ ID NO.: SEQ ID NO.: 10 shows the heavy chain complementarity determining region HCDR1, HCDR2, HCDR3 of mAb005c;
  • the heavy chain variable region further comprises a FR region of a human source or a FR region of a murine source.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 7.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 15.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 23.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27.
  • a heavy chain of an antibody having the heavy chain variable region of the first aspect of the invention.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining AXL binding affinity.
  • the light chain variable region comprises the following complementarity determining region:
  • SEQ ID NO.: 4 SEQ ID NO.: 5
  • SEQ ID NO.: 6 light chain complementarity determining region of the mAb002c LCDR1, LCDR2, LCDR3; or
  • SEQ ID NO.: 12 SEQ ID NO.: 13, SEQ ID NO.: 14 light chain complementarity determining region LCDR1, LCDR2, LCDR3;
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 8.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 16.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 24.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.:28, SEQ ID NO.:29, SEQ ID NO.:30, SEQ ID NO.:31.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.:32, SEQ ID NO.:33, SEQ ID NO.:34, SEQ ID NO.:35.
  • a light chain of an antibody having the light chain variable region of the third aspect of the invention.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • an antibody having:
  • the antibody has: the heavy chain of the second aspect of the invention; and/or the light chain of the fourth aspect of the invention.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the CDR regions of the humanized antibody comprise 1, 2, or 3 amino acid changes.
  • the animal is a non-human mammal, preferably a mouse, a sheep, or a rabbit.
  • the antibody is a diabody, or a single chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the number of amino acids added, deleted, modified and/or substituted does not exceed 40%, preferably 20%, more preferably 10%, of the total amino acid number of the initial amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 7, preferably from 1 to 3, more preferably one.
  • the at least one amino acid sequence added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
  • the derivative sequence added, deleted, modified and/or substituted for at least one amino acid has the function of inhibiting cell surface AXL or recombinant AXL protein.
  • the antibody is in the form of a drug conjugate.
  • the antibody to AXL (AXL protein, such as human extracellular domain, AXL-ECD) of 0.04 to affinity EC 50 of 0.5 nM, preferably 0.04 to 0.1 nM, more preferably from 0.04 to 0.05 nM.
  • the antibody has an affinity EC 50 for tumor cell surface AXL of 0.1 to 1.5 nM, preferably 0.1 to 1 nM, more preferably 0.1 to 0.2 nM.
  • the antibody-drug conjugate has an IC 50 effect on tumor cells highly expressed by AXL of from 0.01 to 1 nM, preferably from 0.01 to 0.1 nM, more preferably 0.01 to 0.05 nM.
  • a recombinant protein having:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • a CAR construct wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to AXL, and the scFv has the first aspect of the invention The heavy chain variable region of one aspect and the light chain variable region of the third aspect of the invention.
  • a recombinant immune cell is provided, the immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.
  • the immune cells are selected from the group consisting of NK cells, T cells.
  • the immune cells are from a human or non-human mammal (e.g., a mouse).
  • an antibody drug conjugate comprising:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an anti-ALX antibody
  • D is a drug
  • subscript p is a value selected from 1-10, preferably 1-8.
  • the coupled moiety (D) is a cytotoxic drug
  • the cytotoxic drug is: microtubule-targeted drug and/or DNA-targeted drug and/or topoisomerase inhibition Agent.
  • the microtubule-targeting drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansin derivative DM1, and tubulysin. .
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • DM1 maytansin derivative DM1
  • tubulysin maytansin derivative DM1
  • the DNA targeting drug is selected from the group consisting of docamycin, pyrrolo[2,1-c][1,4]benzodiazepine (PBD).
  • the topoisomerase inhibitor is selected from the group consisting of 7-ethyl-10-hydroxycamptothecin (SN38), exenatide (Exatecan), and the like.
  • the antibody moiety is coupled to the coupling moiety by a chemical bond or a linker.
  • the linker (LU) is selected from the group consisting of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinate (MCC), Maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-val-cit-PAB), CL2A (US20140170063, CN201480041766.2) and disubstituted maleimide linkers (CN201611093699.6, CN201711169847.2).
  • MCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinate
  • MCC Maleimidocaproyl
  • mc-val-cit-PAB 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl
  • CL2A US20140170063, CN201480041766.2
  • disubstituted maleimide linkers
  • the antibody drug conjugate cytotoxic to tumor cells is highly expressed AXL IC 50 of 0.01 ⁇ 1nM, preferably of 0.01 ⁇ 0.1nM, more preferably is 0.01 ⁇ 0.05nM.
  • an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, The light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the invention
  • the detection reagent, test plate or kit is used to:
  • the detection reagent, test plate or kit is used to diagnose AXL related diseases.
  • the medicament is for treating or preventing tumors, tumor migration, or tumor resistance that are highly expressed by AXL.
  • the tumor resistance includes: drug resistance of the tumor immunotherapy drug, drug resistance of the tumor targeted therapy drug, drug resistance of conventional tumor chemotherapy, and insensitivity to radiation therapy.
  • the medicament is for use in a group selected from the group consisting of:
  • AXL that specifically binds to tumor cells, and/or immune/stromal cells in the tumor microenvironment
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the AXL-related disease is selected from the group consisting of cancer, autoimmune disease, metabolic related disease, infectious disease, or a combination thereof.
  • the cancer comprises a solid tumor, a blood cancer.
  • the cancer is a tumor with high expression of AXL.
  • the AXL high expression tumor is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, glioma, melanoma, leukemia, lymphoma. Or a combination thereof.
  • the cancer is a drug resistant tumor.
  • the AXL high expression tumor refers to the ratio of the level L1 of the AXL transcript and/or protein in the tumor tissue to the level L0 of the transcript and/or protein in the normal tissue, L1/L0 ⁇ 2 Preferably ⁇ 3.
  • the metabolic related diseases include: diabetes, foodborne obesity, and fat inflammation.
  • the infectious disease comprises: bacterial and viral infections.
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, and the light chain of the third aspect of the invention
  • the variable region, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, the immune cell of the eighth aspect of the invention, the invention The antibody drug conjugate of the ninth aspect, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the fourth aspect of the invention a light chain, or an antibody of the fifth aspect of the invention;
  • the invention provides a vector comprising the polynucleotide of the twelfth aspect of the invention.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector or genome of the thirteenth aspect of the present invention, wherein the multinuclear according to the twelfth aspect of the present invention is integrated Glycosylate.
  • a method of in vitro detection (including diagnostic or non-diagnostic) of AXL in a sample comprising the steps of:
  • test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody of the fifth aspect of the invention or the invention
  • the immunoconjugate of the ninth aspect comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody of the fifth aspect of the invention or the invention.
  • a kit comprising:
  • a first container comprising the antibody of the fifth aspect of the invention.
  • the kit contains the test plate of the sixteenth aspect of the invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a nineteenth aspect of the invention provides a method of treating an AXL-related disease, the method comprising: administering to a subject in need thereof the antibody of the fifth aspect of the invention, the antibody-drug conjugate of the antibody, Or a CAR-T cell expressing the antibody, or a combination thereof.
  • the method further comprises administering to the subject in need of other drugs or treatments for combination therapy.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiation therapy.
  • the anti-tumor immunotherapeutic agent comprises PD-1, PD-L1 mAb.
  • a method of preparing a chimeric antibody comprising the steps of:
  • a method of preparing a humanized antibody comprising the steps of:
  • the nucleotide sequence of the CDR region of the heavy chain variable region of the first aspect of the invention and/or the light chain variable region of the third aspect of the invention is implanted into a nucleoside comprising a FR region of a human antibody
  • the acid sequence template is then cloned into an expression vector containing the human antibody constant region, and the humanized antibody is expressed by transfecting the animal cells.
  • a method of inhibiting tumor cell growth and migration comprising the steps of: administering to a subject in need thereof the antibody of the fifth aspect of the invention, antibody-drug coupling of said antibody Or a CAR-T cell expressing the antibody, or a combination thereof.
  • a method of inhibiting growth of a tumor in a model animal comprising the steps of: administering to the subject in need thereof the antibody of the fifth aspect of the invention, the antibody-drug couple of the antibody A conjugate, or a CAR-T cell expressing the antibody.
  • the medicament may be administered alone or in combination, including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiation therapy.
  • Figure 1 shows the discovery of an anti-human AXL antibody of the present invention.
  • Figure 1A shows the original anti-human AXL monoclonal antibody culture supernatant detected by flow cytometry (FACS) on human AXL-highly expressed MDA-MB-231 (AXL-P), AXL - Binding activity of low expressed MDA-MB-453 (AXL-N) breast cancer cells.
  • Figure 1B shows the numbering of six monoclonal antibodies (mAb001, mAb002, mAb003, mAb004, mAb005, mAb006) and subtype identification of antibodies after purification.
  • Figure 2 is a graph showing the results of agarose gel electrophoresis of PCR amplification of mAb001, mAb002, mAb005, mAb006 heavy chain variable region (VH) and light chain variable region (VL) fragments.
  • VH heavy chain variable region
  • VL light chain variable region
  • FIG 3 is a 4 individuals expressing HEK293T cells - mouse chimeric antibody (chimeric antibody) mAb001c, mAb002c, mAb005c, mAb006c, and then purified using MabSelect TM SuRe TM map column.
  • FIG 4 is an ELISA measuring human - mouse chimeric antibody mAb001c, mAb002c, mAb005c, mAb006c to AXL-ECD binding affinity (Binding affinity EC 50).
  • Figure 5 shows the breast cancer (MDA231, Hs587T, MDA453), lung cancer (NCI-H1299, Calu-1, NCI-H460), pancreatic cancer (SW1990, Capan-2, Panc-1) by Western blot. , Capan-2) cell line expression level of AXL protein.
  • Figure 6 is a graph showing the expression level of AXL mRNA (ratio to -actin) in a plurality of tumor cell lines (breast cancer, lung cancer, glioma, melanoma) and human normal tissues.
  • Figure 7 is a graph showing the high-invasion, high-metastasis Basal-type contrast tube of AXL mRNA in 51 human breast cancer cell line gene expression databases (Neve RM et al., Cancer Cell 2006; 10: 515-27). Expression levels in a population of Luminal-type breast cancer cells.
  • Figure 8 is a graph showing the expression level of AXL mRNA in epithelial type versus interstitial lung cancer cell line in the CCLE database.
  • Figure 9 shows mAb002c (5g/mL) against AXL-high expression (NCI-H1299, LCLC-103H, CaLu-1, MDA-MB-231, Hs578T) or low expression (MDA-MB-453) tumor cell surface AXL Combination level.
  • Figure 10 shows the results of binding of the chimeric antibodies mAb001c, mAb002c, mAb005c, mAb006c to MDA-MB-231 cell surface AXL (Binding affinity EC 50 ), using 1x10 5 cells mixed with the indicated concentration of antibody, and incubated for 1 hour. FACS detection analysis was performed by flow cytometry (FACSCalibur).
  • Figure 11 shows the binding affinities of the chimeric antibodies mAb001c, mAb002c, mAb005c to NCI-H1299 cell surface AXL (Binding affinity EC 50 ), using 1x10 5 cells mixed with the indicated concentration of antibody, incubated for 1 hour and then used Flow cytometry (FACSAria II) was performed for FACS analysis.
  • Figure 13 shows the binding affinity of humanized antibody series Hu002-1 to Hu002-24 to MDA-MB-231 cell surface AXL (Binding affinity EC 50 ), using 1x10 5 cells mixed with the indicated concentration of antibody, incubating 1 After the hour, FACS detection analysis was performed using a flow cytometer (FACSCalibur).
  • Figure 14 shows the binding affinity of humanized antibody series Hu002-1 to Hu002-5 to LCLC-103H cell surface AXL (Binding affinity EC 50 ), using 1x10 5 cells mixed with the indicated concentration of antibody, and incubated for 1 hour. FACS detection analysis was performed by flow cytometry.
  • FIG. 15 shows that Hu002-2 binds to MDA-MB-231 cells resulting in internalization to intracellular lysosomes.
  • the antibody (5 g/mL) was incubated with the cells at 4 ° C for 1 hour, or at 37 ° C for 4 hours, and placed under laser confocal microscopy.
  • Figure 16 shows the inhibition of AXL protein expression by Western blot after LCLC-103H cells were treated with Hu002-2 or Hu002-2-BL20-MMAE for 24 or 48 hours.
  • Figure 17 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate AXL107-vc-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 18 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate AXL107-BL20-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 19 is a mass spectrum of monoclonal antibody AXL107.
  • Figure 20 is a mass spectrum of the antibody drug conjugate AXL107-vc-MMAE.
  • Figure 21 is a mass spectrum of the antibody drug conjugate AXL107-BL20-MMAE.
  • Figure 22 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate mAb002c-vc-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 23 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate mAb002c-BL20-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 24 is a mass spectrum of the monoclonal antibody mAb002c.
  • Figure 25 is a mass spectrum of the antibody drug conjugate mAb002c-vc-MMAE.
  • Figure 26 is a mass spectrum of the antibody drug conjugate mAb002c-BL20-MMAE.
  • Figure 27 is a hydrophobic interaction chromatography (HIC) map of the antibody drug conjugate Hu002-2-BL20-MMAE.
  • HIC hydrophobic interaction chromatography
  • Figure 28 is a mass spectrum of the antibody drug conjugate Hu002-2-BL20-MMAE.
  • Figure 29 is a mass spectrum of the humanized monoclonal antibody Hu002-2.
  • Fig. 30 shows the results of detection of the inhibitory activity (IC 50 ) of mAb002c-ADC and AXL107-ADC against MDA-MB-453 cells in vitro.
  • Figure 31 shows the results of detection of the inhibitory activity (IC 50 ) of mAb002c-ADC and AXL107-ADC against MDA-MB-231 cells in vitro.
  • Figure 32 shows the results of detection of the inhibitory activity (IC 50 ) of mb002c-ADC and AXL107-ADC against Hs578T cells in vitro.
  • Fig. 33 shows the results of detection of the inhibitory activity (IC 50 ) of mAb002c-ADC and AXL107-ADC on Calu-1 cell proliferation in vitro.
  • Figure 34 shows the results of detection of inhibitory activity (IC 50 ) of mAb002c-ADC and AXL107-ADC against LCLC-103H cell proliferation in vitro.
  • Figure 35 shows the results of detection of the inhibitory activity (IC 50 ) of U87MG cells in vitro by mAb002c-ADC and AXL107-ADC.
  • Figure 36 shows the results of detection of the inhibitory activity (IC 50 ) of the MDA-MB-231 cells in vitro by the humanized Hu002 series antibody-conjugated BL20-MMAE.
  • Fig. 37 shows the results of detection of the inhibitory activity (IC 50 ) of the proliferation of Hs578T cells by the humanized Hu002 series antibody-conjugated BL20-MMAE.
  • Figure 38 shows the results of detection of the inhibitory activity (IC 50 ) of U87MG cells in vitro by humanized Hu002 series antibody-conjugated BL20-MMAE ADC.
  • FIG 39 is a humanized antibody conjugate Hu002 series of ADC BL20-MMAE LCLC-103H vitro cell proliferation inhibitory activity (IC 50) of the detection result.
  • Figure 40 shows the in vivo antitumor efficacy of the chimeric antibody mAb002c antibody coupled with vc-MMAE, BL20-MMAE ADC (both 5 mg/kg) in the U87MG glioma model, and the results showed that BL20-MMAE compared to vc -MMAE has superior in vivo therapeutic effects.
  • Figure 41 shows the in vivo antitumor efficacy of humanized Hu002-1, Hu002-4 antibody-conjugated BL20-MMAE (3 mg/kg), AXL107-vc-MMAE (3 mg/kg) in the U87MG glioma model.
  • Figure 42 shows in vivo antitumor efficacy results of humanized Hu002-2, Hu002-5 antibody conjugated to BL20-MMAE ADC (3 mg/kg; twice a week) in the U87MG glioma model.
  • Figure 43 shows in vivo antitumor drugs in human lung cancer model of human lung Hu002-1, Hu002-4 antibody conjugated to BL20-MMAE (3 mg/kg, 1 mg/kg; twice a week) in LCLC-103H lung cancer model. Effect results.
  • Figure 44 is an in vivo antitumor drug in a LCLC-103H lung cancer model by humanized Hu002-2 and Hu002-5 antibodies conjugated to BL20-MMAE ADC (3 mg/kg, 1 mg/kg; twice a week) Effect results.
  • Figure 45 shows the in vivo resistance of humanized Hu002-1, Hu002-2, and Hu002-5 antibodies to BL20-MMAE ADCs (both 1 mg/kg; twice a week) in LCLC-103H lung cancer model. Tumor efficacy results.
  • Figure 46 shows that humanized Hu002-2 and Hu002-5 antibodies were coupled to BL20-MMAE ADC (1 mg/kg, 0.5 mg/kg) and AXL107-vc-MMAE (1 mg/kg) twice a week for 2 times. In vivo anti-tumor efficacy results in the LCLC-103H lung cancer model after drug administration.
  • Figure 47 is a graph showing the results of humanized Hu002-2-BL20-MMAE (5 mg/kg; single administration) against LCLC-103H large volume tumors (800 mm 3 is the volume at the time of initial administration) which can cause tumor regression.
  • Figure 48 is a graph showing the results of humanized Hu002-2-BL20-MMAE (10 mg/kg; single administration) against LCLC-103H large volume tumors (1800 mm 3 is the volume at the time of initial administration) which can cause tumor regression.
  • Figure 49 is a graph showing the detection of FACS-binding activity of HUK293T transiently expressing mouse AXL protein by Hu002-2; Hu002-2 or AXL107 showed a weak binding activity to murine AXL compared to human-derived AXL.
  • Figure 50 is the binding affinity of Hu002-2 to cynomolgus monkey AXL.
  • Figure 50A shows the protein expression level of HEK293T transient transgenic monkey AXL vector after 24 hours by Western blot.
  • Figure 50B shows that HEK293T transiently expresses monkey AXL 24 hours after receiving cells, and its binding affinity for Hu002-2 is detected by FACS. (Binding affinity EC 50 ).
  • the inventors have unexpectedly obtained six anti-AXL monoclonal antibodies, which are named mAb001 to mAb006, through extensive and intensive research.
  • human-mouse chimeric antibodies were constructed by selecting mAb001 (IgG1- ⁇ ), mAb002 (IgG1- ⁇ ), mAb005 (IgG1- ⁇ ), mAb006 (IgG2b- ⁇ ), and named them as mAb001c and mAb002c, respectively.
  • mAb005c, mAb006c After further testing the above antibodies, the results were as follows:
  • the chimeric antibody can bind to the AXL antigen with high specificity, and the EC 50 of the ELISA is 0.092 nM, 0.073 nM, 0.103 nM, and 0.101 nM, respectively;
  • a chimeric antibody having a high binding affinity for tumor cells is highly expressed strains AXL, FACS is determined EC 50 0.174nM ⁇ 1.5nM, gene sequencing and display mAb006c mAb005c complementarity determining region (CDR) of Highly overlapping, so the subsequent study of mAb006c was terminated;
  • the drug conjugate (ADC) of the antibody has excellent characteristics, that is, has no obvious toxic and side effects on AXL-normally expressed cells, and has extremely high killing activity on AXL-highly expressed tumor cells, and cell proliferation
  • the inhibition IC 50 value is 0.01 nM to 0.07 nM;
  • the AXL-ADC product obtained by the novel linker of the present invention has the advantages of high uniformity and further improvement of stability in vitro and in vivo.
  • the present invention has been completed on this basis.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a variable region having a monoclonal antibody from a murine, and a chimeric antibody from a constant region of a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent No. 4,816,397, incorporated herein by reference in its entirety herein.
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety.
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention provides three broad classes of highly specific and high affinity antibodies targeting AXL comprising heavy and light chains comprising a heavy chain variable region (VH) amino acid sequence comprising a light chain Variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the heavy chain variable region (VH) amino acid sequence, the light chain variable region (VL) amino acid sequence comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the following polypeptide sequences:
  • HCDR1 is SEQ ID NO.: 1: DFYIN, SEQ ID NO.: 9: SYYIH, or SEQ ID NO.: 17: SGYWS;
  • HCDR2 is SEQ ID NO.: 2: WIYPGSGNTKYNEKFKG, SEQ ID NO.: 10: WIYPGSDNTKYNEKFKD, or SEQ ID NO.: 18: YMTYSGATYYNPSLKS;
  • HCDR3 is SEQ ID NO.: 3: STGFFDY, SEQ ID NO.: 11: NYYDYDGGTWFPY, or SEQ ID NO.: 19: GGNSYFFDY;
  • LCDR1 is SEQ ID NO.: 4: SASSSIGYMY, SEQ ID NO: 12: RASQDINYYLN, or SEQ ID NO.: 20: RASENIYSNLA;
  • LCDR2 is SEQ ID NO.: 5: LTSNLAS, SEQ ID NO.: 13: YTSRLHS, or SEQ ID NO.: 21: AATNLAD;
  • LCDR3 is SEQ ID NO.: 6: QQWSSNPPT; SEQ ID NO.: 14: QQGNTLPWT, or SEQ ID NO.: 22: QHFWGTPLT;
  • A7 A sequence having an AXL binding affinity by adding, deleting, modifying and/or substituting at least one amino acid sequence of any one of the above amino acid sequences.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.
  • the antibody has an inhibitory effect on the cell surface and recombinant AXL protease which is rapidly endocytosed by the cells into the lysosome.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, preferably a humanized antibody, and more preferably a fully humanized antibody.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a chimeric antibody, a humanized antibody, a CDR grafted and/or a modified antibody that targets human AXL.
  • VH CDR1, CDR2, CDR3 are each independently selected from the group consisting of SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or selected from the group consisting of SEQ ID NO .: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, or one or more sequences selected from the group consisting of SEQ ID NO.: 17, SEQ ID NO.: 18, SEQ ID NO.: 19, Or they have been added, deleted, modified and/or substituted for at least one amino acid sequence having AXL binding affinity;
  • VL CDR1, CDR2, CDR3 are independently selected from SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, or selected from SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, or selected from SEQ ID NO.: 20, SEQ ID NO.: 21, SEQ ID NO. Any one or more of the sequences of 22, or a sequence thereof having an AXL binding affinity by addition, deletion, modification
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably It is 1-2.
  • the antibody is the original murine antibody mAb001, mAb002, mAb003, mAb004, mAb005, mAb006.
  • the antibody is a human-mouse chimeric antibody mAb001c, mAb002c, mAb005c, mAb006c.
  • the antibodies are humanized antibodies Hu002c-1, Hu002c-2, Hu002c-3, Hu002c-4, Hu002c-5, Hu002c-6, Hu002c-7, Hu002c-8, Hu002c-9 Hu002c-10, Hu002c-11, Hu002c-12, Hu002c-13, Hu002c-14, Hu002c-15, Hu002c-16, Hu002c-17, Hu002c-18, Hu002c-19, Hu002c-20, Hu002c-21, Hu002c -22, Hu002c-23, Hu002c-24.
  • the three broad classes of antibodies of the present invention can be used in combination for the construction of CAR constructs, recombinant immune cells comprising CAR constructs, antibody drug conjugates, and the like, and can also be used for (a) preparation of detection reagents, detection plates or Kit; and/or (b) a medicament for the prevention and/or treatment of AXL related diseases.
  • Serial number Sequence name Serial number Sequence name SEQ ID NO.: 1 mAb002HCDR1 SEQ ID NO.: 21 mAb001LCDR2 SEQ ID NO.: 2 mAb002HCDR2 SEQ ID NO.: 22 mAb001LCDR3 SEQ ID NO.: 3 mAb002HCDR3 SEQ ID NO.: 23 mAb001-VH SEQ ID NO.: 4 mAb002LCDR1 SEQ ID NO.: 24 mAb001-VL SEQ ID NO.: 5 mAb002LCDR2 SEQ ID NO.: 25 mAb002-VH_HuG0 SEQ ID NO.: 6 mAb002LCDR3 SEQ ID NO.: 26 mAb002-VH_HuG1 SEQ ID NO.: 7 mAb002-VH SEQ ID NO.: 27 mAb002-VH_HuG2 SEQ ID NO.: 8 mAb002-VL SEQ ID NO.: 28 mAb002-VK_Hu
  • the monkey AXL protein sequence and the mouse AXL protein sequence are also referred to in the present invention, Genebank ID is XP_014979499.1 (monkey) and Genebank ID: NP_033491.2 (mouse), respectively.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem., 107: 220 (1980).
  • the antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention also provides an antibody-drug conjugate (ADC) based on an antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-conjugated drug comprises the antibody, and an effector molecule, which is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a therapeutically active drug.
  • the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or a combination of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between an effector molecule and an antibody, such as glutaraldehyde or the like.
  • the coupling agent using a carboxyl group may be any one or more of an cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazine coupling agent (coupling site is an acylhydrazine).
  • Certain residues on the antibody are used to link to a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes). , stabilizers (such as ethylene glycol polymers) and therapeutic agents.
  • imaging agents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as ethylene glycol polymers
  • therapeutic agents such as ethylene glycol polymers
  • the antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly via a linker.
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC comprises a linker between the drug and the antibody.
  • the linker can be a degradable or non-degradable linker.
  • Degradable linkers are typically susceptible to degradation under the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as barium splices) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
  • the linker Prior to attachment to an antibody, the linker has an reactive reactive group capable of reacting with certain amino acid residues, and attachment is achieved by reactive reactive groups.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (eg, iodine, bromine or chlorinated); haloesters (eg, iodine, bromine or chlorinated) Halogenated methyl ketone (eg iodine, bromine or chlorinated), benzyl halide (eg iodine, bromine or chlorinated); vinyl sulfone, pyridyl disulfide; mercury derivative such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker can include, for example, a maleimide attached to the antibody via
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker binds the antibody to the drug, and the drug has a functional group that can bond to the linker.
  • the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group which may be bonded to a linker.
  • the drug is directly attached to the linker, the drug has a reactive group that is reactive prior to attachment to the antibody.
  • Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , vinca alkaloids, etc.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( Taxanes), benzodiazepines or benzodiazepine containing drugs (eg pyrrolo[1,4]benzodiazepines (PBDs), porphyrin benzodiazepines Classes (indolinobenzodiazepines) and oxazolidinobenzodiazepines and vinca alkaloids, 7-ethyl-10-hydroxycamptothecin (SN38), exenatide (Exatecan) ) and its analogs.
  • typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two or more step process. For example, a cysteine residue is reacted with a reactive moiety of the linker in a first step, and in a subsequent step, a functional group on the linker reacts with the drug to form an ADC.
  • a functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety.
  • a portion based on an azide compound can be used to specifically react with a reactive alkynyl group on a drug moiety.
  • the drug is covalently bound to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for amines) And alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols).
  • the invention also provides a method of making an ADC, which can further comprise: binding the antibody to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • the present invention relates to antibody-drug conjugates, and more particularly to AXL antibody-drug conjugates having therapeutic applications.
  • the anti-AXL antibody can be conjugated to a chemotherapeutic or small molecule toxin by a specific linker.
  • the invention also relates to methods of treating mammalian cells or related pathological conditions using anti-AXL antibody-drug conjugates.
  • the DAR value distribution of T-DM1 (average DAR value of 3.5) is 0-8.
  • the DAR value distribution of T-DM1 (average DAR value of 3.5) is 0-8.
  • the resulting conjugate is not a uniform product, and is composed of a plurality of components, and the DAR value of the main component thereof is 0, 2, 4, 6, 8, and the components corresponding to each of the specific DAR values have isomers formed due to the difference in the attachment sites.
  • the heterogeneity of the antibody drug conjugate product can result in pharmacokinetic properties, potency, and toxicity heterogeneity between the various member components. For example, components with higher DAR values are cleared faster in the body and result in higher toxicity.
  • the purpose of the fixed-point coupling of the existing antibodies by a simple chemical method is to save a lot of manpower, material and financial resources, and thus is more attractive.
  • related researches are: CN200480019814.4 applied by Polylites Co., Ltd.; WO2014197871A2 applied by Igenica Biotherapeutics; CN201380025774.3 applied by Sorrento Medical Co., Ltd.; Shanghai New Concept Biomedical Technology Co., Ltd. CN201310025021.4 and so on.
  • Genmabu reported a class of antibody conjugates targeting AXL (CN201580045131.4), which are also antibody drug conjugates based on traditional coupling techniques.
  • the site-directed coupling of antibody-drug conjugates targeting AXL can be achieved by simple chemical methods, which can improve the uniformity of the drug and save a lot of manpower, material and financial resources in process and quality control. At the same time, it may also improve the stability of the conjugate, the efficacy and safety of the drug.
  • the present invention employs a novel class of linker structures (a new class of disubstituted maleimide linkers CN201611093699.6, CN201711169847.2 developed by the inventors in the early stage) and is applied to target AXL antibodies for coupling.
  • the linker may be coupled to all or part of the light chain-heavy chain of the antibody and the heavy chain-heavy chain disulfide-reduced cysteine thiol group, and the targeted AXL antibody drug coupling obtained by the coupling method is used.
  • a narrower drug/antibody ratio (DAR) distribution compared to traditional antibody drug conjugates.
  • the structure of the AXL antibody-drug conjugate having the disubstituted maleimide linker is as shown in Formula Ia, Ib:
  • Ar' is selected from the group consisting of substituted or unsubstituted C6-C10 arylene, substituted or unsubstituted 5-12 membered heteroarylene;
  • L 1 is -O(CH 2 CH 2 O) n - attached to the Ar' group, wherein n is selected from any of 1-20.
  • L 2 is a chemical bond, or an AA-PAB structure; wherein AA is a polypeptide fragment consisting of 2-4 amino acids, and PAB is p-aminobenzylcarbamoyl;
  • CTD is a cytotoxic small molecule drug that is bonded to L 2 via an amide bond.
  • Ab is an antibody that targets AXL.
  • the present invention provides a coupling method for coupling a small molecule of a toxin to a targeted AXL antibody via a specific linker, which substantially increases the lethality of the antibody against tumor cells without altering the affinity of the antibody.
  • the invention provides a linker or coupling reagent comprising a diarylthiomaleimide unit and a coupling group.
  • the diarylthiomaleimide unit is used to crosslink the sulfhydryl group between the antibody chains (after reduction), while the coupling group is used to couple with the small molecule drug or drug-linker unit.
  • These ADCs are homogeneous due to the bidentate binding of the diarylthiomaleimide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody. It is more stable than an ADC with a single-toothed joint. Thus they will have a increased in vivo half-life, reduce the amount of systemically released cytotoxin, and be safer than the ADC with a single-toothed linker.
  • the produced drug-linker unit is coupled to the antibody via the linker to form a partial interchain cross-linking conjugate.
  • the antibody/antibody ratio (DAR) distribution of the antibody drug conjugate prepared by the method of the present invention is narrower than that of the conventional antibody drug conjugate, thereby greatly improving product uniformity and pharmacological property uniformity.
  • the antibody drug conjugate can be used to target delivery of a drug to a target cell population, such as a tumor cell.
  • the antibody drug conjugate can specifically bind to the cell surface protein, and the resulting conjugate is then endocytosed by the cell. Within the cell, the drug is released as an active drug to produce efficacy.
  • Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that bind to an antigen; or antibody Fc fusion proteins; or proteins.
  • a "drug” is a highly active drug (see definitions), and in some cases, the drug may be polyethylene glycol.
  • the coupled product provided by the present invention although still a mixture, has a narrow DAR distribution range compared to the antibody drug conjugate obtained by conventional coupling.
  • the average DAR value is close to 4, which is close to the range of optimal DAR values (2-4) for optimal antibody drug conjugates.
  • the preparation route of the antibody drug conjugate is as follows.
  • the interchain disulfide bond of the antibody is reduced to yield 2n (e.g., 8) sulfhydryl groups.
  • the substituted maleimide linker-drug conjugate of the present invention (the compound of formula Ic) is cross-linked with the reduced antibody thiol to form the corresponding antibody drug conjugate, wherein the antibody drug conjugate is present as follows One or two forms of the illustration.
  • a typical preparation method comprises: diluting the antibody stock solution to 2-10 mg/mL with a reaction buffer, adding a 140-200 times excess molar ratio of dithiothreitol (DTT), or adding a 6.0-20 fold excess molar ratio.
  • DTT dithiothreitol
  • Tris(2-carboxyethyl)phosphine hydrochloride the reaction solution is stirred at 10-35 ° C for 2-48 hours;
  • the reaction buffer may be a buffer prepared in the following ratio: 50 mM phosphoric acid Potassium hydrogen-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate - citric acid / 150 mM Sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM boric acid - borax / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide/150 m
  • reaction solution is cooled to 0-10 ° C. If DTT reduction is used, excess DTT is removed by desalting column or ultrafiltration after completion of the reduction reaction, and then substituted maleimide compound (previously dissolved in 10 mg/ml) is added. Acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA), and ensure that the volume of organic solvent in the reaction solution does not exceed 15%, even The reaction was stirred at 0-37 ° C for 2-4 hours. If TCEP reduction is used, it is also possible to directly add a substituted maleimide compound for coupling without removing the remaining TCEP.
  • ACN acetonitrile
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • DMA diethyl acetamide
  • the coupling reaction mixture was purified by filtration using a sodium succinate/NaCl buffer or a histidine-acetic acid/sucrose gel using a desalting column, and a peak sample was collected based on the UV280 ultraviolet absorption value. Or ultrafiltration several times.
  • the bacteria were then sterilized by filtration and the resulting product was stored at a low temperature.
  • the temperature is from -100 to 60 ° C, and the pore size of the filtration device is preferably from 0.15 to 0.3 ⁇ m.
  • the drug antibody coupling ratio (DAR) of the obtained antibody drug conjugate was relatively uniform.
  • DAR drug antibody coupling ratio
  • the ADC product homogeneity is very high (typically DAR dominant products (such as DAR is about 4) account for at least 60%, at least 70%, of at least 70% of all ADCs. 80%, at least 90% or higher).
  • DAR hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • IEC ion exchange Chromatography
  • the antibody-drug conjugate provided by the present invention can be targeted to a specific cell population and bind to a cell surface specific protein (antigen), the drug can be released into the cell in an active form by endocytosis or drug infiltration.
  • the antibody-drug conjugates of the invention can be used to treat a disease of interest, and the antibody-drug conjugates mentioned above can be administered to a subject (e.g., a human) by a suitable route in a therapeutically effective amount.
  • a subject in need of treatment can be a patient at risk or suspected of having a condition associated with the activity or amount of expression of a particular antigen. Such patients can be identified by routine physical examination.
  • compositions can be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implantation.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • it can be administered to the subject of injectable or biodegradable materials and methods by administration of an injectable depot route, for example using 1-, 3-, or 6 months.
  • the injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, Liquid polyethylene glycol, etc.).
  • the water-soluble antibody can be administered by infusion by a drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient.
  • Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • An intramuscular preparation for example, a sterile preparation in the form of a suitable soluble salt of the antibody, a pharmaceutically acceptable excipient which can be dissolved and administered, such as a water exchange injection, 0.9% saline, or a 5% glucose solution.
  • delivery can be by conventional methods in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.
  • Other methods include the use of various transport and carrier systems through the use of conjugates and biodegradable polymers.
  • compositions of the present invention comprise a safe and effective amount of an antibody-drug conjugate of the invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of a solution, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the effective amount of the antibody-drug conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, patient's weight, patient's immunity Status, route of administration, etc.
  • the antibody-drug conjugate of the present invention when administered at a dose of about 0.0001 mg to 50 mg/kg of animal body weight per day (preferably 0.001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained.
  • a dose of about 0.0001 mg to 50 mg/kg of animal body weight per day preferably 0.001 mg to 10 mg/kg of animal body weight
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 5 to 500 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibodies of the invention or their ADCs can be used in detection applications, for example for detecting samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples, and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may include, for example, a resected sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
  • the antibody of the invention may be immobilized on a test plate.
  • the invention also provides the use of an antibody of the invention, for example for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of AXL related diseases.
  • AXL-related diseases include tumorigenesis, growth and/or metastasis, tumor resistance-related diseases, inflammation, metabolism-related diseases, and the like.
  • the tumor includes, but is not limited to, breast cancer (such as triple negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, and knot.
  • breast cancer such as triple negative breast cancer
  • lung cancer such as non-small cell lung cancer
  • pancreatic cancer malignant glioma
  • gastric cancer liver cancer
  • esophageal cancer esophageal cancer
  • kidney cancer esophageal cancer
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Lytle syndrome, psoriasis arthropathy, infectious arthritis, tuberculous arthritis, viral joints Inflammation, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolic related diseases include, but are not limited to, diabetes, foodborne obesity, and fat inflammation.
  • antibodies described herein novel and excellent biological activity in particular, preferably according AXL antibody has a high affinity (ELISA assay which EC 50 of 0.04 ⁇ 0.05nM). Furthermore, the preferred antibody has a good binding affinity for the tumor cell surface AXL (FACS determined EC 50 of 0.07 ⁇ 0.14nM), can be used as therapeutic antibody targeting of AXL.
  • the humanized antibody of the present invention not only has an activity comparable to or higher than that of a murine antibody, but also has lower immunogenicity.
  • the antibody-drug conjugate (ADC) of the present invention has specific AXL-dependent antitumor activity; the preferred humanized antibody drug conjugate (ADC) is free of AXL-normally expressed cells. It has obvious toxic and side effects, and has extremely high killing activity against AXL-highly expressed tumor cells.
  • the cell proliferation inhibition test measures IC 50 of 0.01 nM to 0.05 nM.
  • the novel linker provided by the present invention can be coupled to a targeted AXL antibody by a simple chemical method, and the DAR value distribution of the AXL antibody drug conjugate obtained by using the linker is very high compared with the conventional coupling method. It is narrow, so the product produced is highly uniform, and the obtained single component of cross-linking (DAR is 4) accounts for more than 80%.
  • the in vitro tumor cell proliferation inhibitory activity of cross-linking is more than that of traditional mcVC-PAB.
  • the properties of the drug, such as activity and safety, are improved or maintained.
  • the maleimide-based disulfide bridge bridging has better stability, and the introduction of a substituent at the Ar' site can mediate the reaction rate of maleimide ring-opening hydrolysis and slow down the opening of maleimide.
  • the subsequent cyclization secondary hydrolysis reaction is less prone to in vivo oxime ether exchange and ring-opening secondary hydrolysis reaction after ring opening, further enhancing the stability of the AXL antibody-drug conjugate in vitro and in vivo.
  • Preferred antibodies and antibody-drug conjugates of the invention have superior in vitro and in vivo anti-tumor therapeutic effects compared to prior art AXL07-vc-MMAE.
  • AXL-ECD The extracellular domain of human AXL protein (AXL-ECD) was first prepared as an antigen.
  • NCBI NCBI: NP_068713.2 amino acid position 33 to 449, using gene cloning technology and mammalian vector expression system to obtain C-terminus polyhistidine-tagged antigen, the specific amino acid sequence is as follows ( SEQ ID NO.: 36):
  • mice were immunized with human AXL extracellular domain protein expressed and prepared in HEK293T cells in an amount of 50 ⁇ g/head to prepare immune spleen cells; mouse myeloma cells (SP2/0) and feeder cells were prepared at appropriate time. Need for integration.
  • the spleen cells and SP2/0 cells are fused by PEG-mediated fusion, PEG is removed, resuspended in HAT complete medium containing feeder cells, inoculated into 96-well plates, and cultured by ELISA/FACS method. Positive well screening was performed. Finally, the cells of the positive wells were cloned and cultured by limiting dilution method, and the cells with high titer, good morphology and monoclonal growth were screened by ELSIA or FASCS, and the subcloning screening was continued until the positive cloning rate was 100 for three consecutive screenings. %, the cell strain can be expanded and constructed.
  • the cell culture supernatant was collected, filtered through a 0.22 ⁇ m filter, and the resulting culture supernatant was added at a constant rate to a previously equilibrated Protein A resin column.
  • the equilibration column was then eluted with 0.1 M sodium citrate buffer, and the eluate was collected and quantified for SDS-PAGE electrophoresis, SEC-HPLC, and endotoxin detection.
  • the obtained purified antibody was dispensed and stored at -80 ° C until use.
  • Step 3 Determination of the biological activity and specificity of targeting human AXL murine monoclonal antibodies:
  • the selected six hybridoma monoclonal antibodies were assayed for biological activity and specificity.
  • Figure 1A the supernatant of the monoclonal cell culture solution was detected by flow cytometry (FACS).
  • FACS flow cytometry
  • Six monoclonal antibodies specifically bind to human AXL-highly expressed MDA-MB-231 cells. (AXL-P), but no significant binding activity to AXL-low expressed MDA-MB-453 cells (AXL-N).
  • FIG. 1B the purified antibody sample was used for subtype detection, and mAb001 to mAb005 were identified as IgG1/k, and mAb006 was IgG2b/k.
  • Step 4 The purified antibody samples were diluted gradient detected by ELISA, as shown in Table 1, mAb001 ⁇ mAb006 to AXL-ECD has excellent binding affinity, wherein, mAb001, mAb002, mAb005, mAb006 both the EC 50 ⁇ 0.1nM.
  • Table-1 ELISA activity targeting human AXL original murine antibody
  • mAb001, mAb002, mAb005 and mAb006 were preferentially selected for antibody sequencing.
  • Primers were designed to amplify heavy chain (VH), light chain (VL) variable region fragments (see Figure 2) by conventional PCR techniques, cloned into vectors, and sequenced.
  • the following heavy chain variable region (VH), light chain variable region (VL) amino acid sequence, and complementarity determining region (CDR) information were obtained by conventional sequencing and analysis by Kabat database (http://www.bioinf.org.uk).
  • the underline “_” shows the CDR-1/2/3 amino acid sequence.
  • variable region sequences Three sets of variable region sequences are by genetic recombination techniques (see SEQ ID NO.: 7, SEQ ID NO.: 15, SEQ ID NO.: 23, SEQ ID NO.: 8, SEQ ID NO.: 16, SEQ ID NO .: 24) was cloned into a vector containing the human IgG1 heavy chain constant region and Kappa chain constant region, was sequenced correctly, using chimeric antibodies transfection and mammalian expression systems (FreeStyle TM 293T cells) the Construction of expression And purified (see Figure 3), the obtained human-mouse chimeric antibodies, numbering, heavy chain and light chain composition are listed in Table-2.
  • the humanized antibody AXL07 disclosed in the invention patent application CN201580045131.4 was prepared in the same manner as a control.
  • AXL protein extracellular domain (AXL-ECD) was diluted to 1 ⁇ g/mL with a coating solution, coated with an ELISA plate, 100 ⁇ L/well, 4 ° C, overnight. Wash off the excess antigen, block with 1% BSA for 2h at room temperature, then add 3 times serial dilution of each monoclonal antibody, 100L / well, incubate for 1h at room temperature; wash away unbound antibody, add appropriate concentration of horseradish peroxidase Labeled anti-mouse secondary antibody, 100 ⁇ L/well, incubated for 0.5 h at room temperature.
  • the unbound secondary antibody was washed away, reacted with TMB coloring solution for about 15 min, 1N HCL was added, 50 ⁇ L/well, the color reaction was terminated, and the absorbance was measured at 450 nm, and the data was analyzed.
  • mAb001c, mAb002c, mAb005c, and mAb006c have strong affinity for AXL-ECD.
  • the specific EC 50 values are shown in Table-3.
  • the affinity of mAb002c for AXL-ECD is slightly higher than that of the control.
  • Example 5 AXL protein is highly expressed in various tumor cells
  • Mammary gland cell lines (MDA-MB-231, Hs578T, MDA-MB-453), lung cancer cell lines (NCI-H1299, Calu-1, NCI-H460), pancreatic cancer cell lines (SW1990) , Capan-2, Panc-1, Canpan-1), the total protein of the cells was prepared, and after accurate quantification, the expression level of AXL protein was detected by Western blot. The results are shown in Figure 5. The AXL protein is abnormally activated in most of the breast, lung, and pancreatic cancer cell lines tested.
  • AXL mRNA levels in tumor cell cohorts (eg, breast cancer, lung cancer, glioma, melanoma) relative to normal human tissues. This study also analyzes the expression levels of AXL mRNA in different molecular types of breast cancer (eg, luminal type versus basal type), lung cancer of different malignant degrees (eg, epithelial type versus interstitial type).
  • the results are shown in Figure 6.
  • the AXL-targeted antibody of the present invention has a remarkable effect in the diagnosis, prevention and treatment of the AXL-targeted antibody of the present invention in the diagnosis, prevention and treatment of triple-negative breast cancer, lung cancer, and glioma.
  • the results are shown in Figure 8.
  • the average AXL mRNA expression level of the high metastatic/interstitial (EMT-high) lung cancer cell line was significantly higher than that of the low metastatic/epithelial (EMT-low) lung cancer cell line, and has statistical significane.
  • EMT-high high metastatic/interstitial
  • EMT-low low metastatic/epithelial
  • the AXL-targeted antibody of the present invention has a more significant effect in the diagnosis and treatment of high metastatic, drug-resistant, advanced lung cancer.
  • the binding of the chimeric antibody mAb002c to cell surface AXL was measured. 3x10 5 tumor cells were mixed with the antibody (final concentration 5 ⁇ g/mL), then incubated at 4 ° C for 1 hour, the cells were washed twice with PBS to remove unbound primary antibody, and 200 ⁇ L (2 ⁇ g/mL) of PE labeled was added.
  • the secondary antibody was incubated at 4 ° C for 30 min, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200 ⁇ L of PBS, and the binding affinity of the test antibody to cell surface AXL was determined by flow cytometry FACSCalibur (Binding) Affinity), or overall binding fluorescence intensity (MFI) of different tumor cells at the same antibody concentration.
  • the test results are shown in Figure 9.
  • the chimeric antibody mAb002c specifically recognizes tumor cells that bind to AXL-high expression, and the fluorescence intensity of the binding rate is NCI-H1299, LCLC-103H, MDA-MB-231, Hs578T, respectively.
  • AXL-low expressed tumor cells MDA-MB-453 showed weak binding fluorescence intensity. Comparing the binding rate (MFI) of NCI-H1299 and LCLC-103H to the antibody and the binding rate of MDA-MB-453 to the antibody, the difference in the binding rate of mAb002c was 127-fold and 91-fold, respectively.
  • AXL-highly expressed triple-negative breast cancer cell MDA-MB-231 was used as a target cell, and 100 ⁇ L of a test antibody diluted from 200 nM to 0.091 nM in a 3-fold gradient was used as a primary antibody, and cultured in suspension with 100 ⁇ L of RPMI-1640 serum-free, respectively.
  • mAb001c, mAb002c, mAb005c and mAb006c have strong affinity for MDA-MB-231 cells, and the affinity of mAb002c for MDA-MB-231 cells is significantly higher. Control antibody AXL107.
  • Table-4 Binding activity of chimeric antibodies to MDA-MB-231 cells
  • AXL-highly expressed lung cancer NCI-H1299 as a target cell, 100 ⁇ L of a 3-fold gradient dilution test was used as a primary antibody and mixed with 1 ⁇ 10 5 NCI-H1299 cells suspended in 100 ⁇ L of RPMI-1640 serum-free medium. The procedure was the same as above. Finally, the cells were resuspended in 200 ⁇ L of PBS, and the binding affinity of the test antibody to cell surface AXL was determined by flow cytometry FACSAria II.
  • mAb001c, mAb002c, mAb005c, and mAb006c have strong affinity for NCI-H1299 cells.
  • the affinity of mAb002c for NCI-H1299 cells is significantly higher than that of the control antibody AXL107.
  • the humanized template matching the non-CDR region of mAb002 was searched and selected in the Germline database, and then the CDR region of the antibody was transplanted onto the selected humanized template, replacing the CDR region of the human template, and then constant with IgG1.
  • the region is recombined, and based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDR and the CDR region and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation.
  • variable regions of three humanized heavy chains SEQ ID NO.: 25, SEQ ID NO.: 26, SEQ ID NO.: 27
  • 8 humanized light chains Variable region (SEQ ID NO.: 28, SEQ ID NO.: 29, SEQ ID NO.: 30, SEQ ID NO.: 31, SEQ ID NO.: 32, SEQ ID NO.: 33, SEQ ID NO .:34, SEQ ID NO.: 35).
  • the designed humanized variable region sequence was cloned into a vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region by genetic recombination technology, and after sequencing, the transfection technique and the mammalian expression system (FreeStyleTM 293 ) were utilized.
  • the humanized antibody expression vector to be constructed. These humanized heavy and light chains were separately expressed in combination, and finally 24 humanized antibodies were obtained in the mAb002c group, and the corresponding heavy and light chain combinations of each antibody are shown in Table-6.
  • the humanized antibodies Hu002c-1 to Hu002c-24 have strong binding affinity to the AXL-ECD protein, and the EC 50 value is 0.043 nM to 0.082 nM.
  • the 24 humanized antibodies in Table-6 were serially diluted, and their affinity for surface AXL of MDA-MB-231 cells was measured by flow cytometry.
  • the experimental method is as described in Example 8.
  • the humanized antibody has high binding affinity to MDA-MB-231 cell surface AXL, and the EC 50 value is 0.073 nM to 0.17 nM, indicating affinity. Both were higher than the control AXL107 (0.43 nM).
  • the 4 humanized antibodies in Table-6 were serially diluted, and their affinity for surface AXL of LCLC-103H cells was measured by flow cytometry.
  • the experimental method is as described in Example 8.
  • the experimental results are shown in Figure 14.
  • the humanized antibodies Hu002-1, Hu002-2, Hu002-4, Hu002-5 have high binding affinity to LCLC-103H cell surface AXL, and the EC 50 values are respectively 0.28 nM, 0.37 nM, 0.49 nM, 0.36 nM indicated that the affinity was higher than the control AXL107 (0.80 nM).
  • Example 12 Humanized antibody binds to tumor cells resulting in endocytosis to intracellular lysosomes
  • AXL-highly expressed mammary gland cell line MDA-MB-231 was used as the target cell, and 50% density of MDA-MB-231 was plated in a laser confocal culture dish for 16 h, then 5 ⁇ g/mL was added (diluted in 10%).
  • AXL humanized antibody Hu002-2 of fetal bovine serum (1640 medium) was incubated at 37 ° C for 4 hours or 4 ° C for 1 hour (as a control); PBS was washed 3 times to remove antibody not bound to cells, using 4% paraformaldehyde (diluted in PBS) was fixed at room temperature for 30 min; washed three times with PBS, and permeabilized with 0.4% Triton X-100 (diluted in PBS) for 10 min at room temperature; after washing with PBS for 3 times, LAMP-2 was used.
  • the rabbit anti-human antibody was incubated at 37 ° C for 1 hour to label the position of the cell lysosome; the unbound antibody was washed with PBS, and the R-PE-labeled goat anti-human and Alexa 488 secondary antibody were incubated at 37 ° C for 30 min; The unbound secondary antibody was washed away with PBS, stained with DAPI for 10 min to locate the nucleus, and then the endocytosis of the antibody was detected using a Fluorescence microscope (Leica, 20 ⁇ ).
  • AXL-highly expressed lung cancer cell LCLC-103H was used as a target cell, and spread in a 12-well plate at 16% confluence; after adherence for 16 hours, the medium was changed to subtype control hIgG1 containing PBS and 2 ⁇ g/mL, respectively.
  • Hu002-2, or antibody-drug conjugate (ADC) Hu002-2-BL20-MMAE (prepared from Example 15 below) serum-free medium, two wells; respectively, 24 hours and 48 hours of protein sample incubation ; Western blotting was used to detect changes in the amount of AXL protein expression in the cells.
  • mc-VC-PAB-MMAE purchased from Shanghai Qianyuan Chemical, pre-dissolved in DMA
  • the antibody conjugate was named AXL107-vc-MMAE.
  • the AXL107 stock solution was replaced with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4)/150 mM sodium chloride (NaCl)/2 mM ethylenediaminetetraacetic acid (EDTA) in a pH 7.0 reaction buffer to adjust the concentration.
  • NaH2PO4-Na2HPO4 sodium dihydrogen phosphate-disodium hydrogen phosphate
  • EDTA ethylenediaminetetraacetic acid
  • the excess TCEP was removed by a G25 desalting column, and then an appropriate amount of diethyl acetamide (DMA) was added to the collected reduced antibody, and a compound 6c-4 in a molar excess ratio was added (10 mg/ml was previously dissolved in DMA). ), to ensure that the volume of DMA in the reaction system does not exceed 10%, and the coupling is carried out by stirring at 20 ° C for 2.0 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a Tris-hydrochloric acid/sucrose gel of pH 7.5, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized via a 0.22 micron pore size filtration device and stored at -80 ° C.
  • the resulting antibody conjugate was designated AXL107-BL20-MMAE.
  • 6.0 eq of mc-VC-PAB-MMAE (purchased from Shanghai Qianyuan Chemical, pre-dissolved in DMA) was directly added without purification, and reacted at 0 ° C for 1 hour, and the reaction was terminated by adding cysteine.
  • the mAb002c stock solution was replaced with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4)/150 mM sodium chloride (NaCl)/2 mM ethylenediaminetetraacetic acid (EDTA) in a pH 7.0 reaction buffer to make the concentration
  • NaH2PO4-Na2HPO4 sodium dihydrogen phosphate-disodium hydrogen phosphate
  • EDTA ethylenediaminetetraacetic acid
  • the excess TCEP was removed by a G25 desalting column, and then an appropriate amount of diethyl acetamide (DMA) was added to the collected reduced antibody, and a compound 6c-4 in a molar excess ratio was added (10 mg/ml was previously dissolved in DMA). ), to ensure that the volume of DMA in the reaction system does not exceed 10%, and the coupling is carried out by stirring at 20 ° C for 2.0 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a Tris-hydrochloric acid/sucrose gel of pH 7.5, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized via a 0.22 micron pore size filter device and stored at -80 ° C.
  • the resulting antibody conjugate was named mAb002c-BL20-MMAE.
  • the preparation method of the humanized series antibody Hu002-BL20-MMAE is the same as the preparation method of the above mAb002c-BL20-MMAE.
  • the humanized antibody Hu002-2-BL20-MMAE as an example, the results are shown in Figures 27, 28 and 29, the mass spectrum of the antibody Hu002-2 (Fig. 52) and its antibody conjugate Hu002-2-BL20-MMAE HIC and mass spectrum (Fig. 27, Fig. 28) showed that the antibody Hu002-2 was coupled to form the antibody conjugate Hu002-2-BL20-MMAE, and the molecular weight of the conjugate was consistent with the expected value. 4.0.
  • Example 16 In vitro antitumor activity of AXL chimeric antibody-drug conjugate (AXL-ADC) against triple-negative breast cancer cells, lung cancer cells and glioma cells with high expression of AXL
  • the cell lines used in this example were purchased from the American Type Culture Collection (ATCC) or the Chinese Academy of Sciences Cell Bank and cultured according to the corresponding instructions, including: MDA-MB-453, MDA-MB-231, Hs578T, Calu- 1. NCI-H1299, LCLC-103H, NCI-H292, NCI-H441, NCI-H2228, NCI-H460, U87MG.
  • Cell proliferation test The cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 600-2,500 cells per well (depending on the growth rate of different cells), 150 ⁇ L/well, set 37 After incubation for 5-12 hours at °C and 5% CO2, different concentrations of AXL-ADCs were added, and 3 replicate wells and corresponding vehicle control and blank control wells were set for each drug concentration. After 4-6 days, the culture was decanted.
  • the solution was added to the MTS reaction solution (purchased from Promega, cat#G3581) at 100 ⁇ L/well, and reacted at 37 ° C to the desired color depth and the multi-plate reader (BioTek Synergy II) was used to determine the cell viability (OD490nm) of each group.
  • survival rate (OD administration - OD blank) / (OD control - OD blank) x 100%.
  • Each proliferation assay was independently repeated 3-4 times. It said data analysis software by GraphPad Prism 5, and IC 50 value was calculated on different cell lines of drug, respectively.
  • the experimental results show that the AXL antibody-drug conjugates mAb002c-vc-MMAE and mAb002c-BL20-MMAE of the present invention have high AXL-targeting specific cytotoxic activity, ie, AXL-lowly expressed MDA-MB- 453 cells had no significant proliferation inhibition (Figure 30), whereas AXL-highly expressed triple-negative breast cancer MDA-MB-231 (Figure 31), Hs578T ( Figure 32), lung cancer Calu-1 ( Figure 33), LCLC -103H (FIG. 34) and of U87MG glioma (FIG. 35) showed strong cell proliferation inhibitory activity, IC 50 value of 0.03nM ⁇ 0.07nM (table -9).
  • the antibody-drug conjugates mAb002c-vc-MMAE and mAb002c-BL20-MMAE of the present invention have significantly stronger inhibitory activity against AXL-highly expressed tumor cells than the control antibody-drug conjugate AXL107-vc-MMAE, AXL107-BL20-MMAE.
  • the AXL humanized series antibody-drug conjugate Hu002-1/2/4/5/7/16-BL20-MMAE of the present invention has a high AXL-target specificity.
  • Cytotoxic activity ie no significant proliferation inhibition effect on AXL-lowly expressed MDA-MB-453 cells, but on AXL-high expression of triple negative breast cancer MDA-MB-231 (Fig. 36), Hs578T (Fig. 37 ), glioma U87MG (Fig. 38) and lung cancer LCLC-103H (Fig. 39) showed strong proliferation inhibitory activity with IC 50 values ranging from 0.013 nM to 0.05 nM (Table-10).
  • LCLC-103H cell suspension 200 ⁇ L of a 5x10 6 U87MG, LCLC-103H cell suspension was inoculated subcutaneously into the female immunodeficient mice (Balb/cnude, 6-8 weeks old).
  • the dose of 0.5 mg/kg was administered once a week in the tail vein for a total of 2 weeks; hIgG1-BL20-MMAE was also set as a negative control. Tumor volume and nude mouse body weight were measured 2-3 times per week and recorded to plot tumor growth curves.
  • the chimeric antibody mAb002c, four preferred humanized antibodies Hu002-1, Hu002-2, Hu002-4, Hu002-5 were coupled to vc-MMAE or BL20-MMAE, respectively, while the prior art AXL107-vc-MMAE was used.
  • AXL107-vc-MMAE was used for comparison, in vivo antitumor activity evaluation was performed.
  • the preparation of the humanized AXL-ADC is described in Example 15.
  • the anti-tumor effect of mAb002c-BL20-MMAE or mAb002c-vc-MMAE at a dose of 5 mg/kg in the U87MG tumor model was very significant and similar during the administration, but mAb002c-vc was stopped 3 weeks later.
  • the tumor-recovery growth of the MMAE group has been shown to be significantly lower than that of the mAb002c-BL20-MMAE group, indicating that the BL20-MMAE linker is more advantageous in vivo.
  • the four preferred AXL-ADCs of the present invention showed dose-related therapeutic effects at doses of 3 mg/kg and 1 mg/kg, and can cause And its significant tumor regression, indicating that LCLC-103H tumors are highly sensitive to the treatment of AXL-ADC.
  • the LCLC-103H tumor was grown in vivo to a dose of 800 mg 3 at a dose of 5 mg/kg of Hu002-2-BL20-MMAE, and the tumor was completely regressed on the 34th day of treatment.
  • LCLC-103H tumors were grown in vivo to a dose of 10 mg/kg of Hu002-2-BL20-MMAE at a dose of 1800 mm 3 , and the tumor regression rate was observed on the 20th day of treatment. 90%.
  • the full-length gene sequence of the artificial cynomolgus monkey and mouse is constructed into the mammalian expression vector pcDNA3.1 to prepare a positive expression vector plasmid;
  • HEK293T cells spread them at 50% density in culture dishes; confluence is about 80% after overnight incubation at 37 °C, and transiently transfect 2 ⁇ g of monkey/mouse prepared above using liposome Lipo2000 (Invitrogen) -AXL vector plasmid; proteins were collected for Western blot analysis 24 hours after transfection, and cells were harvested for FACS detection of binding activity of Hu002-2 to HEK293T cell surface monkey/mouse AXL. See Example 8 for the FACS test method.
  • the AXL humanized antibody Hu002-2 was similar to the control antibody AXL107, and had poor binding ability to the mouse AXL on the surface of HEK-293T, and did not reach the binding saturation platform at the highest antibody concentration (200 nM). .
  • the heavy chain and light chain variable region sequences (VH/VL) of the AXL107 antibody disclosed in the invention patent application No. CN201580045131 artificially synthesized the heavy chain and light chain variable regions thereof and cloned into the heavy chain constant region containing human IgG1 and Kappa chain constant region vector, expressing the sequence in the correct FreeStyle TM 293T cells and purified to give AXL107 system (Example 3), then turn it into a preparation of antibody AXL107 - drug conjugate (Example 14).
  • AXL107, AXL107-vc-MMAE, AXL107-BL20-MMAE were added as reference drugs to the study of the present invention.
  • VH AXL107-heavy chain variable region (VH) SEQ ID NO.: 37
  • VL AXL107-light chain variable region (VL) SEQ ID NO.: 38
  • mAb002c-BL20-MMAE has higher material uniformity than the prior art AXL107-vc-MMAE (Fig. 17, Fig. 20), and the ratio of single distributed component (DAR4) More than 90%;

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Abstract

本发明公开了一种全新的靶向AXL的单克隆抗体以及抗体-药物偶联物。本发明还公开了制备所述抗体、抗体-药物偶联物的方法。本发明的AXL抗体能够高效，高特异性地结合纯化的人AXL蛋白和多种肿瘤细胞表面的AXL，所述人源化抗体同样具有很高的亲和力及很低的免疫原性，所述AXL抗体-药物偶联物对AXL高表达的肿瘤具有显著的抗肿瘤作用。

Description

靶向AXL的抗体及抗体-药物偶联物及其制备方法和用途 技术领域
本发明涉及药物领域，更具体地，涉及靶向于AXL的抗体及抗体药物偶联物(ADC)、其制备方法和用途。
背景技术
AXL是受体酪氨酸激酶亚家族-TAM家族成员之一，TAM家族包括Tyro-3、Axl和Mer，它们的配体均为生长停滞特异性基因6(Gas6)所编码的蛋白分子。AXL与Gas6结合后被激活，从而活化其下游如PI3K/AKT，RAS/ERK和β-Catenin/TCF等信号转导通路，从而调节细胞的增殖、凋亡、趋化、黏附和识别等多种生理功能。
研究发现AXL在多种癌症中呈激活表达状态，如肺癌、乳腺癌、前列腺癌、甲状腺癌、子宫内膜癌、卵巢癌和肾癌等多种肿瘤组织中，参与肿瘤细胞上皮间质转化、血管生成、凋亡和免疫调节等多种机制，并与不良预后(Cancer Cell 2015,27:533-46)和许多情况下的耐药(Oncotarget 2015,6:15321-31；Cancer Res.2013,19:279-90)相关，包括EGFR抑制剂难治的肺癌(Nat.Genet.2012,44:852-60)、PI3K抑制剂耐药的头颈癌(Cancer Cell 2015,27:533-46)、抗HER2耐药的乳腺癌(Biochem Soc Trans.2014,42:822-30)，舒尼替尼耐药的肾癌(Oncogene 2016,35:2687-97)和ALK抑制剂耐药的神经母细胞瘤(Oncogene 2016,35:3681-91)。此外，AXL的表达与传统化疗和放疗的获得性耐药相关(Theranostics 2016,6:1205-19)。抑制AXL后耐药细胞对细胞毒性药物和靶向抑制剂的敏感性增强(Nat.Commun.2016,7:13898)。
鉴于AXL在肿瘤靶向治疗中的重要性和作为潜在的药物靶标，期望研究开发更多具有良好特性的特异性结合AXL的抗体。
本发明人前期研究也发现与正常组织相比，AXL在肿瘤组织中异常激活表达，尤其是在高侵袭、高转移的基底样和/或三阴性乳腺癌、转移性肺癌、胰腺癌等，并且与其他靶点相比，靶向AXL的抗体可被快速大量的内化，可见AXL可能是一个更为优选的抗体-药物偶联物(antibody-drug conjugate，ADC)开发靶点。然而，目前国际上，尤其国内尚缺乏高特异性的针对人AXL的抗体药物偶联物。
抗体药物偶联物一般由三部分组成：抗体或抗体类配体，小分子药物，和将配体与药物偶联起来的连接子。目前进入临床试验的抗体药物偶联物结构中，高活性的细胞毒性药物通常是通过连接子连接在配体表面的赖氨酸残基，或者抗体铰链区域的半胱氨酸残基(由链间二硫键部分还原得到)上，最佳的药物/配体比值(DAR)为2-4。抗体表面大量的赖氨酸残基(超过80个)以及偶联反应的非选择性，导致偶联数目和位点的不确定性，进而导致生成的抗体药物偶联物的不均一性。根马布公司报导了一类靶向AXL的抗体偶联物(CN201580045131.4)，也是基于传统偶联技术的抗体药物偶联物。
此外，抗体药物偶联物的作用机制看似简单，但一个抗体药物偶联物是否能成为安全有效的药物是非常复杂和不可预测的，依赖于多种因素，例如：
1)靶点的特性：靶点是否能有效内吞，靶点的表达水平，靶点是否在癌细胞和正常细胞中有足够的表达水平差异，靶点是否会被分泌或脱落至细胞外部分而到血液中。
2)单抗的特性：单抗对于靶点的特异性是否足够好(与其它蛋白无交叉反应)，单抗稳定性和与靶点结合后内吞速率和程度等。
3)接头的特性：接头需要在血液中足够稳定，同时接头的变化对于ADC上链接的药物数量和区域位置均会有改变，最终将导致整个ADC药物的安性性和有效性的变化。
可以看出，ADC药物的研发需要大量的实验摸索和验证，其安全性和有效性是无法在实验前预测的。
综上，本领域迫切需要开发亲和力高、免疫原性低、稳定性好的靶向AXL的抗体及抗体药物偶联物。
发明内容
本发明提供了一种靶向人源AXL的抗体，其具有阻断AXL的生物活性，具有抑制肿瘤生长和转移活性，并可以减少抗肿瘤治疗耐药性的出现。
本发明还提供了靶向AXL的抗体药物偶联物，所述偶联物对AXL高表达的肿瘤细胞具有显著抗肿瘤作用。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:1所示的CDR1，
SEQ ID NO.:2所示的CDR2，和
SEQ ID NO.:3所示的CDR3；
或者，
SEQ ID NO.:9所示的CDR1，
SEQ ID NO.:10所示的CDR2，和
SEQ ID NO.:11所示的CDR3；
或者，
SEQ ID NO.:17的CDR1，
SEQ ID NO.:18所示的CDR2，和
SEQ ID NO.:19所示的CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留AXL结合亲和力的衍生序列。
在另一优选例中，所述重链可变区包括以下互补决定区：
SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:3所示mAb002c的重链互补决定区HCDR1、HCDR2、HCDR3；或
SEQ ID NO.:9、SEQ ID NO.:10、SEQ ID NO.:11所示mAb005c的重链互补决定区HCDR1、HCDR2、HCDR3；或
SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:19所示mAb001c的重链互补决定区HCDR1、HCDR2、HCDR3。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区具有SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:15所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:23所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.:25、SEQ ID NO.:26、SEQ ID NO.:27所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区包括以下三个互补决定区CDR：
或者，
SEQ ID NO.:4所示的CDR1'，
SEQ ID NO.:5所示的CDR2'，和
SEQ ID NO.:6所示的CDR3'；
或者，
SEQ ID NO.:12所示的CDR1'，
SEQ ID NO.:13所示的CDR2'，和
SEQ ID NO.:14所示的CDR3'；
或者，
SEQ ID NO.:20所示的CDR1'，
SEQ ID NO.:21所示的CDR2'，和
SEQ ID NO.:22所示的CDR3'；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留AXL结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区包括以下互补决定区：
SEQ ID NO.:4、SEQ ID NO.:5、SEQ ID NO.:6所示mAb002c的轻链互补决定区LCDR1、LCDR2、LCDR3；或
SEQ ID NO.:12、SEQ ID NO.:13、SEQ ID NO.:14所示mAb005c的轻链互补决定区LCDR1、LCDR2、LCDR3；或
SEQ ID NO.:20、SEQ ID NO.:21、SEQ ID NO.:22所示mAb001c的轻链互补决定区LCDR1、LCDR2、LCDR3。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:16所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:24所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30、SEQ ID NO.:31所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有本发明第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)本发明第一方面所述的重链可变区；和/或
(2)本发明第三方面所述的轻链可变区；
或者，所述抗体具有：本发明第二方面所述的重链；和/或本发明第四方面所述的轻链。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述人源化抗体的CDR区包含1、2、或3个氨基酸的变化。
在另一优选例中，所述的动物为非人哺乳动物，较佳地为鼠、羊、兔。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量，不超过初始氨基酸序列总氨基酸数量的40％，较佳地为20％，更佳地为10％。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个，较佳地为1-3个，更佳地为1个。
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸的衍生序列具有抑制细胞表面AXL或重组AXL蛋白的功能。
在另一优选例中，所述的抗体为药物偶联物形式。
在另一优选例中，所述抗体对AXL(如人AXL蛋白胞外区,AXL-ECD)的亲和力EC 50为0.04～0.5nM，较佳地为0.04～0.1nM，更佳地为0.04～0.05nM。
在另一优选例中，所述抗体对肿瘤细胞表面AXL的亲和力EC 50为0.1～1.5nM，较佳地为0.1～1nM，更佳地为0.1～0.2nM。
在另一优选例中，所述抗体-药物偶联物(AXL-ADC)对AXL高表达的肿瘤细胞的毒性作用IC 50为0.01～1nM，较佳地为0.01～0.1nM,更佳地为0.01～0.05nM。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
本发明的第七方面，提供了一种CAR构建物，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于AXL的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
本发明的第八方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第七方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第九方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019086475-appb-000001
其中：
Ab是抗ALX的抗体，
LU是接头；
D是药物；
而且下标p是选自1-10，较佳地1-8的值。
在另一优选例中，所述的偶联部分(D)为细胞毒性药物，并且所述的细胞毒性药物为：微管靶向药物和/或DNA靶向药物和/或拓扑异构酶抑制剂。。
在另一优选例中，所述的微管靶向药物选自下组：单甲基澳瑞他汀E(MMAE)、单甲基澳瑞他汀F(MMAF)、美登素衍生物DM1和tubulysin。
在另一优选例中，所述的DNA靶向药物选自下组：多卡霉素、吡咯并[2,1-c][1,4]苯二氮卓(PBD)。
在另一优选例中，所述的拓扑异构酶抑制剂选自下组：7-乙基-10-羟基喜树碱(SN38)、依沙替康(Exatecan)及其类似物。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或连接子进行偶联。
在另一优选例中，所述的连接子(LU)选自下组：4-(N-马来酰亚胺基甲基)环己烷-1-甲酸琥酸亚胺酯(MCC)、马亚酰亚胺基己酰基(MC)、6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧基羰基(mc-val-cit-PAB)、CL2A(US20140170063,CN201480041766.2)和双取代马来酰亚胺类连接子(CN201611093699.6，CN201711169847.2)。
在另一优选例中，所述抗体药物偶联物对AXL高表达的肿瘤细胞的毒性作用IC 50为0.01～1nM，较佳地为0.01～0.1nM,更佳地为0.01～0.05nM。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗 体药物偶联物、或其组合，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗AXL相关疾病的药物。
在另一优选例中，所述检测试剂、检测板或试剂盒用于：
(1)检测样品中的AXL蛋白；和/或
(2)检测肿瘤细胞中内源性的AXL蛋白；和/或
(3)检测表达AXL蛋白的肿瘤细胞。
在另一优选例中，所述的检测试剂、检测板或试剂盒用于诊断AXL相关疾病。
在另一优选例中，所述的药物用于治疗或预防AXL高表达的肿瘤、肿瘤迁移、或肿瘤耐药。
在另一优选例中，所述的肿瘤耐药包括：肿瘤免疫治疗药物的耐药、肿瘤靶向治疗药物的耐药、常规肿瘤化疗的耐药，放射治疗的不敏感。
在另一优选例中，所述的药物用于选自下组的用途：
(a)特异结合肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的AXL；
(b)抑制肿瘤/肿瘤微环境中过度活化的AXL生物功能；
(c)抑制肿瘤细胞迁移或转移；
(d)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(e)抗体依赖的细胞介导的细胞毒性作用(ADCC)。
在另一优选例中，所述AXL相关疾病选自下组：癌症、自身免疫疾病、代谢相关疾病、感染疾病、或其组合。
在另一优选例中，所述的癌症包括实体瘤、血液癌。
在另一优选例中，所述的癌症为AXL高表达的肿瘤。
在另一优选例中，所述的AXL高表达的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、卵巢癌、前列腺癌癌、直肠癌、脑胶质瘤、黑色素瘤、白血病、淋巴瘤、或其组合。
在另一优选例中，所述的癌症为耐药性肿瘤。
在另一优选例中，所述的AXL高表达的肿瘤指肿瘤组织中AXL转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
在另一优选例中，所述代谢相关疾病包括：糖尿病、食源性肥胖和脂肪炎症。
在另一优选例中，所述感染疾病包括：细菌和病毒感染。
本发明的第十一方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)本发明第六方面所述的重组蛋白；
(3)本发明第七方面所述的CAR构建物。
本发明的第十三方面，提供了一种载体，所述的载体含有本发明第十二方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有本发明第十三方面所述的载体或基因组中整合有本发明第十二方面所述的多核苷酸。
本发明的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中AXL的方法，所述方法包括步骤：
(1)在体外，将所述样品与本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在AXL。
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有本发明第五方面所述的抗体或本发明第九方面所述的免疫偶联物。
本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有本发明第十六方面所述的检测板。
本发明的第十八方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是本发明第五方面所述的抗体或本发明第六方面所述的重组蛋白。
本发明的第十九方面，提供了一种治疗AXL相关疾病的方法，所述方法包括：给需要的对象施用本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在另一优选例中，所述的方法还包括：给需要的对象施用其他药物或治疗方法进行联合治疗。
在另一优选例中，所述的其他药物或治疗方法包括：抗肿瘤免疫治疗药物、肿瘤靶向药物、肿瘤化疗药物、肿瘤放射治疗。
在另一优选例中，所述的抗肿瘤免疫治疗药物包括PD-1、PD-L1单抗。
在本发明的第二十方面，提供了一种制备嵌合抗体的方法，包括步骤：
将本发明第一方面所述的重链可变区和/或本发明第三方面所述的轻链可变区的核苷酸序列克隆入含有人抗体恒定区的核苷酸序列的表达载体后，通过转染动物细胞表达人-鼠嵌合抗体。
在本发明的第二十一方面，提供了一种制备人源化抗体的方法，包括步骤：
将本发明第一方面所述的重链可变区和/或本发明第三方面所述的轻链可变区中的CDR区的核苷酸序列植入含人源抗体FR区的核苷酸序列模板，再将其克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。
在本发明的第二十二方面，提供了一种抑制肿瘤细胞生长和迁移的方法，包括步骤：给需要的对象施用本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在本发明的第二十三方面，提供了一种抑制肿瘤在模型动物体内生长的方法，包括步骤：给需要的对象施用本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞。
在另一优选例中，所述药物可以实施单独给药，或组合用药包括肿瘤免疫疗法、肿瘤靶向药物、细胞毒性药物、放射治疗。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一赘述。
附图说明
图1为本发明的抗人AXL抗体的发现。图1A为流式细胞荧光分选仪(FACS)检测原始发现的抗人AXL单克隆抗体(original hybridoma)培养上清对人源AXL-高表达的MDA-MB-231(AXL-P)、AXL-低表达的MDA-MB-453(AXL-N)乳腺癌细胞的的结合活性。图1B为6个单克隆抗体的编号(mAb001、mAb002、mAb003、mAb004、mAb005、mAb006)以及对纯化后抗体的亚型鉴定。
图2为PCR扩增mAb001、mAb002、mAb005、mAb006重链可变区(VH)、轻链可变区(VL)片段琼脂糖凝胶电泳结果图。VH/VL片段测序鉴定后用于克隆和组装人-鼠嵌合抗体表达载体。
图3为HEK293T细胞表达的4个人-鼠嵌合抗体(chimeric antibody)mAb001c、mAb002c、mAb005c、mAb006c，然后采用MabSelect TMSuRe TM柱子的纯化图谱。
图4为ELISA测定人-鼠嵌合抗体mAb001c、mAb002c、mAb005c、mAb006c对AXL-ECD的结合亲和力(Binding affinity EC 50)。
图5为免疫印迹实验(Western blot)检测所示乳腺癌(MDA231、Hs587T、MDA453),肺癌(NCI-H1299、Calu-1、NCI-H460)，胰腺癌(SW1990、Capan-2、Panc-1、Capan-2)细胞株中AXL蛋白的表达水平。
图6为分析对比多个肿瘤细胞株(乳腺癌、肺癌、脑胶质瘤、黑色素瘤)与人正常组织中AXLmRNA的表达水平(与-actin的比值)。
图7为分析51株人乳腺癌细胞株基因表达数据库(Neve RM et al.,Cancer Cell 2006；10:515-27)中AXL mRNA在高侵袭、高转移的基底型(Basal-type)对比管腔型(Luminal-type)乳腺癌细胞株群中的表达水平。
图8为分析CCLE数据库中上皮型相对于间质型肺癌细胞株中AXLmRNA的表达水平。
图9为mAb002c(5g/mL)针对AXL-高表达(NCI-H1299、LCLC-103H、CaLu-1、MDA-MB-231、Hs578T)或低表达(MDA-MB-453)肿瘤细胞表面AXL的结合水平。
图10为嵌合抗体mAb001c、mAb002c、mAb005c、mAb006c对MDA-MB-231细胞表面AXL的 结合亲和力(Binding affinity EC 50)检测结果，采用1x10 5细胞与所示浓度梯度的抗体混合，孵育1小时后用流式细胞仪(FACSCalibur)进行FACS检测分析。
图11为嵌合抗体mAb001c、mAb002c、mAb005c对NCI-H1299细胞表面AXL的结合亲和力(Binding affinity EC 50)检测结果，采用1x10 5细胞与所示浓度梯度的抗体混合，孵育1小时后小时后用流式细胞仪(FACSAria II)进行FACS检测分析。
图12为ELISA测定人源化抗体系列Hu002-1～Hu002-24对AXL-ECD的结合亲和力(Binding affinity EC 50)。
图13为人源化抗体系列Hu002-1～Hu002-24对MDA-MB-231细胞表面AXL的结合亲和力(Binding affinity EC 50)检测结果，采用1x10 5细胞与所示浓度梯度的抗体混合，孵育1小时后用流式细胞仪(FACSCalibur)进行FACS检测分析。
图14为人源化抗体系列Hu002-1～Hu002-5对LCLC-103H细胞表面AXL的结合亲和力(Binding affinity EC 50)检测结果，采用1x10 5细胞与所示浓度梯度的抗体混合，孵育1小时后用流式细胞仪进行FACS检测分析。
图15为Hu002-2与MDA-MB-231细胞结合导致内吞(Internalization)至细胞内溶酶体。所述抗体(5g/mL)与细胞孵育4℃ 1小时，或37℃ 4小时后置于激光共聚焦显微镜观察结果
图16为LCLC-103H细胞经Hu002-2或Hu002-2-BL20-MMAE处理24或48小时后，采用免疫印迹(Western blot)检测其对AXL蛋白表达的抑制作用。
图17为抗体药物偶联物AXL107-vc-MMAE的疏水作用层析(HIC)图谱。
图18为抗体药物偶联物AXL107-BL20-MMAE的疏水作用层析(HIC)图谱。
图19为单克隆抗体AXL107的质谱图谱。
图20为抗体药物偶联物AXL107-vc-MMAE的质谱图谱。
图21为抗体药物偶联物AXL107-BL20-MMAE的质谱图谱。
图22为抗体药物偶联物mAb002c-vc-MMAE的疏水作用层析(HIC)图谱。
图23为抗体药物偶联物mAb002c-BL20-MMAE的疏水作用层析(HIC)图谱。
图24为单克隆抗体mAb002c的质谱图谱。
图25为抗体药物偶联物mAb002c-vc-MMAE的质谱图谱。
图26为抗体药物偶联物mAb002c-BL20-MMAE的质谱图谱。
图27为抗体药物偶联物Hu002-2-BL20-MMAE的疏水作用层析(HIC)图谱。
图28为抗体药物偶联物Hu002-2-BL20-MMAE的质谱图谱。
图29为人源化单克隆抗体Hu002-2的质谱图谱。
图30为mAb002c-ADC、AXL107-ADC对MDA-MB-453细胞体外增殖的抑制活性(IC 50)的检测结果。
图31为mAb002c-ADC、AXL107-ADC对MDA-MB-231细胞体外增殖的抑制活性(IC 50)的检测结果。
图32为mAb002c-ADC、AXL107-ADC对Hs578T细胞体外增殖的抑制活性(IC 50)的检测结果。
图33为mAb002c-ADC、AXL107-ADC对Calu-1细胞体外增殖的抑制活性(IC 50)的检测结果。
图34为mAb002c-ADC、AXL107-ADC对LCLC-103H细胞体外增殖的抑制活性(IC 50)的检测结果。
图35为mAb002c-ADC、AXL107-ADC对U87MG细胞体外增殖的抑制活性(IC 50)的检测结果。
图36为人源化Hu002系列抗体偶联BL20-MMAE的ADC对MDA-MB-231细胞体外增殖的抑制 活性(IC 50)的检测结果。
图37为人源化Hu002系列抗体偶联BL20-MMAE的ADC对Hs578T细胞体外增殖的抑制活性(IC 50)的检测结果。
图38为人源化Hu002系列抗体偶联BL20-MMAE的ADC对U87MG细胞体外增殖的抑制活性(IC 50)的检测结果。
图39为人源化Hu002系列抗体偶联BL20-MMAE的ADC对LCLC-103H细胞体外增殖的抑制活性(IC 50)的检测结果。
图40为嵌合抗体mAb002c抗体分别偶联vc-MMAE、BL20-MMAE的ADC(均为5mg/kg)在U87MG脑胶质瘤模型中的体内抗肿瘤药效，结果显示BL20-MMAE相比vc-MMAE具有更优异的体内治疗效果。
图41为人源化Hu002-1、Hu002-4抗体偶联BL20-MMAE(3mg/kg)、AXL107-vc-MMAE(3mg/kg)在U87MG脑胶质瘤模型中的体内抗肿瘤药效。
图42为人源化Hu002-2、Hu002-5抗体分别偶联BL20-MMAE的ADC(3mg/kg；每周1次共2次)在U87MG脑胶质瘤模型中的体内抗肿瘤药效结果。
图43为人源化Hu002-1、Hu002-4抗体分别偶联BL20-MMAE的ADC(3mg/kg、1mg/kg；每周1次共2次)在LCLC-103H肺癌模型中的体内抗肿瘤药效结果。
图44为人源化Hu002-2、Hu002-5抗体分别偶联BL20-MMAE的ADC(3mg/kg、1mg/kg；每周1次共2次)在LCLC-103H肺癌模型中的体内抗肿瘤药效结果。
图45为人源化Hu002-1、Hu002-2、Hu002-5抗体分别偶联BL20-MMAE的ADC(均为1mg/kg；每周1次共2次)在LCLC-103H肺癌模型中的体内抗肿瘤药效结果。
图46为人源化Hu002-2、Hu002-5抗体分别偶联BL20-MMAE的ADC(1mg/kg、0.5mg/kg)和AXL107-vc-MMAE(1mg/kg)每周1次共2次给药后在LCLC-103H肺癌模型中的体内抗肿瘤药效结果。
图47为人源化Hu002-2-BL20-MMAE(5mg/kg；单次给药)针对LCLC-103H大体积肿瘤(800mm 3为起始给药时体积)能导致肿瘤消退的结果。
图48为人源化Hu002-2-BL20-MMAE(10mg/kg；单次给药)针对LCLC-103H大体积肿瘤(1800mm 3为起始给药时体积)能导致肿瘤消退的结果。
图49为Hu002-2对HEK293T瞬转表达鼠源AXL蛋白后的FACS结合活性的检测；相比人来源的AXL，Hu002-2或AXL107对鼠源AXL显示出很微弱的结合活性。
图50为Hu002-2对食蟹猴AXL的结合亲和力。图50A为免疫印迹实验(Western blot)检测HEK293T瞬转猴AXL载体24小时后的蛋白表达水平；图50B为HEK293T瞬转表达猴AXL24小时后收取细胞，采用FACS检测其结合Hu002-2的结合亲和力(Binding affinity EC 50)。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得了6个抗AXL单克隆抗体，分别命名为mAb001～mAb006。依据活性测试结果，选择mAb001(IgG1-κ)、mAb002(IgG1-κ)、mAb005(IgG1-κ)、mAb006(IgG2b-κ)构建人-鼠嵌合抗体，将其分别命名为mAb001c、mAb002c、mAb005c、mAb006c。进一步测试上述抗体后获得结果如下：
第一、所述的嵌合抗体均能够高特异性地结合AXL抗原，ELISA测定其EC 50分别为0.092nM、0.073nM、0.103nM、0.101nM；
第二、所述的嵌合抗体针对多株AXL高表达的肿瘤细胞具有极高的结合亲和力，FACS测定其EC 50为0.174nM～1.5nM，基因测序显示mAb006c与mAb005c的互补决定区(CDR)高度重叠，故终止了对mAb006c的后续研究；
第三、基于mAb002c设计的一系列人源化抗体具有更高的AXL蛋白结合亲和力及细胞结 合亲和力；ELISA测定其EC 50为0.045nM～0.08nM；FACS测定其EC 50为0.09nM～0.14nM。
第四、所述抗体的药物偶联物(ADC)具有优异的特性，即对AXL-正常表达的细胞无明显毒副作用，而对AXL-高表达的肿瘤细胞具有极高的杀伤活性，细胞增殖抑制IC 50值为0.01nM～0.07nM；
第五、通过本发明的新型连接子得到的AXL-ADC产品具有均一性高、进一步提高体外和体内稳定性的优势。
第六、本发明中优选的抗体和抗体-药物偶联物相比现有技术具有更优异、更持续的体内抗肿瘤治疗效果。
在此基础上完成了本发明。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不 同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗AXL的抗体
本发明提供3大类靶向AXL的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)氨基酸序列、轻链可变区(VL)氨基酸序列包含具有以下多肽序列的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
a1)HCDR1为SEQ ID NO.:1：DFYIN，SEQ ID NO.:9:SYYIH，或SEQ ID NO.:17：SGYWS；
a2)HCDR2为SEQ ID NO.:2：WIYPGSGNTKYNEKFKG，SEQ ID NO.:10:WIYPGSDNTKYNEKFKD，或SEQ ID NO.:18：YMTYSGATYYNPSLKS；
a3)HCDR3为SEQ ID NO.:3：STGFFDY，SEQ ID NO.:11:NYYDYDGGTWFPY，或SEQ ID NO.:19：GGNSYFFDY；
a4)LCDR1为SEQ ID NO.:4：SASSSIGYMY，SEQ ID NO:12:RASQDINYYLN，或SEQ ID NO.:20：RASENIYSNLA；
a5)LCDR2为SEQ ID NO.:5：LTSNLAS，SEQ ID NO.:13：YTSRLHS，或SEQ ID NO.:21：AATNLAD；
a6)LCDR3为SEQ ID NO.:6：QQWSSNPPT；SEQ ID NO.:14：QQGNTLPWT，或SEQ ID NO.:22：QHFWGTPLT；
a7)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有AXL结合亲和力的序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
优选地，所述的抗体具有抑制细胞表面及重组AXL蛋白酶功能，所述抗体能快速被细胞内吞进入溶酶体。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人-动物嵌合抗体、优选为人源化抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人AXL的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID NO.:1～3、SEQ ID NO.:9～11、SEQ ID NO.:17～19中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有AXL结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID NO.:4～6、SEQ ID NO.:12～14、SEQ ID NO.:20～22中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有CD73结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:1、SEQ ID NO.:2、SEQ ID NO.:3，或选自SEQ ID NO.:9、SEQ ID NO.:10、SEQ ID NO.:11，或选自SEQ ID NO.:17、SEQ ID NO.:18、SEQ ID NO.:19中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有AXL结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:4、SEQ ID NO.:5、SEQ ID NO.:6，或选自SEQ ID NO.:12、SEQ ID NO.:13、SEQ ID NO.:14，或选自SEQ ID NO.:20、SEQ ID NO.:21、SEQ ID NO.:22中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有AXL结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述的抗体为原始的鼠源抗体mAb001、mAb002、mAb003、mAb004、mAb005、mAb006。
在另一优选例中，所述的抗体为人-鼠嵌合抗体mAb001c、mAb002c、mAb005c、mAb006c。
在另一优选例中，所述的抗体为人源化抗体Hu002c-1、Hu002c-2、Hu002c-3、 Hu002c-4、Hu002c-5、Hu002c-6、Hu002c-7、Hu002c-8、Hu002c-9、Hu002c-10、Hu002c-11、Hu002c-12、Hu002c-13、Hu002c-14、Hu002c-15、Hu002c-16、Hu002c-17、Hu002c-18、Hu002c-19、Hu002c-20、Hu002c-21、Hu002c-22、Hu002c-23、Hu002c-24。
本发明的3大类抗体可以联合应用，用于构建CAR构建物、包含CAR构建物的重组的免疫细胞、抗体药物偶联物等用途，还可以用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗AXL相关疾病的药物。
本发明序列表中涉及的各序列的代表含义如下表B所示。
表B
序列编号 序列名称 序列编号 序列名称
SEQ ID NO.:1 mAb002HCDR1 SEQ ID NO.:21 mAb001LCDR2
SEQ ID NO.:2 mAb002HCDR2 SEQ ID NO.:22 mAb001LCDR3
SEQ ID NO.:3 mAb002HCDR3 SEQ ID NO.:23 mAb001-VH
SEQ ID NO.:4 mAb002LCDR1 SEQ ID NO.:24 mAb001-VL
SEQ ID NO.:5 mAb002LCDR2 SEQ ID NO.:25 mAb002-VH_HuG0
SEQ ID NO.:6 mAb002LCDR3 SEQ ID NO.:26 mAb002-VH_HuG1
SEQ ID NO.:7 mAb002-VH SEQ ID NO.:27 mAb002-VH_HuG2
SEQ ID NO.:8 mAb002-VL SEQ ID NO.:28 mAb002-VK_HuG0
SEQ ID NO.:9 mAb005HCDR1 SEQ ID NO.:29 mAb002-VK_HuG1
SEQ ID NO.:10 mAb005HCDR2 SEQ ID NO.:30 mAb002-VK_HuG2
SEQ ID NO.:11 mAb005HCDR3 SEQ ID NO.:31 mAb002-VK_HuG3
SEQ ID NO.:12 mAb005LCDR1 SEQ ID NO.:32 mAb002-VK_HuG4
SEQ ID NO.:13 mAb005LCDR2 SEQ ID NO.:33 mAb002-VK_HuG5
SEQ ID NO.:14 mAb005LCDR3 SEQ ID NO.:34 mAb002-VK_HuG6
SEQ ID NO.:15 mAb005-VH SEQ ID NO.:35 mAb001-VK_HuG7
SEQ ID NO.:16 mAb005-VL SEQ ID NO.:36 人AXL蛋白的胞外区
SEQ ID NO.:17 mAb001HCDR1 SEQ ID NO.:37 AXL107-VH
SEQ ID NO.:18 mAb001HCDR2 SEQ ID NO.:38 AXL107-VL
SEQ ID NO.:19 mAb001HCDR3 SEQ ID NO.:39 猴AXL蛋白序列
SEQ ID NO.:20 mAb001LCDR1 SEQ ID NO.:40 小鼠AXL蛋白序列
本发明中还涉及了猴AXL蛋白序列和小鼠AXL蛋白序列，Genebank ID分别是XP_014979499.1(猴)和Genebank ID:NP_033491.2(小鼠)。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文 库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物、检测试剂、稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合 适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)、7-乙基-10-羟基喜树碱(SN38)、依沙替康(Exatecan)及其类似物等。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗 体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019086475-appb-000002
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
AXL抗体-药物偶联物
本发明涉及抗体-药物偶联物，更具体地说，本发明涉及具有治疗应用的AXL抗体-药物偶联物。可以通过特定连接子将抗AXL抗体与化疗药或者小分子毒素偶联。本发明还涉及及使用抗AXL抗体-药物偶联物治疗哺乳动物细胞或相关病理性情况的方法。
由于抗体表面大量的赖氨酸残基(超过80个)以及偶联反应的非选择性，导致偶联数目和位点的不确定性，进而导致生成的抗体药物偶联物的不均一性。例如，T-DM1(平均DAR值为3.5)的DAR值分布为0-8。同样，尽管抗体铰链区的链间二硫键只有四对，但为达到最佳平均DAR值(2-4)的要求，需要部分还原链间二硫键。由于现有的还原剂(DTT,TCEP等)无法选择性地还原链间二硫键，因此生成的偶联物也不是均一的产物，由多种组分组成，其主要组分的DAR值为0,2,4,6,8，而且对应每一种特定DAR值的组分都存在由于连接位点不同而形成的异构体。抗体药物偶联物产品的不均一性可以导致各成员组分间药物动力学性质，效价以及毒性的不均一性。例如，具有较高DAR值的组分在体内被清除得更快，并导致更高的毒性。
针对以上偶联技术存在的问题，通过简单的化学方法对现有抗体实现定点偶联目的，会节省大量的人力物力财力，因此更加富有吸引力。其中已有相关的研究包括：波利泰里克斯有限公司申请的CN200480019814.4；Igenica Biotherapeutics公司申请的WO2014197871A2；索伦托医疗有限公司申请的CN201380025774.3；上海新理念生物医药科技有限公司申请的CN201310025021.4等。然而上述技术存在偶联试剂合成路线较长、偶联试剂化学稳定性不佳、抗体偶联物电泳图较为杂乱，提示偶联过程中可能存在副反应、现有方案并未解决体内循环过程中的巯基交换(逆迈克尔加成反应)等问题。
根马布公司报导了一类靶向AXL的抗体偶联物(CN201580045131.4)，也是基于传统偶联技术的抗体药物偶联物。
针对以上偶联技术存在的问题，通过简单的化学方法对靶向AXL的抗体-药物偶联物实现定点偶联，可提高药物的均一性，在工艺与质量控制方面节省了大量的人力物力财力，同时还可能提高偶联物的稳定性、药效与安全性等成药性质。
本发明采用一类新型连接子结构(发明人前期所开发的一类新型双取代马来酰亚胺类连接子CN201611093699.6，CN201711169847.2)，并应用于靶向AXL抗体进行偶联，该连 接子可以全部/部分交叉偶联抗体的轻链-重链及重链-重链二硫键还原的半胱氨酸巯基上，且应用此种偶联方法得到的靶向AXL抗体药物偶联物，与传统抗体药物偶联物相比，具有更窄的药物/抗体比值(DAR)分布。具有该双取代马来酰亚胺类连接子的AXL抗体-药物偶联物的结构如式Ia、Ib所示：
Figure PCTCN2019086475-appb-000003
其中，
Ar'选自下组：取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；
L 1为连接于Ar'基团上的-O(CH 2CH 2O) n-，其中n选自1-20中任一整数。
L 2为化学键，或AA-PAB结构；其中，AA为2-4个氨基酸组成的多肽片断，PAB为对-氨基苄基氨甲酰基；
CTD为通过酰胺键键合于L 2的细胞毒类小分子药物。
m为3.8-4.2；
Ab为靶向AXL的抗体。
本发明提供了一种偶联方法，将毒素小分子通过特定连接物偶联到靶向AXL抗体上，在不改变抗体亲和性的基础上大幅提高抗体对肿瘤细胞的杀伤力。
本发明提供了连接子或偶联试剂，包含二芳硫基马来酰亚胺单元和一个偶联基团。二芳硫基马来酰亚胺单元用于交联抗体链间的巯基基团(还原后)，而偶联基团用于与小分子药物或药物-连接子单元偶联。由于该二芳硫基马来酰亚胺单元与抗体中的开放半胱氨酸-半胱氨酸二硫键的两个硫原子的二齿结合(bidentate binding)，因此这些ADC是均质的并比含有单齿接头的ADC具有更强的稳定性。因此它们将具有增长的体内半衰期，减少全身性释放的细胞毒素的量，并且比具有单齿接头的ADC更加安全的药物性质。
在另一个方面，所产生的药物-连接子单元通过该连接子与抗体偶联，生成部分链间交联的偶联物。与传统的抗体药物偶联物相比，应用本发明方法制备的抗体药物偶联物的药物/抗体比值(DAR)分布更窄，从而大幅提升了产品均一性及药理学特性均一性。该抗体药物偶联物可用于靶向输送药物到达目标细胞群体，例如肿瘤细胞。抗体药物偶联物可以特异性的与细胞表面蛋白结合，所产生的结合物随即被细胞内吞。在细胞内，药物以活性药物的方式释放出来产生功效。抗体包括嵌合抗体，人源化抗体，人抗体；可与抗原结合的抗体片段；或者抗体Fc融合蛋白；或者蛋白。“药物”是高活性药物(见定义部分)，在 某种情况下，药物可以是聚乙二醇。
本发明提供的偶联产品，尽管仍然是混合物，但与传统方式偶联得到的抗体药物偶联物相比，其DAR分布范围很窄。其平均DAR值接近4，接近最佳抗体药物偶联物平均DAR值(2-4)范围。此外，偶联产品极少不含有裸抗(DAR＝0)，这一组分对细胞毒杀不起作用。同时，偶联产品也不含有重度偶联产品(DAR＝8),这一组分在体内的清除速度很快，相对于低DAR的组分而言。因此，本发明提供的抗体药物偶联物产品非均一性得到很大的改善。
AXL抗体-药物偶联物的制备
抗体药物偶联物制备路线如下所示。抗体链间二硫键被还原，产生2n个(如8个)巯基基团。本发明的取代马来酰亚胺类连接子-药物缀合物(式Ic化合物)与还原后的抗体巯基交联，生成相应的抗体药物偶联物，其中该抗体药物偶联物存在如下所示中的一种或二种形式。
Figure PCTCN2019086475-appb-000004
其中，所述的式Ic化合物选自下组：
Figure PCTCN2019086475-appb-000005
Figure PCTCN2019086475-appb-000006
Figure PCTCN2019086475-appb-000007
等。
一种典型的制备方法包括：将抗体原液用反应缓冲液稀释至2-10mg/mL，加入140-200倍过量摩尔比的二硫苏糖醇(DTT)，或加入6.0-20倍过量摩尔比的三(2-羧乙基)膦盐酸盐 (TCEP)，反应液于10-35℃搅动2-48小时；在此所述反应缓冲液可以是按以下比例制备的缓冲液：50mM磷酸二氢钾-氢氧化钠(KH 2PO 4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9和PBS//1mM二乙基三胺五乙酸(DTPA)，pH 6-9。
将上述反应液冷至0-10℃，若采用DTT还原，需在还原反应完成后过脱盐柱或超滤除去过量的DTT，再加入取代马来酰亚胺类化合物(预先10mg/ml溶在乙腈(ACN)、二甲亚砜(DMSO)、二甲基甲酰胺(DMF)或二乙基乙酰胺(DMA)中)，并保证反应液中有机溶剂的体积占比不超过15％，偶联反应于0-37℃搅动2-4小时。若采用TCEP还原，也可不需除去剩余TCEP，直接加入取代马来酰亚胺类化合物进行偶联。
采用脱盐柱将偶联反应混合物用琥珀酸钠/NaCl缓冲液或组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。或超滤数遍。然后过滤除菌，所得产物低温保存。优选温度为-100-60℃，过滤装置的孔径优选0.15-0.3微米。
所得抗体药物偶联物的药物抗体偶联比(DAR)较为均一。采用本发明不同取代马来酰亚胺连接头(连接子片断)时，ADC产物均一性非常高(通常DAR优势产物(如DAR约为4)占所有ADC的至少60％，至少70％，至少80％，至少90％或更高)。对于DAR有一定差别的ADC，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
药物组合物和施用方法
由于本发明提供的抗体-药物偶联物，可以靶向瞄准特殊的细胞群体，与细胞表面特异蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内，因此，本发明的抗体-药物偶联物可以用于治疗目标疾病，上面提到的抗体-药物偶联物可以以治疗有效量，通过合适的途径给予受试者(例如人)。需要治疗的受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。
常规方法，已知的医学领域的普通技术人员，可以用于施用药物组合物给受试者，这取决于疾病的要治疗的类型或疾病的部位。此组合物还可以通过其它常规途径，例如，口服，肠胃外给药，通过吸入喷雾，局部，直肠，经鼻，口腔，阴道或通过植入进行给药。本文所用的术语“肠胃外”包括皮下，皮内，静脉内，肌内，关节内，动脉内，滑膜内，胸骨内，鞘内，病灶内和颅内注射或输注技术。此外，它可以施用到通过施用可注射的贮库途径，例如使用1-，3-，或6个月的贮库可注射或可生物降解的材料和方法的主题。
注射组合物可以含有各种载体如植物油，二甲基乙酰胺(dimethylactamide)，二甲基甲酰胺，乳酸乙酯，碳酸乙酯，肉豆蔻酸异丙酯，乙醇，多元醇(甘油，丙二醇，液体聚乙二醇，等等)。对于静脉内注射，水溶性抗体可以通过点滴方法，由此含有抗体和生理上可接受的赋形剂的药物制剂输注给药。生理上可接受的赋形剂可以包括，例如，5％葡萄糖，0.9％盐水，林格溶液或其它合适的赋形剂。肌内制剂，例如，抗体的一个合适的可溶盐形式的无菌制剂，可以溶解和施用的药用赋形剂诸如水换注射液，0.9％盐水，或5％葡萄 糖溶液。
当用本发明的抗体-药物偶联物治疗时，可以通过本领域常规的方法进行递送。例如，它可以通过使用脂质体，水凝胶，环糊精，生物可降解的纳米胶囊，或生物粘附性微球被引入到细胞中。或者，所述核酸或载体可在本地通过直接注射或通过使用输注泵递送。其它方法包括通过使用缀合物和生物可降解的聚合物的使用各种运输和载体系统。
本发明的药物组合物含有安全有效量的本发明的抗体-药物偶联物以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成溶液剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。
本发明所述的抗体-药物偶联物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的双功能抗体偶联物的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的抗体-药物偶联物每天以约0.0001mg-50mg/kg动物体重(较佳的0.001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的治疗剂联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
应用
本发明还提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗AXL相关的疾病的药物。所述AXL相关的疾病包括肿瘤发生、生长和/或转移、肿瘤耐药相关疾病、炎症、代谢相关疾病等。
本发明抗体、ADC或CAR-T等的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是AXL高表达的肿瘤。所述肿瘤包括(但并不限于)：乳腺癌(如三阴性乳腺癌)、肺癌(如非小细胞肺癌)、胰腺癌、恶性脑胶质瘤、胃癌、肝癌、食道癌、肾癌、结直肠癌、膀胱癌、前列腺癌、子宫内膜癌、卵巢癌、宫颈癌、白血病、骨髓癌、血管肉瘤等；尤其是三阴性乳腺癌、非小细胞肺癌、胰腺癌、恶性脑胶质瘤，更优选为三阴性乳腺癌和/或非小细胞肺癌。
(ii)诊断、预防和/或治疗自身免疫疾病。所述自身免疫疾病包括(但并不限于)：系统性红斑狼疮、类风湿关节炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症。
(iii)诊断、预防和/或治疗炎症。所述炎症包括(但并不限于)：风湿性关节炎、骨关节炎、强直性脊柱炎、痛风、莱特尔综合征、牛皮癣性关节病、感染性关节炎、结核性关节炎、病毒性关节炎、真菌性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病、特发性肺纤维化。
(iv)诊断、预防和/或治代谢相关疾病。所述代谢相关疾病包括(但并不限于)：糖尿病、食源性肥胖和脂肪炎症。
本发明的主要优点包括：
1、本发明所描述的抗体具有新颖和优异的生物活性，具体地，所述的优选抗体具有很高的AXL的亲和力(ELISA测定其EC 50为0.04～0.05nM)。此外，所述的优选抗体对肿瘤细胞表面的AXL具有良好的结合亲合力(FACS测定其EC 50为0.07～0.14nM),可用做靶向AXL的治疗抗体。
2、本发明所述的人源化抗体不仅具有与鼠源抗体相当或更高的的活性，而且具有更低的免疫原性。
3、本发明所述的抗体-药物偶联物(ADC)具有特异AXL-依赖的抗肿瘤活性；所述的优选人源化抗体的药物偶联物(ADC)对AXL-正常表达的细胞无明显毒副作用，而对AXL-高表达的肿瘤细胞具有极高的杀伤活性，细胞增殖抑制试验测定其IC 50为0.01nM～0.05nM。
4、本发明提供的新型连接子，可通过简单的化学方法与靶向AXL抗体偶联，与传统的偶联方式相比，应用这种连接子得到的AXL抗体药物偶联物DAR值分布非常窄，因此生成的产品均一性高，获得的交联物单一分布的组份(DAR为4)占比80％以上，交联物的体外肿瘤细胞增殖抑制活性较传统mcVC-PAB交联生物学活性、安全性等成药性质方面有所提高或保持。
5、本发明基于马来酰亚胺的二硫链桥接具有更好的稳定性，Ar’部位引入取代基可以调解马来酰亚胺开环水解的反应速度与减缓马来酰亚胺开环后的环合二次水解反应，在体内不易发生巯醚交换和开环后的环合二次水解反应，进一步加强了AXL抗体-药物偶联物在体外和体内的稳定性。
6、与现有技术AXL07-vc-MMAE相比，本发明的优选抗体及抗体-药物偶联物具有更优异的体外和体内抗肿瘤治疗效果。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。细胞株为常规的市售产品或购自ATCC，质粒均为市售产品。
实施例1 靶向人AXL单克隆抗体的发现和制备
步骤①杂交瘤细胞的制备：
首先制备人AXL蛋白的胞外区(AXL-ECD)作为抗原。参照NCBI:NP_068713.2氨基酸的第33位到第449位，采用基因克隆技术和哺乳动物载体表达体系获得碳末端多组氨酸标记(C-terminus polyhistidine-tagged)的抗原，具体氨基酸序列如下(SEQ ID NO.:36)：
Figure PCTCN2019086475-appb-000008
采用上述在HEK293T细胞表达和制备的人AXL胞外区蛋白免疫Balb/c小鼠，用量为50μg/只，以制备免疫脾细胞；适时的制备鼠骨髓瘤细胞(SP2/0)和饲养细胞以备融合之需。
待上述三种细胞准备完毕，通过PEG介导融合免疫脾细胞和SP2/0细胞，去除PEG，用含有饲养细胞的HAT完全培养基重悬，接种到96孔板中培养，通过ELISA/FACS法进行阳性孔筛选。最后再对阳性孔的细胞通过有限稀释法进行克隆化培养，通过ELSIA或FASCS筛选效价高、形态好、呈单克隆生长的细胞继续进行亚克隆筛选，直到连续三次筛选阳性克隆率全为100％，即可对该细胞株进行扩大培养和建库。
步骤②靶向人AXL鼠源单克隆抗体的纯化：
将步骤①中筛选出来的杂交瘤细胞在滚瓶中扩大培养14天后，收集细胞培养上清，经0.22μm滤膜过滤后，将所得培养上清恒速加入事先平衡好的Protein A树脂柱中，并用0.1M Tris-HCl(PH＝8.0,含有1.5M NaCl)平衡柱子。然后采用0.1M柠檬酸钠缓冲液洗脱平衡柱，收集洗脱液并定量并进行SDS-PAGE电泳、SEC-HPLC及内毒素检测。所得纯化抗体分装、-80℃冻存备用。
步骤③靶向人AXL鼠源单克隆抗体的生物活性和特异性的确定：
经过反复筛选，对选定的6个杂交瘤单克隆抗体进行生物活性和靶向特异性测定定。如图1A所示，采用流式细胞荧光分选仪(FACS)检测单克隆细胞培液的上清，6个单克隆均可以特异性的结合人源AXL-高表达的MDA-MB-231细胞(AXL-P)，而对AXL-低表达的MDA-MB-453细胞(AXL-N)无明显结合活性。如图1B所示，采用纯化后的抗体样品进行亚型检测，mAb001～mAb005均鉴定为IgG1/k，mAb006为IgG2b/k。
步骤④采用纯化的抗体样品，通过梯度稀释后进行ELISA检测，如表-1所示，mAb001～mAb006对AXL-ECD具有优异的结合亲和力，其中,mAb001、mAb002、mAb005、mAb006的EC 50均为<0.1nM。
表-1：靶向人AXL原始鼠源抗体的ELISA活性
Figure PCTCN2019086475-appb-000009
实施例2 抗体测序、互补决定区(CDR)的鉴定
基于优异的特异性及亲和力，优先选取mAb001、mAb002、mAb005、mAb006进行抗体测序鉴定。设计引物通过常规PCR技术扩增重链(VH)、轻链(VL)可变区片段(见图2)，克隆入载体，测序。采用常规测序并通过Kabat数据库分析(http://www.bioinf.org.uk)得到以下重链可变区(VH)、轻链可变区(VL)氨基酸序列、互补决定区(CDR)信息，下划线“_”所示为CDR-1/2/3氨基酸序列。经基因测序后注意到mAb006c与mAb005c的CDR序列高度相似，不再单独列出。
SEQ ID NO.:7mAb002重链可变区(VH)氨基酸序列
Figure PCTCN2019086475-appb-000010
SEQ ID NO.:15mAb005重链可变区(VH)氨基酸序列
Figure PCTCN2019086475-appb-000011
SEQ ID NO.:23mAb001重链可变区(VH)氨基酸序列
Figure PCTCN2019086475-appb-000012
SEQ ID NO.:8mAb002轻链可变区(VL)氨基酸序列
Figure PCTCN2019086475-appb-000013
SEQ ID NO.:16mAb005轻链可变区(VL)氨基酸序列
Figure PCTCN2019086475-appb-000014
SEQ ID NO.:24mAb001轻链可变区(VL)氨基酸序列
Figure PCTCN2019086475-appb-000015
实施例3 人-鼠嵌合抗体的制备
通过基因重组技术将3组可变区序列(参见SEQ ID NO.:7、SEQ ID NO.:15、SEQ ID NO.:23、SEQ ID NO.:8、SEQ ID NO.:16、SEQ ID NO.:24)克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后,采用转染技术和哺乳动物表达系统(FreeStyle TM293T细胞)将构建的嵌合型抗体表达和纯化(见图3)，所获得的人-鼠嵌合型抗体，编号、重链及轻链组成由表-2列出。用同样方法制备发明专利申请CN201580045131.4中公开的人源化抗体AXL07作为对照。
表-2：人-鼠嵌合抗体的制备
Figure PCTCN2019086475-appb-000016
实施例4 嵌合抗体对人AXL蛋白亲和力的ELISA测定
用包被液将AXL蛋白胞外区(AXL-ECD)稀释成1μg/mL，包被ELISA板，100μL/孔，4℃，过夜。洗去多余抗原，用1％BSA于室温封闭2h，然后加入3倍梯度稀释的各单克隆抗体，100L/孔，室温孵育1h；洗去未结合的抗体，加入合适浓度辣根过氧化物酶标记的抗鼠的二抗，100μL/孔，室温孵育0.5h。洗去未结合的二抗，加入TMB显色液反应大约15min，加入1N HCL，50μL/孔，终止显色反应，然后在450nm处测定其吸光度，并分析数据。
检测结果如图4所示，mAb001c、mAb002c、mAb005c、mAb006c对AXL-ECD有很强的亲和性，具体的EC 50值如表-3所示，mAb002c对AXL-ECD的亲和力略高于对照抗体AXL107。
表-3：嵌合抗体的ELISA测试活性
Figure PCTCN2019086475-appb-000017
实施例5 AXL蛋白在多种肿瘤细胞中呈高表达
针对多种不同分子分型的乳腺细胞株(MDA-MB-231、Hs578T、MDA-MB-453),肺癌细胞株(NCI-H1299、Calu-1、NCI-H460)，胰腺癌细胞株(SW1990、Capan-2、Panc-1、Canpan-1)，制备细胞总蛋白，精确定量后，通过免疫印迹试验(Western blot)检测AXL蛋白的表达水平。结果如图5显示，AXL蛋白在大多数被检测的乳腺癌、肺癌、胰腺癌细胞株呈异常激活表达。
实施例6 人肿瘤与正常组织基因表达数据库分析
通过下载CCLE(Cancer cell line encyclopedia)数据库、G-Tex(人正常组织)数据库、和51株人乳腺癌细胞株数据库(Neve RM et al.,Cancer Cell 2006；10:515-27)的基因表达信息，进行分析AXLmRNA水平在肿瘤株群组(如乳腺癌、肺癌、脑胶质瘤、黑色素瘤)相对于人正常组织的表达情况。本实施还分析对比了不同分子分型的乳腺癌(例如：管腔型相对于基底型)、不同恶性程度的肺癌(例如：上皮型相对于间质型)中AXLmRNA的表达水平。
结果如图6显示，对比CCLE数据库及G-Tex数据库，高侵袭性的乳腺癌、肺癌、胶质瘤和黑色素瘤细胞株的平均AXL mRNA表达水平显著高于正常组织。本发明以AXL为靶点的抗体在诊断、预防及治疗本发明以AXL为靶点的抗体在诊断、预防和治疗三阴性乳腺癌、肺癌、胶质瘤的应用中将具有显著的效果。
结果如图7显示，相比管腔型(Luminal-type)乳腺癌细胞株，高侵袭、高转移的基底型(Basal-type)乳腺癌细胞株的平均AXLmRNA表达水平显著高于管腔型，且具有统计学意义。鉴于基底型乳腺癌是临床上“三阴性”乳腺癌的主要来源，本发明以AXL为靶点的抗体在诊断、预防和治疗三阴性乳腺癌的应用中将具有更为显著的效果。
结果如图8显示，相比于低转移型/上皮型(EMT-low)肺癌细胞株，高转移型/间 质型(EMT-high)肺癌细胞株的平均AXL mRNA表达水平显著升高，且具有统计学意义。鉴于高转移型肺癌在临床上多为耐药型、预后较差，本发明以AXL为靶点的抗体在诊断及治疗高转移、耐药、晚期肺癌的应用中将有更为显著的效果。
实施例7 FACS检测肿瘤细胞表面AXL蛋白与嵌合抗体的特异性结合
采用AXL-高表达的非小细胞肺癌细胞NCI-1299、LCLC-103H、Calu-1，高表达的三阴性乳腺癌细胞MDA-MB-231、Hs578T，同时采用AXL-低表达的乳腺癌细胞MDA-MB-453作为靶细胞，测定嵌合抗体mAb002c对细胞表面AXL的结合情况。采用3x10 5个肿瘤细胞与抗体混匀(最终浓度5μg/mL)，然后于4℃孵育1小时，PBS洗涤细胞两次以去除未结合的一抗，再加入200μL(2μg/mL)PE标记的二抗置4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μL PBS中，通过流式细胞仪FACSCalibur测定受试抗体对细胞表面AXL的结合亲和力(Binding affinity)，或在同一抗体浓度下对不同肿瘤细胞的总体结合荧光强度(MFI)。
测试结果如图9所示，嵌合抗体mAb002c能特异性识别结合AXL-高表达的肿瘤细胞，结合率荧光强度顺序依次为NCI-H1299、LCLC-103H、MDA-MB-231、Hs578T，而对AXL-低表达的肿瘤细胞MDA-MB-453显示微弱的结合荧光强度。将NCI-H1299、LCLC-103H与抗体的结合率(MFI)对比MDA-MB-453与抗体的结合率，可以得出mAb002c的结合率差异分别为127倍与91倍。
实施例8 测定嵌合抗体对肿瘤细胞表面AXL的结合亲和力
采用AXL-高表达的三阴性乳腺癌细胞MDA-MB-231作为靶细胞，将100μL按照3倍梯度从200nM稀释到0.091nM的受试抗体作为一抗，分别与悬浮于100μLRPMI-1640无血清培养基中的1x10 5个MDA-MB-231混匀，然后于4℃孵育1h，PBS洗涤细胞两次以去除未结合的一抗，再将靶细胞与200μL，2μg/mL，PE标记的二抗4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μLPBS中，通过流式细胞仪FACSCalibur测定受试抗体对细胞表面AXL的结合亲和力(Binding affinity)。
测试结果如图-10、表-4所示，mAb001c、mAb002c、mAb005c、mAb006c对MDA-MB-231细胞有很强的亲和性，其中，mAb002c对MDA-MB-231细胞的亲和力明显高于对照抗体AXL107。
表-4：嵌合抗体对MDA-MB-231细胞的结合活性
Figure PCTCN2019086475-appb-000018
采用AXL-高表达的肺癌NCI-H1299作为靶细胞，将100μL按照3倍梯度稀释的受试作为一抗与悬浮于100μLRPMI-1640无血清培养基中的1x10 5个NCI-H1299细胞混匀，操作方法同上，最后将细胞重悬在200μLPBS中，通过流式细胞仪FACSAria II测定受试抗体对细胞表面AXL的结合亲和力(Binding affinity)。
测试结果如图-11、表-5所示，mAb001c、mAb002c、mAb005c、mAb006c对NCI-H1299 细胞有很强的亲和性，其中，mAb002c对NCI-H1299细胞的亲和力明显高于对照抗体AXL107。
表-5：嵌合抗体对NCI-H1299细胞的结合活性
Figure PCTCN2019086475-appb-000019
实施例9 人源化抗体的制备
在Germline数据库中检索并选取与mAb002非CDR区匹配最好的人源化模板，然后将抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与IgG1恒定区重组，同时以鼠源抗体的三维结构为基础，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残基进行回复突变。
具体地，mAb002c的人源化实施获得3个人源化重链的可变区(SEQ ID NO.:25、SEQ ID NO.:26、SEQ ID NO.:27)，以及8个人源化轻链的可变区(SEQ ID NO.:28、SEQ ID NO.:29、SEQ ID NO.:30、SEQ ID NO.:31、SEQ ID NO.:32、SEQ ID NO.:33、SEQ ID NO.:34、SEQ ID NO.:35)。
SEQ ID NO.:25mAb002_VHg0
Figure PCTCN2019086475-appb-000020
SEQ ID NO.:26mAb002_VHg1
Figure PCTCN2019086475-appb-000021
SEQ ID NO.:27mAb002_VHg2
Figure PCTCN2019086475-appb-000022
SEQ ID NO.:28mAb002_VKg0
Figure PCTCN2019086475-appb-000023
SEQ ID NO.:29mAb002_VKg1
Figure PCTCN2019086475-appb-000024
SEQ ID NO.:30mAb002_VKg2
Figure PCTCN2019086475-appb-000025
SEQ ID NO.:31mAb002_VKg3
Figure PCTCN2019086475-appb-000026
SEQ ID NO.:32mAb002_VKg4
Figure PCTCN2019086475-appb-000027
Figure PCTCN2019086475-appb-000028
SEQ ID NO.:33mAb002_VKg5
Figure PCTCN2019086475-appb-000029
SEQ ID NO.:34mAb002_VKg6
Figure PCTCN2019086475-appb-000030
SEQ ID NO.:35mAb002_VKg7
Figure PCTCN2019086475-appb-000031
通过基因重组技术将所设计的人源化可变区序列克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后，利用转染技术和哺乳动物表达系统(FreeStyle TM293细胞)将构建的人源化抗体表达载体。分别组合表达这些人源化的重链及轻链，最终mAb002c组获得了了24个人源化抗体，各抗体相应的重链和轻链组合如表-6所示。
表-6：人源化抗体的制备
Figure PCTCN2019086475-appb-000032
实施例10 人源化抗体对AXL-ECD的结合亲和力
将表-6中的24个人源化抗体梯度稀释，采用ELISA法测定其对AXL-ECD蛋白的亲和力，实验方法参照实施例4。
实验结果如图12、表-7所示,所述人源化抗体Hu002c-1～Hu002c-24均对AXL-ECD蛋白具有很强的结合亲和力，EC 50值为0.043nM～0.082nM。
表-7：人源化抗体的ELISA测试活性
Figure PCTCN2019086475-appb-000033
Figure PCTCN2019086475-appb-000034
实施例11 人源化抗体对肿瘤细胞AXL的结合亲和力
将表-6中的24个人源化抗体梯度稀释，通过流式细胞仪测定其对MDA-MB-231细胞表面AXL的亲和力，实验方法参照实施例8。
实验结果图13、表-8所示所示，所述人源化抗体对MDA-MB-231细胞表面AXL具有很高的结合亲和活性，EC 50值为0.073nM～0.17nM，表明其亲和力均高于对照AXL107(0.43nM)。
表-8：人源化抗体对MDA-MB-231细胞的结合活性
Figure PCTCN2019086475-appb-000035
将表-6中的4个人源化抗体梯度稀释，通过流式细胞仪测定其对LCLC-103H细胞表面AXL的亲和力，实验方法参照实施例8。
实验结果如图14所示所示，人源化抗体Hu002-1、Hu002-2、Hu002-4、Hu002-5对LCLC-103H细胞表面AXL具有很高的结合亲和活性，EC 50值分别为0.28nM、0.37nM、0.49nM、0.36nM，表明其亲和力均高于对照AXL107(0.80nM)。
实施例12 人源化抗体与肿瘤细胞结合导致内吞至细胞内溶酶体
采用AXL-高表达的乳腺细胞株MDA-MB-231作为靶细胞，铺制50％密度的MDA-MB-231于激光共聚焦培养皿中培养16h后，加入5μg/mL(稀释于含10％胎牛血清的1640培养基)的 AXL人源化抗体Hu002-2，分别于37℃孵育4小时或4℃孵育1小时(作为对照)；PBS洗涤3次以除去未与细胞结合的抗体，采用4％多聚甲醛(稀释于PBS)室温固定30min；PBS洗涤3次，使用0.4％Triton X-100(稀释于PBS)室温下透化处理细胞10min；PBS洗涤3次后，使用LAMP-2(兔抗人)抗体37℃条件下孵育1小时，以标记细胞溶酶体的位置；使用PBS洗洗去未结合的抗体，37℃孵育R-PE标记的羊抗人及Alexa 488二抗30min；PBS洗去未结合的二抗，用DAPI染色10min以定位细胞核，之后使用Fluorescence microscope(Leica,20×)检测抗体的内吞情况。
结果如图15所示，Hu002-2能快速并大幅度的被MDA-MB-231细胞内吞至溶酶体。该结果表明，本发明的抗体适合用于制备抗体-药物偶联物(ADC)，提示AXL-ADC将具有良好的ADC药物特性，可用于广谱和高特异性靶向AXL-阳性肿瘤治疗药物的前景。
实施例13 人源化抗体与肿瘤细胞结合导致降低肿瘤AXL的蛋白表达水平
采用AXL-高表达的肺癌细胞LCLC-103H作为靶细胞，按照16％汇合度铺制于12孔板中；贴壁16小时后，换液为分别含PBS、2μg/mL的亚型对照hIgG1、Hu002-2、或抗体-药物偶联物(ADC)Hu002-2-BL20-MMAE(由下述实施例15制备)的无血清培养基，各两孔；分别收取孵育24小时与48小时蛋白样品；使用免疫印迹法检测细胞中AXL蛋白表达量的变化。
结果如图16所示，给予PBS或hIgG1后，细胞整体的AXL表达量无明显变化，而给药Hu002-2或其ADC后，LCLC-103H细胞中AXL表达显著下调，可能是由于抗体或ADC与细胞结合并内吞后在溶酶体中降解导致。
实施例14 AXL107-vc-MMAE、AXL107-BL20-MMAE的制备
在靶向AXL的人源化抗体AXL107原液中加入PBS/EDTA(pH＝7.4)缓冲液使其浓度在20mg/ml，然后用2.6eq的TCEP于25℃还原2小时，取出后置于冰上冷却，未纯化直接加入6.0eq的mc-VC-PAB-MMAE(购自上海皓元化学，预先溶在DMA中)，0℃反应1小时，加半胱氨酸终止反应。采用G25脱盐柱除去过量的小分子，并置换至20mM枸橼酸-枸橼酸钠/6％蔗糖，pH 6.6的缓冲液中，经0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为AXL107-vc-MMAE。
结果如图17、19、20所示，人源化抗体AXL107的质谱图谱(图19)与其抗体偶联物AXL107-vc-MMAE的HIC和质谱图谱(图17、图20)均表明，抗体AXL107经偶联反应后，形成了抗体偶联物AXL107-vc-MMAE，偶联物的分子量与预期值相符，平均DAR值约为4.0。
将AXL107原液置换至50mM磷酸二氢钠-磷酸氢二钠(NaH2PO4-Na2HPO4)/150mM氯化钠(NaCl)/2mM乙二胺四乙酸(EDTA)，pH 7.0的反应缓冲液中，使其浓度为10mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动4小时。采用G25脱盐柱除去过量的TCEP，然后在收集得到的还原抗体中加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物Ic-4(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于20℃搅动2.0小时进行偶联。采用脱盐柱将偶联反应混合物用pH 7.5的Tris-盐酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为AXL107-BL20-MMAE。
结果如图18、19、21所示，人源化抗体AXL107的质谱图谱(图19)与其抗体偶联物AXL107-BL20-MMAE的HIC和质谱图谱(图18、图21)均表明，抗体AXL107经偶联反应后，形成了抗体偶联物AXL107-BL20-MMAE，偶联物的分子量与预期值相符，DAR约为4.0。
实施例15 mAb002c-vc-MMAE、mAb002c-BL20-MMAE、人源化抗体系列Hu002-BL20-MMAE的制备
在靶向AXL的嵌合抗体mAb002c原液中加入PBS/EDTA(pH＝7.4)缓冲液使其浓度在20mg/ml，然后用2.6eq的TCEP于25℃还原2小时，取出后置于冰上冷却，未纯化直接加入6.0eq的mc-VC-PAB-MMAE(购自上海皓元化学，预先溶在DMA中)，0℃反应1小时，加半胱氨酸终止反应。采用G25脱盐柱除去过量的小分子，并置换至20mM枸橼酸-枸橼酸钠/6％蔗糖，pH 6.6的缓冲液中，经0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为mAb002c-vc-MMAE。
结果如图22、24、25所示，抗体mAb002c的质谱图谱(图24)与其抗体偶联物mAb002c-vc-MMAE的HIC和质谱图谱(图22、图25)均表明，抗体mAb002c经偶联反应后，形成了抗体偶联物mAb002c-vc-MMAE，偶联物的分子量与预期值相符，平均DAR值约为4.0。
将mAb002c原液置换至50mM磷酸二氢钠-磷酸氢二钠(NaH2PO4-Na2HPO4)/150mM氯化钠(NaCl)/2mM乙二胺四乙酸(EDTA)，pH 7.0的反应缓冲液中，使其浓度为10mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动4小时。采用G25脱盐柱除去过量的TCEP，然后在收集得到的还原抗体中加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物Ic-4(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于20℃搅动2.0小时进行偶联。采用脱盐柱将偶联反应混合物用pH 7.5的Tris-盐酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为mAb002c-BL20-MMAE。
结果如图23、24、26所示，抗体mAb002c的质谱图谱(图24)与其抗体偶联物mAb002c-BL20-MMAE的HIC和质谱图谱(图23、图26)均表明，抗体mAb002c经偶联反应后，形成了抗体偶联物mAb002c-BL20-MMAE，偶联物的分子量与预期值相符，DAR约为4.0。
人源化系列抗体Hu002-BL20-MMAE的制备方法与上述mAb002c-BL20-MMAE的制备方法相同。以人源化抗体Hu002-2-BL20-MMAE为例，结果如图27、28、29所示，抗体Hu002-2的质谱图谱(图52)与其抗体偶联物Hu002-2-BL20-MMAE的HIC和质谱图谱(图27、图28)表明，抗体Hu002-2经偶联反应后，形成了抗体偶联物Hu002-2-BL20-MMAE，偶联物的分子量与预期值相符，DAR约为4.0。
实施例16 AXL嵌合抗体-药物偶联物(AXL-ADC)针对AXL高表达的三阴性乳腺癌细胞、肺癌细胞和脑胶质瘤细胞的体外抗肿瘤活性
本实例所使用细胞系购自于美国典型培养物保藏中心(ATCC)或中国科学院细胞库,并按照相应的说明进行培养，包括：MDA-MB-453、MDA-MB-231、Hs578T、Calu-1、NCI-H1299、LCLC-103H、NCI-H292、NCI-H441、NCI-H2228、NCI-H460、U87MG。
细胞增殖测试：将上述处于对数生长期的细胞，分别以每孔600-2,500个细胞的密度(依不同细胞的生长速率而定)接种至96孔细胞培养板中，150μL/孔，置37℃、5％CO2培养约5-12小时后，分别加入不同浓度的AXL-ADCs，每个药物浓度设置3个复孔及相应的溶媒对照和空白对照孔，作用4-6天后，倾去培养液，加入MTS反应液(购自Promega,cat#G3581)，100μL/孔，于37℃反应至预期颜色深浅后置多功能酶标仪(BioTek Synergy II)测定每组的细胞活力(OD490nm)，并按照以下公式计算细胞存活率：存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。每个增殖试验设置独立重复3-4次。通过GraphPad Prism 5软件分析上述数据,并分别计算药物在不同细胞株上的IC 50值。
实验结果表明，本发明的AXL抗体-药物偶联物mAb002c-vc-MMAE、mAb002c-BL20-MMAE具有很高的AXL-靶向特异性细胞毒活性，即对AXL-低表达的MDA-MB-453细胞无明显的增殖抑制作用(图30)，而对AXL-高表达的三阴性乳腺癌MDA-MB-231(图31)、Hs578T(图32)、肺癌Calu-1(图33)、LCLC-103H(图34)和胶质瘤U87MG(图35)细胞均显示很强的增殖抑制活性，IC 50值为0.03nM～0.07nM(表-9)。并且，本发明的抗体-药物偶联物mAb002c-vc-MMAE、mAb002c-BL20-MMAE对AXL-高表达的各肿瘤细胞的抑制活性明显强于对照抗体-药物偶联物AXL107-vc-MMAE、AXL107-BL20-MMAE。
表-9：嵌合抗体-药物偶联物的体外抗肿瘤活性
Figure PCTCN2019086475-appb-000036
实施例17 人源化AXL-ADCs的体外抗肿瘤活性
类似地参照实施例16的检测方法，本发明的AXL人源化系列抗体-药物偶联物Hu002-1/2/4/5/7/16-BL20-MMAE具有很高的AXL-靶向特异性细胞毒活性，即对AXL-低表达的MDA-MB-453细胞无明显的增殖抑制作用，而对AXL-高表达的三阴性乳腺癌MDA-MB-231(图36)、Hs578T(图37)、胶质瘤U87MG(图38)和肺癌LCLC-103H(图39)细胞均显示很强的增殖抑制活性，IC 50值为0.013nM～0.05nM(表-10)。
表-10：人源化AXL-ADC的体外抗肿瘤活性
Figure PCTCN2019086475-appb-000037
实施例18 AXL-ADC的体内抗肿瘤活性
分别将200μL含有5x10 6U87MG、LCLC-103H细胞悬液接种至雌性免疫缺陷小鼠(Balb/cnude，6-8周龄)背部皮下。待肿瘤体积体积达到100～300mm 3且能够观察到明显的肿瘤生长时，根据肿瘤体积大小及裸鼠体重随机分组(n＝6～8)，分别采用25mg/kg、3mg/kg、1mg/kg、0.5mg/kg的剂量，每周尾静脉给药一次，共计给药2周；同时设置hIgG1-BL20-MMAE为阴性对照。每周测量2-3次肿瘤体积及裸鼠体重并记录以绘制肿瘤生长曲线。实验结束后，统计并分析实验数据，绘制肿瘤生长曲线及裸鼠体重变化曲线，手术取出皮下移植瘤并称重，其中肿瘤体积(V)计算公式为：V＝L x W 2/2，其中L、W分别表示肿瘤的长、宽。
采用嵌合抗体mAb002c、4种优选的人源化抗体Hu002-1、Hu002-2、Hu002-4、Hu002-5分别偶联vc-MMAE或BL20-MMAE，同时以现有技术AXL107-vc-MMAE作对比进行体内抗肿瘤活性评价。人源化AXL-ADC的制备参照实施例15。
如图40所示，在U87MG肿瘤模型中剂量为5mg/kg的mAb002c-BL20-MMAE或mAb002c-vc-MMAE在给药期间抗肿瘤效果非常显著且类似，但当停药3周后mAb002c-vc-MMAE组已显现肿瘤回复生长，其抑瘤效果明显低于mAb002c-BL20-MMAE组，说明BL20-MMAE连接子在体内更具有优越性。
如图41、图42所示，在U87MG肿瘤模型中，当以剂量为3mg/kg的4个优选人源化AXL-ADC与AXL107-vc-MMAE进行体内药效检测时，观察到均具有优异的抗肿瘤活性；并且本发明优选的AXL-ADC相比AXL107-vc-MMAE能够给予更加显著的体内肿瘤治疗效果。
对以上描述的三次U87MG体内药效试验的结果予以汇总和统计(表-11)。
表-11：AXL-ADC的体内抗肿瘤活性(U87MG模型)
Figure PCTCN2019086475-appb-000038
如图43、图44所示，在LCLC-103H肿瘤模型中，本发明4个优选的AXL-ADC在给药剂量3mg/kg、1mg/kg时均显示呈剂量相关的治疗效果，并且能导致及其显著的肿瘤消退，这说明LCLC-103H肿瘤对AXL-ADC的治疗具有高度敏感性。
如图45所示，在LCLC-103H肿瘤的重复试验中，给予1mg/kg剂量的Hu002-1-BL20-MMAE、Hu002-2-BL20-MMAE、Hu002-5-BL20-MMAE后均能导致显著的肿瘤消退，并且其中Hu002-2-BL20-MMAE对肿瘤的消退效果最显著。
对以上描述的三次LCLC-03H体内药效试验的结果予以汇总和统计(表-12)。
表-12：AXL-ADC的体内抗肿瘤活性(LCLC-103H模型)
Figure PCTCN2019086475-appb-000039
如图46、表-13所示，在LCLC-103H的另一重复试验中，给药剂量1mg/kg的Hu002-2-BL20-MMAE、Hu002-5-BL20-MMAE再次被确认能诱导肿瘤消退，其效果显著优于同剂量的AXL107-vc-MMAE；同时，0.5mg/kg Hu002-2-BL20-MMAE也具一定的抑瘤效果。
表-13：AXL-ADC的体内抗肿瘤活性(LCLC-103H模型)
Figure PCTCN2019086475-appb-000040
实施例19 人源化AXL-ADC对大体积肿瘤的消退活性
基于LCLC-103H肺癌对AXL-ADC的高度敏感性，本实施例研究了AXL-ADC对特大体积肿瘤的活性；待肿瘤生长体积达到1000-2000mm 3时给药治疗，观察对肿瘤生长抑制或肿瘤消退的情况，试验方法参见实施例18。
结果如图47所示，LCLC-103H肿瘤在体内生长至体积达到800mm 3时单次给药5mg/kg剂量的Hu002-2-BL20-MMAE，治疗第34天可观察到肿瘤已完全消退。
结果如图48所示，类似地，LCLC-103H肿瘤在体内生长至体积达到1800mm 3时单次给药10mg/kg剂量的Hu002-2-BL20-MMAE，治疗第20天可见肿瘤消退率达>90％。
实施例20 人源化AXL抗体对小鼠AXL和食蟹猴AXL的结合活性测定
1、采用猴全长AXL的蛋白序列(Genebank ID:XP_014979499.1；894个氨基酸)、小鼠全长AXL的蛋白序列(Genebank ID:NP_033491.2；888个氨基酸)。具体氨基酸序列分别由SEQ ID NO.：39、SEQ ID NO.：40列出；
2、将人工合成的食蟹猴、小鼠全长基因序列构建至哺乳动物表达载体pcDNA3.1中，制备阳性表达载体质粒；
3、选择HEK293T细胞，将其铺制50％密度于培养皿中；37℃培养过夜后其汇合度约为80％，使用脂质体Lipo2000(Invitrogen)瞬时转染2μg上述制备的猴/小鼠-AXL载体质粒；转染24小时后分别收取蛋白用于免疫印迹(Western blot)分析，同时收取细胞用于FACS检测Hu002-2与HEK293T细胞表面猴/鼠AXL的结合活性。FACS检测试验方法参见实施例8。
结果如图49所示，AXL人源化抗体Hu002-2与对照抗体AXL107的作用类似，对HEK-293T表面的鼠AXL结合能力较差，在抗体最高浓度(200nM)时仍未达到结合饱和平台。
结果如图50所示，通过免疫印迹法证明猴AXL瞬时转染效率及人源化抗体Hu002-2对其的结合能力(图50A)；同时经由FACS检测，Hu002-2对HEK-293T细胞表面食蟹猴AXL有优异的结合亲和力，EC 50为0.135nM(图50B)，这个结果支持了将食蟹猴用于临床前研究模型以评估Hu002系列抗体的毒性、药代和毒代的合理性。
实施例21 所述抗体、抗体-药物偶联物与现有技术的比较
以发明专利申请号CN201580045131中公开的AXL107抗体的重链、轻链可变区序列(VH/VL)，人工合成其重链、轻链可变区并克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后在FreeStyle TM293T细胞体系表达和纯化得到AXL107(实施例3)，之后又将其制备成AXL107的抗体-药物偶联物(实施例14)。将AXL107、AXL107-vc-MMAE、AXL107-BL20-MMAE作为参比药物加入本发明的研究。
AXL107-重链可变区(VH)SEQ ID NO.:37
Figure PCTCN2019086475-appb-000041
AXL107-轻链可变区(VL)SEQ ID NO.:38
Figure PCTCN2019086475-appb-000042
对比试验的活性结果总结如下：
1、结果如表-3、表-4、表-5、表-7、表-8所示，与AXL107相比，本发明的mAb005c具有与之相当的靶标亲和力，而mAb002c及其人源化Hu002系列的多个抗体则具有更高的肿瘤细胞AXL的亲和力，预期更高的肿瘤抑制活性；
2、结果如表-9、表-10所示，基于更高的肿瘤细胞亲和力，mAb002c-vc-MMAE、mAb002c-BL20-MMAE，以及相应的人源化Hu002抗体所制备的AXL-ADC均显示出更强的AXL-靶向特异的体外抗肿瘤作用；
3、结果如表-11(图41)、表-13(图46)所示，与体外结果保持一致，本发明优选的多个人源化AXL-ADC在体内具有优异的抗肿瘤效果，其活性优于现有技术AXL107-vc-MMAE；
4、结果如图23、图26所示，mAb002c-BL20-MMAE较现有技术AXL107-vc-MMAE(图17、图20)具有更高的物质均一性，单一分布组份(DAR4)占比达到90％以上；
5、进一步的肿瘤体内药效试验直接对比了BL20-MMAE、vc-MMA联接子的治疗效果，结果表明了BL20-MMAE较vc-MMAE更具优异性(图40、表-11)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.:1所示的CDR1，
    SEQ ID NO.:2所示的CDR2，和
    SEQ ID NO.:3所示的CDR3；
    或者，
    SEQ ID NO.:9所示的CDR1，
    SEQ ID NO.:10所示的CDR2，和
    SEQ ID NO.:11所示的CDR3；
    或者，
    SEQ ID NO.:17的CDR1，
    SEQ ID NO.:18所示的CDR2，和
    SEQ ID NO.:19所示的CDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留AXL结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.:4所示的CDR1'，
    SEQ ID NO.:5所示的CDR2'，和
    SEQ ID NO.:6所示的CDR3'；
    或者，
    SEQ ID NO.:12所示的CDR1'，
    SEQ ID NO.:13所示的CDR2'，和
    SEQ ID NO.:14所示的CDR3'；
    或者，
    SEQ ID NO.:20所示的CDR1'，
    SEQ ID NO.:21所示的CDR2'，和
    SEQ ID NO.:22所示的CDR3'；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留AXL结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  7. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于AXL的结合区，并且所述scFv具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区。
  8. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求7所述的CAR构建物。
  9. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶、或其组合；
    较佳地，所述的抗体部分与所述的偶联部分通过化学键或连接子进行偶联，
    更佳地，所述的连接子选自下组：4-(N-马来酰亚胺基甲基)环己烷-1-甲酸琥酸亚胺酯(MCC)、马亚酰亚胺基己酰基(MC)、6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧基羰基(mc-val-cit-PAB)和双取代马来酰亚胺类连接子。
  10. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的 重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求6所述的重组蛋白、如权利要求8所述的免疫细胞、如权利要求9所述的抗体药物偶联物、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗AXL相关疾病的药物。
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