HK40016110A - Axl-targeting antibody, antibody-drug conjugate, preparation method therefor, and use thereof - Google Patents

Description

AXL-targeted antibody, antibody-drug conjugate, and preparation methods and applications thereof

Technical Field

The invention relates to the field of medicines, and in particular relates to an AXL-targeted antibody and an Antibody Drug Conjugate (ADC), and a preparation method and application thereof.

Background

AXL is one of the members of the receptor tyrosine kinase subfamily, the TAM family, which includes Tyro-3, AXL and Mer, and their ligands are protein molecules encoded by the growth arrest specific gene 6(Gas 6). AXL is activated after being combined with Gas6, thereby activating signal transduction pathways such as PI3K/AKT, RAS/ERK and beta-Catenin/TCF and the like at the downstream of the AXL, so as to regulate various physiological functions such as proliferation, apoptosis, chemotaxis, adhesion and recognition of cells.

AXL was found to be in an activated expression state in a variety of cancers, such as lung, breast, prostate, thyroid, endometrial, ovarian, and renal cancers in a variety of tumor tissues, involved in a variety of mechanisms including epithelial-mesenchymal transition, angiogenesis, apoptosis, and immunoregulation of tumor cells, and associated with poor prognosis (Cancer Cell 2015, 27: 533-46) and in many cases resistance (Cancer 2015, 6: 15321-31; Cancer res.2013, 19: 279-90), including EGFR inhibitor refractory lung Cancer (nat. genet.2012, 44: 852-60), PI3K inhibitor resistant head and neck Cancer (Cancer 2015, 27: 533-46), anti-HER 2 resistant breast Cancer (Biochem sons. trans.2014, 42: 822-30), sunitinib resistant renal Cancer (Oncogene 2016, 35: 2687-97), and ALK inhibitor resistant neuroblastoma (Oncogene 2016, 35: 3681-91). Furthermore, the expression of AXL is associated with acquired resistance to traditional chemotherapy and radiotherapy (Theranostics 2016, 6: 1205-19). Drug-resistant cells have increased sensitivity to cytotoxic drugs and targeted inhibitors after inhibition of AXL (nat. commun.2016, 7: 13898).

In view of the importance of AXL in tumor-targeted therapy and as a potential drug target, research into developing more antibodies with good properties that specifically bind AXL is desired.

The inventor also found that AXL abnormally activates expression in tumor tissues compared with normal tissues, especially in highly invasive, highly metastatic basal-like and/or triple negative breast cancer, metastatic lung cancer, pancreatic cancer, etc., and that antibodies targeting AXL can be internalized rapidly and in large quantities compared with other targets, which indicates that AXL may be a more preferable antibody-drug conjugate (ADC) development target. However, there is currently no highly specific antibody drug conjugate against human AXL at home and abroad, in particular.

Antibody drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker coupling the ligand to the drug. In the structure of antibody drug conjugates currently in clinical trials, highly active cytotoxic drugs are usually attached via a linker to lysine residues on the surface of the ligand, or cysteine residues in the hinge region of the antibody (reduced by the interchain disulfide moiety), with an optimal drug/ligand ratio (DAR) of 2-4. The large number of lysine residues (over 80) on the antibody surface and the non-selectivity of the conjugation reaction lead to uncertainty in the number and position of conjugation, which in turn leads to heterogeneity of the resulting antibody drug conjugates. The root mabu company reports a class of antibody conjugates (CN201580045131.4) targeting AXL, which are also antibody drug conjugates based on traditional conjugation techniques.

Furthermore, the mechanism of action of antibody drug conjugates seems simple, but whether an antibody drug conjugate can be a safe and effective drug is very complex and unpredictable, depending on a number of factors, such as:

1) the characteristics of the target point are as follows: whether the target can be effectively endocytosed, the expression level of the target, whether the target has enough expression level difference in cancer cells and normal cells, and whether the target can be secreted or shed to the extracellular part to enter blood.

2) The characteristics of the monoclonal antibody are as follows: whether the specificity of the monoclonal antibody to the target is good enough (no cross reaction with other proteins), the stability of the monoclonal antibody, the endocytosis rate and degree after the monoclonal antibody is combined with the target, and the like.

3) Characteristics of the joint: the linker needs to be sufficiently stable in the blood, and changes in the linker will change the number and location of the regions of the drug linked to the ADC, which will ultimately lead to changes in the safety and effectiveness of the entire ADC drug.

It can be seen that the development of ADC drugs requires extensive experimental exploration and validation, and their safety and effectiveness are unpredictable prior to experimentation.

In summary, there is an urgent need in the art to develop AXL-targeting antibodies and antibody drug conjugates with high affinity, low immunogenicity, and good stability.

Disclosure of Invention

The invention provides an antibody targeting human AXL, which has the biological activity of blocking AXL, has the activity of inhibiting tumor growth and metastasis, and can reduce the occurrence of anti-tumor treatment drug resistance.

The invention also provides an AXL-targeted antibody drug conjugate, which has a remarkable anti-tumor effect on AXL high-expression tumor cells.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining regions CDRs:

SEQ ID No.:1 is shown as a CDR1 in the figure,

SEQ ID No.:2, and a CDR2, and

SEQ ID No.:3, CDR 3;

alternatively, the first and second electrodes may be,

SEQ ID No.:9 of the CDR1 shown in figure 9,

SEQ ID No.:10, and a CDR2, and

SEQ ID No.:11 CDR 3;

alternatively, the first and second electrodes may be,

SEQ ID No.: 17 of the CDR1 of the signal sequence,

SEQ ID No.: 18, and a CDR2, and

SEQ ID No.: 19 CDR 3;

wherein any one of the amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one amino acid and capable of retaining the AXL binding affinity.

In another preferred embodiment, 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 regions HCDR1, HCDR2, HCDR3 of mAb002 c; or

SEQ ID No.: 9. SEQ ID No.: 10. SEQ ID No.:11 heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 of mAb005 c; or

SEQ ID No.: 17. SEQ ID No.: 18. SEQ ID No.: the heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 of mAb001c shown in FIG. 19.

In another preferred embodiment, the heavy chain variable region further comprises a human FR region or a murine FR region.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence of SEQ ID No.: 7.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence of SEQ ID No.: 15, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence of SEQ ID No.: 23, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence of SEQ ID No.: 25. SEQ ID No.: 26. SEQ ID No.: 27, or a pharmaceutically acceptable salt thereof.

In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.

In a third aspect of the invention, there is provided a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining regions CDRs:

alternatively, the first and second electrodes may be,

SEQ ID No.:4 of the CDR1',

SEQ ID No.:5 CDR2', and

SEQ ID No.:6 CDR 3';

alternatively, the first and second liquid crystal display panels may be,

SEQ ID No.:12 is shown as CDR1',

SEQ ID No.:13, and CDR2' shown in FIG. 13

SEQ ID No.:14 CDR 3';

alternatively, the first and second electrodes may be,

SEQ ID No.: 20, a CDR1' shown in FIG. 20,

SEQ ID No.: 21, and CDR2' shown in FIG. 21

SEQ ID No.: 22 CDR 3';

wherein any one of the amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one amino acid and capable of retaining the AXL binding affinity.

In another preferred embodiment, the light chain variable region comprises the following complementarity determining regions:

SEQ ID No.: 4. SEQ ID No.: 5. SEQ ID No.:6, light chain complementarity determining regions LCDR1, LCDR2, LCDR3 of mAb002 c; or

SEQ ID No.: 12. SEQ ID No.: 13. SEQ ID No.:14, light chain complementarity determining regions LCDR1, LCDR2, LCDR3 of mAb005 c; or

SEQ ID No.: 20. SEQ ID No.: 21. SEQ ID No.: 22, light chain complementarity determining regions LCDR1, LCDR2, LCDR3 of mAb001 c.

In another preferred embodiment, the light chain variable region further comprises an FR region of human or murine origin.

In another preferred embodiment, the light chain variable region has the amino acid sequence of SEQ ID No.: 8.

In another preferred embodiment, the light chain variable region has the amino acid sequence of SEQ ID No.: 16.

In another preferred embodiment, the light chain variable region has the amino acid sequence of SEQ ID No.: 24.

In another preferred embodiment, the light chain variable region has the amino acid sequence of SEQ ID No.: 28. SEQ ID No.: 29. SEQ ID No.: 30. SEQ ID No.: 31, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the light chain variable region has the amino acid sequence of SEQ ID No.: 32. SEQ ID No.: 33. SEQ ID No.: 34. SEQ ID No.: 35.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention;

alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the CDR regions of the humanized antibody comprise 1, 2, or 3 amino acid changes.

In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted is not more than 40%, preferably 20%, more preferably 10% of the total number of amino acids in the original amino acid sequence.

In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 7, preferably 1 to 3, and more preferably 1.

In another preferred embodiment, the at least one amino acid sequence that is added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.

In another preferred embodiment, said derived sequence with at least one amino acid added, deleted, modified and/or substituted has the function of inhibiting cell surface AXL or recombinant AXL proteins.

In another preferred embodiment, the antibody is in the form of a drug conjugate.

In another preferred embodiment, the antibody has an affinity EC for AXL (e.g., the extracellular domain of human AXL protein, AXL-ECD) ₅₀ 0.04-0.5 nM, preferably 0.04-0.1 nM, and more preferably 0.04-0.05 nM.

In another preferred embodiment, the antibody has an affinity, EC, for AXL on the surface of tumor cells ₅₀ Is 0.1 &1.5nM, preferably 0.1-1 nM, more preferably 0.1-0.2 nM.

In another preferred embodiment, the toxic effect IC of the antibody-drug conjugate (AXL-ADC) on AXL-highly expressed tumor cells ₅₀ Is 0.01 to 1nM, preferably 0.01 to 0.1nM, more preferably 0.01 to 0.05 nM.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In a seventh aspect of the invention there is provided a CAR construct, the scFV of the antigen binding region of the monoclonal antibody of the CAR construct being a binding region that specifically binds to AXL and having a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.

In an eighth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention.

In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

In a ninth aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) an antibody moiety selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the antibody drug conjugate ADC has the formula:

wherein:

ab is an antibody against ALX,

LU is a joint;

d is a drug;

and subscript p is a value selected from 1 to 10, preferably 1 to 8.

In another preferred embodiment, the coupling moiety (D) is a cytotoxic drug and the cytotoxic drug is: microtubule-targeted drugs and/or DNA-targeted drugs and/or topoisomerase inhibitors. .

In another preferred embodiment, the microtubule targeting agent is selected from the group consisting of: monomethyl auristatin e (mmae), monomethyl auristatin f (mmaf), maytansine derivative DM1, and tubulysin.

In another preferred embodiment, the DNA targeting drug is selected from the group consisting of: duocarmycin, pyrrolo [2, 1-c ] [1, 4] benzodiazepine (PBD).

In another preferred embodiment, the topoisomerase inhibitor is selected from the group consisting of: 7-ethyl-10-hydroxycamptothecin (SN38), irinotecan (Exatecan), and analogs thereof.

In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.

In another preferred embodiment, the Linker (LU) is selected from the group consisting of: 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid imidate (MCC), Maleimidocaproyl (MC), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), CL2A (US20140170063, CN201480041766.2) and disubstituted maleimide linker (CN201611093699.6, CN 201711169847.2).

In another preferred embodiment, the antibody drug conjugate has toxic effect IC on AXL-highly expressed tumor cells ₅₀ Is 0.01 to 1nM, preferably 0.01 to 0.1nM, more preferably 0.01 to 0.05 nM.

In a tenth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the immune cell of the eighth aspect of the present invention, the antibody drug conjugate of the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and/or (b) preparing a medicament for preventing and/or treating AXL-related diseases.

In another preferred embodiment, the detection reagent, detection plate or kit is used for:

(1) detecting the AXL protein in a sample; and/or

(2) Detecting endogenous AXL protein in tumor cells; and/or

(3) Detecting tumor cells expressing the AXL protein.

In another preferred embodiment, the detection reagent, detection plate or kit is used for diagnosing AXL-associated diseases.

In another preferred embodiment, the medicament is used for treating or preventing AXL-high expressing tumors, tumor migration, or tumor resistance.

In another preferred embodiment, the tumor resistance comprises: drug resistance of tumor immunotherapy drugs, drug resistance of tumor targeted therapy drugs, drug resistance of conventional tumor chemotherapy, and insensitivity of radiotherapy.

In another preferred embodiment, the medicament is for a use selected from the group consisting of:

(a) AXL that specifically binds to tumor cells, and/or immune/stromal cells in the tumor microenvironment;

(b) inhibiting overactive AXL biological function in the tumor/tumor microenvironment;

(c) inhibiting tumor cell migration or metastasis;

(d) inhibiting tumor growth and improving the anti-tumor curative effect of the combined medicine;

(e) antibody-dependent cell-mediated cytotoxicity (ADCC).

In another preferred embodiment, the AXL-associated disease is selected from the group consisting of: cancer, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof.

In another preferred embodiment, the cancer comprises solid tumor and blood cancer.

In another preferred embodiment, the cancer is a tumor with high AXL expression.

In another preferred embodiment, said AXL-highly expressed tumor is selected from the group consisting of: breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer, brain glioma, melanoma, leukemia, lymphoma, or a combination thereof.

In another preferred embodiment, the cancer is a drug-resistant tumor.

In another preferred embodiment, said AXL highly expressed tumor is the ratio of the level of AXL transcript and/or protein L1 in tumor tissue to the level of transcript and/or protein L0 in normal tissue, L1/L0 is ≥ 2, preferably ≥ 3.

In another preferred embodiment, the metabolic-related diseases include: diabetes, food-borne obesity and steatosis.

In another preferred embodiment, the infectious disease comprises: bacterial and viral infections.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In a twelfth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention;

(3) a CAR construct according to the seventh aspect of the invention.

In a thirteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the twelfth aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a fourteenth aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the thirteenth aspect of the invention or having a polynucleotide according to the twelfth aspect of the invention integrated into its genome.

In a fifteenth aspect of the invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of AXL in a sample, said method comprising the steps of:

(1) contacting the sample with an antibody according to the fifth aspect of the invention in vitro;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of AXL in the sample.

In a sixteenth aspect of the present invention, there is provided a detection board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an immunoconjugate according to the ninth aspect of the invention.

In a seventeenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody against the antibody of the fifth aspect of the invention;

alternatively, the kit comprises a detection plate according to the sixteenth aspect of the invention.

In an eighteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

In a nineteenth aspect of the present invention, there is provided a method of treating an AXL-associated disease, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

In another preferred example, the method further comprises: administering to a subject in need thereof an additional agent or treatment for combination therapy.

In another preferred embodiment, the other medicament or treatment comprises: anti-tumor immunotherapy drugs, tumor targeting drugs, tumor chemotherapy drugs and tumor radiotherapy.

In another preferred embodiment, the anti-tumor immunotherapy medicament comprises PD-1 and PD-L1 monoclonal antibodies.

In a twentieth aspect of the present invention, there is provided a method of producing a chimeric antibody, comprising the steps of:

after cloning the nucleotide sequence of the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention into an expression vector containing the nucleotide sequence of a human antibody constant region, the human-mouse chimeric antibody is expressed by transfecting animal cells.

In a twenty-first aspect of the present invention, there is provided a method of preparing a humanized antibody comprising the steps of:

the nucleotide sequence of the CDR region in the heavy chain variable region according to the first aspect of the present invention and/or the light chain variable region according to the third aspect of the present invention is implanted into a nucleotide sequence template containing an FR region of a human antibody, and then cloned into an expression vector containing a constant region of a human antibody, followed by expressing the humanized antibody by transfecting animal cells.

In a twenty-second aspect of the invention, there is provided a method of inhibiting tumor cell growth and migration, comprising the steps of: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

In a twenty-third aspect of the invention, there is provided a method of inhibiting tumor growth in a model animal comprising the steps of: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody.

In another preferred embodiment, the drugs can be administered alone or in combination including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, radiation therapy.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be described in detail.

Drawings

FIG. 1 shows the findings of an anti-human AXL antibody of the present invention. FIG. 1A shows the binding activity of the culture supernatant of anti-human AXL monoclonal antibody (original hybridoma) detected by flow cytometry fluorescence sorter (FACS) on human AXL-highly expressed MDA-MB-231(AXL-P) and AXL-less expressed MDA-MB-453(AXL-N) breast cancer cells. FIG. 1B shows the numbering of the 6 monoclonal antibodies (mAb001, mAb002, mAb003, mAb004, mAb005, mAb006) and the subtype identification of the purified antibodies.

FIG. 2 shows the results of PCR amplification of fragments of heavy chain variable region (VH) and light chain variable region (VL) of mAb001, mAb002, mAb005 and mAb 006. The VH/VL fragment is used for cloning and assembling a human-mouse chimeric antibody expression vector after sequencing and identification.

FIG. 3 shows 4 human-murine chimeric antibodies (chimeric antibodies) mAb001c, mAb002c, mAb005c, mAb006c expressed by HEK293T cells followed by the use of MabSelect ^TM SuRe ^TM Purification profile of column.

FIG. 4 shows the measurement of the Binding affinity of human-murine chimeric antibodies mAb001c, mAb002c, mAb005c, mAb006c to AXL-ECD (Binding affinity EC) by ELISA ₅₀ )。

FIG. 5 shows the expression levels of AXL protein in cell lines of breast cancer (MDA231, Hs587T, MDA453), lung cancer (NCI-H1299, Calu-1, NCI-H460), and pancreatic cancer (SW1990, Capan-2, Panc-1, Capan-2) as detected by Western blot assay (Western blot).

FIG. 6 is a graph showing the analysis and comparison of the expression level of AXLmRNA (ratio to-actin) in human normal tissues against a number of tumor cell lines (breast cancer, lung cancer, brain glioma, melanoma).

FIG. 7 is a graph showing the analysis of the expression level of AXL mRNA in a population of highly invasive and highly metastatic Basal-type (Basal-type) versus Luminal (Luminal-type) breast Cancer Cell lines in a 51-line human breast Cancer Cell line gene expression database (New RM et al, Cancer Cell 2006; 10: 515-27).

Fig. 8 is a graph analyzing the expression level of axlmna in epithelial versus mesenchymal lung cancer cell lines in the CCLE database.

FIG. 9 is the level of mAb002c (5g/mL) binding to AXL on the surface of tumor cells with high (NCI-H1299, LCLC-103H, CaLu-1, MDA-MB-231, Hs578T) or low (MDA-MB-453) expression of AXL.

FIG. 10 shows the Binding affinities of chimeric antibodies mAb001c, mAb002c, mAb005c, mAb006c to AXL on the cell surface of MDA-MB-231 (Binding affinity EC) ₅₀ ) The detection result is 1x10 ⁵ Cells were mixed with antibody at the indicated concentration gradient and analyzed by FACS detection using a flow cytometer (FACSCalibur) after 1 hour of incubation.

FIG. 11 shows the Binding affinities (Binding affinity EC) of the chimeric antibodies mAb001c, mAb002c, mAb005c to AXL on the cell surface of NCI-H1299 ₅₀ ) The detection result is 1x10 ⁵ Cells were mixed with antibody at the indicated concentration gradient and analyzed by FACS detection using a flow cytometer (FACSAria II) after 1 hour of incubation.

FIG. 12 shows the Binding affinity (Binding affinity EC) of humanized antibody series Hu002-1 to Hu002-24 to AXL-ECD measured by ELISA ₅₀ )。

FIG. 13 shows the Binding affinities (Binding affinity EC) of the humanized antibody series Hu002-1 to Hu002-24 to AXL on the MDA-MB-231 cell surface ₅₀ ) The detection result is 1x10 ⁵ Cells were mixed with antibody at the indicated concentration gradient and analyzed by FACS detection using a flow cytometer (FACSCalibur) after 1 hour of incubation.

FIG. 14 shows the Binding affinities (Binding affinity EC) of humanized antibody series Hu002-1 to Hu002-5 to AXL on the surface of LCLC-103H cells ₅₀ ) The detection result is 1x10 ⁵ Cells were mixed with antibody at the indicated concentration gradient and incubated for 1 hour before FACS detection analysis using a flow cytometer.

FIG. 15 shows that Hu002-2 bound to MDA-MB-231 cells resulting in endocytosis (Internalization) to intracellular lysosomes. The antibody (5g/mL) is incubated with the cells for 1 hour at 4 ℃, or is placed in a laser confocal microscope for observation after 4 hours at 37 DEG C

FIG. 16 shows that LCLC-103H cells were treated with Hu002-2 or Hu002-2-BL20-MMAE for 24 or 48 hours and then tested for inhibition of AXL protein expression by immunoblotting (Western blot).

FIG. 17 is a Hydrophobic Interaction Chromatography (HIC) pattern of antibody drug conjugate AXL 107-vc-MMAE.

FIG. 18 is a Hydrophobic Interaction Chromatography (HIC) pattern of antibody drug conjugate AXL107-BL 20-MMAE.

FIG. 19 is a mass spectrum of monoclonal antibody AXL 107.

FIG. 20 is a mass spectrum of antibody drug conjugate AXL 107-vc-MMAE.

FIG. 21 is a mass spectrum of antibody drug conjugate AXL107-BL 20-MMAE.

FIG. 22 is a Hydrophobic Interaction Chromatography (HIC) profile of antibody drug conjugate mAb002 c-vc-MMAE.

FIG. 23 is a Hydrophobic Interaction Chromatography (HIC) profile of antibody drug conjugate mAb002c-BL 20-MMAE.

FIG. 24 is a mass spectrum of monoclonal antibody mAb002 c.

FIG. 25 is a mass spectrum of antibody drug conjugate mAb002 c-vc-MMAE.

FIG. 26 is a mass spectrum of antibody drug conjugate mAb002c-BL 20-MMAE.

FIG. 27 is a Hydrophobic Interaction Chromatography (HIC) profile of antibody drug conjugate Hu002-2-BL 20-MMAE.

FIG. 28 is a mass spectrum of antibody drug conjugate Hu002-2-BL 20-MMAE.

FIG. 29 is a mass spectrum of humanized monoclonal antibody Hu 002-2.

FIG. 30 shows the inhibitory activity (IC) of mAbs 002c-ADC and AXL107-ADC on MDA-MB-453 cell proliferation in vitro ₅₀ ) The detection result of (1).

FIG. 31 shows the inhibitory activity (IC) of mAb002c-ADC and AXL107-ADC on MDA-MB-231 cell proliferation in vitro ₅₀ ) The detection result of (1).

FIG. 32 shows the inhibitory activity of mAb002c-ADC and AXL107-ADC on Hs578T cell proliferation in vitro (IC) ₅₀ ) The detection result of (1).

FIG. 33 shows the inhibitory activity (IC) of mAb002c-ADC and AXL107-ADC on Calu-1 cell proliferation in vitro ₅₀ ) The detection result of (1).

FIG. 34 shows the inhibitory activity (IC) of mAb002c-ADC and AXL107-ADC on LCLC-103H cell proliferation in vitro ₅₀ ) The detection result of (3).

FIG. 35 shows the inhibitory activity of mAb002c-ADC and AXL107-ADC on U87MG cell proliferation in vitro (IC) ₅₀ ) The detection result of (1).

FIG. 36 shows the inhibitory activity of ADC of humanized Hu002 series antibody coupled to BL20-MMAE on MDA-MB-231 cell proliferation in vitro (IC) ₅₀ ) The detection result of (1).

FIG. 37 shows the inhibitory activity of ADC of humanized Hu002 series antibody conjugated BL20-MMAE on the in vitro proliferation of Hs578T cells (IC) ₅₀ ) The detection result of (3).

FIG. 38 shows the inhibitory activity of ADC of humanized Hu002 series antibody coupled with BL20-MMAE on proliferation of U87MG cells in vitro (IC) ₅₀ ) The detection result of (1).

FIG. 39 shows the inhibitory activity (IC) of ADC of humanized Hu002 series antibody coupled BL20-MMAE on in vitro proliferation of LCLC-103H cells ₅₀ ) The detection result of (1).

FIG. 40 shows the in vivo antitumor effect of the ADCs (both 5mg/kg) of the chimeric antibody mAb002c antibody conjugated to vc-MMAE and BL20-MMAE, respectively, in the U87MG brain glioma model, and the results show that BL20-MMAE has more excellent in vivo therapeutic effect than vc-MMAE.

FIG. 41 shows the in vivo antitumor effect of humanized Hu002-1, Hu002-4 antibody conjugate BL20-MMAE (3mg/kg), AXL107-vc-MMAE (3mg/kg) in U87MG brain glioma model.

FIG. 42 shows the in vivo antitumor effect of ADC (3 mg/kg; 2 times per week 1) conjugated to BL20-MMAE of humanized Hu002-2 and Hu002-5 antibodies, respectively, in U87MG brain glioma model.

FIG. 43 shows the in vivo anti-tumor efficacy of humanized Hu002-1 and Hu002-4 antibodies conjugated to BL20-MMAE ADC (3mg/kg, 1 mg/kg; 2 times per week 1) in LCLC-103H lung cancer model.

FIG. 44 shows the in vivo anti-tumor efficacy of humanized Hu002-2 and Hu002-5 antibodies conjugated to ADC (3mg/kg, 1 mg/kg; 2 times per week 1) of BL20-MMAE in LCLC-103H lung cancer model.

FIG. 45 shows the in vivo anti-tumor efficacy of humanized Hu002-1, Hu002-2, Hu002-5 antibodies conjugated to ADC of BL20-MMAE (all at 1 mg/kg; 1 time per week for 2 times) in LCLC-103H lung cancer model.

FIG. 46 is the in vivo anti-tumor efficacy results in LCLC-103H lung cancer model following 2 doses of BL20-MMAE ADC (1mg/kg, 0.5mg/kg) and AXL107-vc-MMAE (1mg/kg) conjugated to humanized Hu002-2 and Hu002-5 antibodies, respectively, for BL20-MMAE 1 time per week.

FIG. 47 is a humanized Hu002-2-BL20-MMAE (5 mg/kg; single dose) directed against LCLC-103H bulk tumor (800 mm) ³ Volume at the time of initial administration) can lead to a result of tumor regression.

FIG. 48 shows humanized Hu002-2-BL20-MMAE (10 mg/kg; single dose) against LCLC-103H bulk tumor (1800 mm) ³ Volume at the time of initial administration) can lead to a result of tumor regression.

FIG. 49 is a measurement of FACS binding activity of Hu002-2 after HEK293T transiently expresses murine AXL protein; hu002-2 or AXL107 showed very weak binding activity to murine AXL compared to human AXL.

FIG. 50 is the binding affinity of Hu002-2 to cynomolgus monkey AXL. FIG. 50A is a graph showing the level of protein expression of HEK293T transiently transfected monkey AXL vector after 24 hours detection by immunoblotting (Western blot); FIG. 50B shows HEK293T transient expression of monkey AXL24 hours after harvesting cells and Binding affinity to Hu002-2 was detected by FACS (Binding affinity EC) ₅₀ )。

Detailed Description

The present inventors have unexpectedly obtained 6 anti-AXL monoclonal antibodies, designated mAb 001-mAb 006, through extensive and intensive studies and extensive screening. Based on the results of the activity tests, mAb001(IgG 1-. kappa.), mAb002(IgG 1-. kappa.), mAb005(IgG 1-. kappa.), mAb006(IgG2 b-. kappa.) were selected to construct human-murine chimeric antibodies, which were designated mAb001c, mAb002c, mAb005c, and mAb006c, respectively. The results obtained after further testing of the above antibodies were as follows:

first and second chimeric antibodies, each of which is capable of binding with high specificity to the AXL antigen and measuring EC in ELISA ₅₀ 0.092nM, 0.073nM, 0.103nM, 0.101nM, respectively;

secondly, the chimeric antibody has extremely high binding affinity to the tumor cells with high expression of the multiple AXL strains, and the EC of the chimeric antibody is measured by FACS ₅₀ The molecular weight of the antigen is 0.174nM to 1.5nM, and the gene sequencing shows that the mAb006c highly overlapped the Complementarity Determining Region (CDR) of mAb005c, thus terminating subsequent studies with mAb006 c;

thirdly, a series of humanized antibodies designed based on mAb002c have higher AXL protein binding affinity and cell binding affinity; ELISA for determining its EC ₅₀ Is 0.045nM to 0.08 nM; measurement of EC by FACS ₅₀ The concentration is 0.09nM to 0.14 nM.

Fourthly, the drug conjugate (ADC) of the antibody has excellent characteristics that no obvious toxic or side effect is caused to cells with normal expression of AXL, the drug conjugate has extremely high killing activity to tumor cells with high expression of AXL, and the cell proliferation is inhibited by IC ₅₀ The value is 0.01nM to 0.07 nM;

fifthly, the AXL-ADC product obtained by the novel linker has the advantages of high uniformity and further improvement of in vitro and in vivo stability.

Sixth, the antibodies and antibody-drug conjugates preferred in the present invention have superior and more sustained in vivo antitumor therapeutic effects than the prior art.

The present invention has been completed on the basis of this finding.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a number of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain 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 the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain lie closely together through the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol. I, 647-. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 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 IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

In general, the antigen binding properties of an antibody can be described by 3 specific regions, called variable regions (CDRs), located in the heavy and light chain variable regions, which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them 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. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using 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, and are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as chimeric antibodies having a variable region derived from a murine monoclonal antibody, and a constant region derived from a human immunoglobulin (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared to the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			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

anti-AXL antibodies

The present invention provides 3 broad classes of highly specific and high affinity antibodies targeting AXL comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.

Preferably, 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 polypeptide sequences:

a1) HCDR1 is SEQ ID No.: 1: DFYIN, SEQ ID No.: 9: SYYIH, or SEQ ID No.: 17: SGYWS;

a2) HCDR2 is SEQ ID No.: 2: WIYPGSGNTKYNEKFKG, SEQ ID No.: 10: WIYPGSDNTKYNEKFKD, or SEQ ID No.: 18: YMTYSGATYYNPSLKS;

a3) HCDR3 is SEQ ID No.: 3: STGFFDY, SEQ ID No.: 11: NYYDYDGGTWFPY, or SEQ ID No.: 19: GGNSYFFDY, respectively;

a4) LCDR1 is SEQ ID No.: 4: SASSSIGYMY, SEQ ID NO: 12: RASQDINYYLN, or SEQ ID No.: 20: RASENIYSNLA, respectively;

a5) LCDR2 is SEQ ID No.: 5: LTSNLAS, SEQ ID No.: 13: YTSRLHS, or SEQ ID No.: 21: AATNLAD;

a6) LCDR3 is SEQ ID No.: 6: QQWSSNPPT, respectively; SEQ ID No.: 14: QQGNTLPWT, or SEQ ID No.: 22: QHFWGTPLT, respectively;

a7) a sequence having AXL binding affinity, wherein at least one amino acid is added, deleted, modified and/or substituted in any one of the amino acid sequences.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

Preferably, the antibody has the function of inhibiting cell surface and recombinant AXL protease, and can be quickly endocytosed into lysosomes by cells.

The antibody of the present invention may be a double-chain 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 derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.

Among them, the animal is preferably a mammal such as a mouse.

The antibodies of the invention may be chimeric, humanized, CDR-grafted and/or modified antibodies targeting human AXL.

In a preferred embodiment of the invention, the aforementioned SEQ ID No.: 1-3, SEQ ID No.: 9-11, SEQ ID No.: 17-19, or a sequence with AXL binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid in the sequence, and is positioned in a CDR region of a heavy chain variable region (VH).

In a preferred embodiment of the invention, the aforementioned SEQ ID No.: 4-6, SEQ ID No.: 12-14, SEQ ID No.: 20-22, or a sequence with CD73 binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid of the sequences, and is positioned in a CDR region of a light chain variable region (VL).

In a more preferred embodiment of the invention, the VH CDR1, CDR2, CDR3 are each independently selected from SEQ ID No.: 1. SEQ ID No.: 2. SEQ ID No.:3, or is selected from SEQ ID No.: 9. SEQ ID No.: 10. SEQ ID No.:11, or selected from SEQ ID No.: 17. SEQ ID No.: 18. SEQ ID No.: 19, or a sequence which is added, deleted, modified and/or substituted by at least one amino acid and has AXL binding affinity; VL CDR1, CDR2, CDR3 are each independently selected from SEQ ID No.: 4. SEQ ID No.: 5. SEQ ID No.:6, or is 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.: 22, or a sequence with AXL binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid in the sequences.

In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the above-mentioned aspect of the present invention, the number of the amino acids to be added, deleted, modified and/or substituted may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, and still more preferably 1 to 2.

In another preferred embodiment, the antibody is the original murine antibodies mAb001, mAb002, mAb003, mAb004, mAb005, mAb 006.

In another preferred embodiment, the antibody is human-murine chimeric antibody mAb001c, mAb002c, mAb005c, mAb006 c.

In another preferred embodiment, the antibody is 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 and Hu002 c-24.

The 3 major antibodies of the invention can be used in combination for constructing CAR constructs, recombinant immune cells containing CAR constructs, antibody drug conjugates, and the like, and can also be used for (a) preparing detection reagents, detection plates or kits; and/or (b) preparing a medicament for preventing and/or treating AXL-related diseases.

The representative meanings of each sequence related in the sequence table of the present invention are shown in the following table B.

TABLE B

Sequence numbering	Sequence name	Sequence numbering	Sequence name
				SEQ ID NO.：1	mAb002 HCDR1	SEQ ID NO.：21	mAb001 LCDR2
SEQ ID NO.：2	mAb002 HCDR2	SEQ ID NO.：22	mAb001 LCDR3
				SEQ ID NO.：3	mAb002 HCDR3	SEQ ID NO.：23	mAb001-VH
SEQ ID NO.：4	mAb002 LCDR1	SEQ ID NO.：24	mAb001-VL
				SEQ ID NO.：5	mAb002 LCDR2	SEQ ID NO.：25	mAb002-VH_HuG0
SEQ ID NO.：6	mAb002 LCDR3	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	mAb005 HCDR1	SEQ ID NO.：29	mAb002-VK_HuG1
SEQ ID NO.：10	mAb005 HCDR2	SEQ ID NO.：30	mAb002-VK_HuG2
				SEQ ID NO.：11	mAb005 HCDR3	SEQ ID NO.：31	mAb002-VK_HuG3
SEQ ID NO.：12	mAb005 LCDR1	SEQ ID NO.：32	mAb002-VK_HuG4
				SEQ ID NO.：13	mAb005 LCDR2	SEQ ID NO.：33	mAb002-VK_HuG5
SEQ ID NO.：14	mAb005 LCDR3	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	Extracellular region of human AXL protein
				SEQ ID NO.：17	mAb001 HCDR1	SEQ ID NO.：37	AXL107-VH
SEQ ID NO.：18	mAb001 HCDR2	SEQ ID NO.：38	AXL107-VL
				SEQ ID NO.：19	mAb001 HCDR3	SEQ ID NO.：39	Monkey AXL protein sequence
SEQ ID NO.：20	mAb001 LCDR1	SEQ ID NO.：40	Mouse AXL protein sequence

The invention also relates to a monkey AXL protein sequence and a mouse AXL protein sequence, wherein the Genebank ID is XP _014979499.1 (monkey) and Genebank ID: NP _033491.2 (mouse).

Production of antibodies

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together.

At present, the DNA sequence encoding the antibody of the invention (or a fragment thereof, or a derivative thereof) has been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, 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 monoclonal antibodies can be determined, for example, by Munson et al, anal. biochem., 107: 220(1980) by Scatchard analysis.

The antibody of the present invention may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, 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 (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

Antibody-drug conjugates

The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.

Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule, to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, 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 invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more 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 which enables the effector molecule and the antibody to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).

Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.

Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., degradation of the linker at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide 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 a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.

Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and attachment is achieved by the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimide compounds, haloamides (e.g., iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; a mercury derivative such as 3, 6-bis- (mercuric methyl) dioxane, and a counter ion such as acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.

The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that can form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.

Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1, 4] benzodiazepines (PBDs), indobenzodiazepines (indoxazepines) and benzodiazepines (oxyphenodiazepines), and vincristine-7-hydroxy-ethyl acetate (irinotecan-38), and analogues thereof.

In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.

Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 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 reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of the drug moiety and the linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.

The present invention also provides a method of preparing an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, the methods of the invention comprise: the antibody is conjugated to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is bound to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

In some embodiments, the antibody drug conjugate ADC has the formula:

wherein:

ab is an antibody, and Ab is an antibody,

LU is a linker;

d is a drug;

and subscript p is a value selected from 1 to 8.

AXL antibody-drug conjugates

The present invention relates to antibody-drug conjugates, more specifically the present invention relates to AXL antibody-drug conjugates having therapeutic applications. anti-AXL antibodies can be conjugated to chemotherapeutic drugs or small molecule toxins through specific linkers. The invention also relates to methods of treating mammalian cells or associated pathological conditions using anti-AXL antibody-drug conjugates.

Due to the large number of lysine residues (over 80) on the antibody surface and the non-selectivity of the conjugation reaction, uncertainty in the number and position of conjugation results, which in turn leads to heterogeneity of the resulting antibody drug conjugates. For example, the DAR value distribution for T-DM1 (average DAR value of 3.5) is 0-8. Similarly, although there are only four pairs of interchain disulfide bonds in the hinge region of an antibody, partial reduction of interchain disulfide bonds is required to achieve the optimum average DAR value (2-4). Since the existing reducing agents (DTT, TCEP, etc.) do not selectively reduce interchain disulfide bonds, the resulting conjugates are also not homogeneous products, consisting of a plurality of components, whose main components have DAR values of 0, 2, 4, 6, 8, and the components corresponding to each specific DAR value are present as isomers due to differences in the attachment sites. Heterogeneity of antibody drug conjugate products can lead to heterogeneity of pharmacokinetic properties, potency, and toxicity among the component parts. For example, components with higher DAR values are cleared more rapidly in vivo and result in higher toxicity.

Aiming at the problems of the coupling technology, the aim of fixed-point coupling of the existing antibody is fulfilled by a simple chemical method, so that a large amount of manpower, material resources and financial resources are saved, and the method is more attractive. Among them, there have been related studies including: CN200480019814.4 filed by boliteix limited; WO2014197871A2, applied by Igenica Biothereutics; CN201380025774.3, filed by sorento medical limited; CN201310025021.4 applied by Shanghai New concept biomedicine science and technology Limited. However, the above-mentioned techniques have problems of long synthetic route of the coupling reagent, poor chemical stability of the coupling reagent, and disordered electrophoresis pattern of the antibody conjugate, which indicates that there may be side reactions during the coupling process, and the problems of thiol exchange (reverse michael addition reaction) during the in vivo circulation process are not solved by the existing solutions.

The root mabu company reports a class of antibody conjugates targeting AXL (CN201580045131.4), which are also antibody drug conjugates based on traditional conjugation techniques.

Aiming at the problems of the coupling technology, the fixed-point coupling is realized on the AXL-targeted antibody-drug conjugate by a simple chemical method, so that the uniformity of the drug can be improved, a large amount of manpower, material resources and financial resources are saved in the aspects of process and quality control, and the stability, the drug effect, the safety and other pharmaceutical properties of the conjugate can be improved.

The invention adopts a novel linker structure (a novel disubstituted maleimide linker CN201611093699.6, CN201711169847.2 developed by the inventor in the earlier stage) and is applied to coupling of the targeting AXL antibody, the linker can be completely/partially cross-coupled on cysteine sulfydryl reduced by a light chain-heavy chain and a heavy chain-heavy chain disulfide bond of the antibody, and the targeting AXL antibody drug conjugate obtained by applying the coupling method has narrower drug/antibody ratio (DAR) distribution compared with the traditional antibody drug conjugate. The structure of the AXL antibody-drug conjugate with the disubstituted maleimide linker is shown as formulas Ia and Ib:

wherein the content of the first and second substances,

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) attached to an Ar' group ₂ CH ₂ O) _n -, where n is selected from any one of integers from 1 to 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-aminobenzyl carbamoyl;

CTD is bonded to L through an amide bond ₂ The cytotoxic small molecule drug of (1).

m is 3.8-4.2;

ab is an antibody targeting AXL.

The invention provides a coupling method, which couples toxin micromolecules to a targeting AXL antibody through a specific connector and greatly improves the killing power of the antibody to tumor cells on the basis of not changing the affinity of the antibody.

The present invention provides linkers or coupling reagents comprising a diarylthiomaleimide unit and a coupling group. The diarylthiomaleimide units are used to crosslink the sulfhydryl groups between antibody chains (after reduction), while the coupling groups are used to couple with small molecule drugs or drug-linker units. These ADCs are homogeneous and more stable than ADCs containing monodentate linkers due to the bidentate binding of the diarylthiomaleimide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody. They will therefore have an increased half-life in vivo, reduce the amount of cytotoxic released systemically, and be safer pharmaceutical properties than ADCs with monodentate linkers.

In another aspect, the resulting drug-linker unit is conjugated to an antibody via the linker, resulting in a conjugate with partial interchain cross-linking. Compared with the traditional antibody drug conjugate, the antibody drug conjugate prepared by the method has narrower drug/antibody ratio (DAR) distribution, thereby greatly improving the product uniformity and the pharmacological property uniformity. The antibody drug conjugate can be used for targeted delivery of drugs to target cell populations, such as tumor cells. The antibody drug conjugate can be specifically bound to a cell surface protein, and the resulting conjugate can then be endocytosed by the cell. Inside the cell, the drug is released in the form of the active drug to produce the effect. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; an antibody fragment that binds to an antigen; or an antibody Fc fusion protein; or a protein. A "drug" is a highly active drug (see definitions section), in some cases the drug may be polyethylene glycol.

The coupling product provided by the invention is still a mixture, but has a narrow DAR distribution range compared with the antibody drug conjugate obtained by the conventional coupling method. The average DAR value is close to 4, and the average DAR value is close to the range of the optimal antibody drug conjugate (2-4). In addition, the conjugate product is rarely free of naked antibody (DAR ═ 0), and this fraction is not effective in cytotoxic action. Also, the coupling product does not contain a heavy coupling product (DAR ═ 8), and this fraction is cleared rapidly in vivo, relative to the low DAR fraction. Therefore, the heterogeneity of the antibody drug conjugate product provided by the invention is greatly improved.

Preparation of AXL antibody-drug conjugates

The preparation route of the antibody drug conjugate is shown below. The interchain disulfide bonds of the antibody are reduced, yielding 2n (e.g., 8) sulfhydryl groups. The substituted maleimide linker-drug conjugate (compound of formula Ic) of the present invention is cross-linked with reduced antibody sulfhydryl groups to produce the corresponding antibody drug conjugate, wherein the antibody drug conjugate exists in one or two forms as shown below.

Wherein the compound of formula Ic is selected from the group consisting of:

and the like.

One typical preparation method includes: diluting the antibody stock solution to 2-10mg/mL by using a reaction buffer solution, adding 140-fold Dithiothreitol (DTT) with an excess molar ratio of 200 times or adding 6.0-20-fold tris (2-carboxyethyl) phosphine hydrochloride (TCEP) with an excess molar ratio, and stirring the reaction solution at 10-35 ℃ for 2-48 hours; the reaction buffer described herein may be a buffer prepared in the following ratio: 50mM potassium dihydrogen phosphate-sodium hydroxide (KH) ₂ PO ₄ -NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM boric acid-borax/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9 and HPBS//1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9.

Cooling the reaction liquid to 0-10 ℃, if DTT reduction is adopted, removing excessive DTT by a desalting column or ultrafiltration after the reduction reaction is finished, adding a substituted maleimide compound (10mg/ml is dissolved in Acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) or diethyl acetamide (DMA) in advance), ensuring that the volume ratio of an organic solvent in the reaction liquid is not more than 15%, and stirring the coupling reaction at 0-37 ℃ for 2-4 hours. If TCEP is adopted for reduction, the substituted maleimide compound can be directly added for coupling without removing the residual TCEP.

The coupling reaction mixture was purified by filtration using sodium succinate/NaCl buffer or histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Or ultrafiltering for several times. Then filtering and sterilizing, and storing the obtained product at low temperature. The temperature is preferably-100-60 deg.C, and the pore size of the filter unit is preferably 0.15-0.3 μm.

The obtained antibody drug conjugate has a uniform drug antibody coupling ratio (DAR). With the differently substituted maleimide linkers (linker fragments) of the present invention, the ADC products are very homogeneous (typically, the DAR predominant product (e.g., DAR of about 4) comprises at least 60%, at least 70%, at least 80%, at least 90% or more of all ADCs). For the ADC with some difference in DAR, if a sample with better homogeneity is required, the following methods can be further used for separation and purification: hydrophobic Interaction Chromatography (HIC), Size Exclusion Chromatography (SEC), Ion Exchange Chromatography (IEC).

Pharmaceutical compositions and methods of administration

Since the antibody-drug conjugate provided by the present invention can be targeted to a specific cell population, and bound to a cell surface specific protein (antigen), so that the drug is released into the cell in an active form by endocytosis or drug infiltration of the conjugate, the antibody-drug conjugate of the present invention can be used for treating a target disease, and the above-mentioned antibody-drug conjugate can be administered to a subject (e.g., human) in a therapeutically effective amount by an appropriate route. The subject in need of treatment can be a patient at risk for, or suspected of having, a condition associated with the activity or expression of a particular antigen. Such patients can be identified by routine physical examination.

Conventional methods, known to those of ordinary skill in the medical arts, may be used to administer a pharmaceutical composition to a subject, depending on the type of disease to be treated or the site of the disease. The composition may also be administered by other conventional routes, e.g., oral, parenteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal or by implantation. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Furthermore, it may be administered to the subject of depot injectable or biodegradable materials and methods by administration of an injectable depot route, for example using 1-, 3-, or 6-month depot.

Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycols, and the like). For intravenous injection, the water-soluble antibody may be administered by a drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. Intramuscular formulations, e.g., sterile formulations of an appropriate soluble salt form of the antibody, may be dissolved and administered with a pharmaceutically acceptable excipient such as a water-change injection, 0.9% saline, or 5% dextrose solution.

When treated with the antibody-drug conjugates of the invention, delivery can be by methods conventional in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector may be delivered locally by direct injection or by use of an infusion pump. Other methods include the use of various delivery and carrier systems through the use of conjugates and biodegradable polymers.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical preparations should be adapted to the mode of administration, and the pharmaceutical compositions of the present invention may be prepared in the form of solutions, 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, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the antibody-drug conjugate of the present invention is administered at a daily dose of about 0.0001mg to 50mg/kg of animal body weight, preferably 0.001mg to 10mg/kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as urgently required by the condition being treated.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 5 to 500 mg. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.

Detection use and kit

The antibodies of the invention or ADCs thereof may be used in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumors, tissue samples prepared by endoscopic methods or needle biopsies of organs.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or 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. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

Applications of

The invention also provides the use of an antibody of the invention, e.g. for the preparation of a diagnostic formulation, or for the preparation of a medicament for the prevention and/or treatment of AXL-related diseases. The AXL related diseases comprise tumorigenesis, growth and/or metastasis, tumor drug resistance related diseases, inflammation, metabolism related diseases and the like.

Uses of the antibodies, ADCs, or CAR-T, etc., of the invention include (but are not limited to):

(i) to diagnose, prevent and/or treat tumorigenesis, growth and/or metastasis, especially AXL-high expressing tumors. Such tumors include (but are not limited to): breast cancer (such as triple negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant brain glioma, gastric cancer, liver cancer, esophageal cancer, renal cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc.; especially triple negative breast cancer, non-small cell lung cancer, pancreatic cancer, malignant brain glioma, more preferably triple negative breast cancer and/or non-small cell lung cancer.

(ii) Diagnosing, preventing and/or treating autoimmune diseases. Such autoimmune diseases include (but are not limited to): systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis.

(iii) Diagnosing, preventing and/or treating inflammation. Such inflammation includes (but is not limited to): rheumatic arthritis, osteoarthritis, ankylosing spondylitis, gout, Laert's syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.

(iv) Diagnosing, preventing and/or treating metabolic-related diseases. Such metabolic-related diseases include (but are not limited to): diabetes, food-borne obesity and steatosis.

The main advantages of the invention include:

1. the antibodies described in the present invention have novel and excellent biological activity, in particular, the preferred antibodies have high affinity for AXL (EC measured by ELISA) ₅₀ 0.04 to 0.05 nM). Furthermore, said preferred antibodies have good binding affinity for AXL on the surface of tumor cells (EC as measured by FACS) ₅₀ 0.07-0.14 nM) and can be used as therapeutic antibodies targeting AXL.

2. The humanized antibody of the invention not only has activity equivalent to or higher than that of a murine antibody, but also has lower immunogenicity.

3. The antibody-drug conjugates (ADCs) of the invention have specific AXL-dependent anti-tumor activity; the drug conjugate (ADC) of the optimized humanized antibody has no obvious toxic or side effect on cells with normal expression of AXL-and has extremely high killing activity on tumor cells with high expression of AXL-and IC of the drug conjugate is measured by a cell proliferation inhibition test ₅₀ Is 0.01nM to 0.05 nM.

4. The novel linker provided by the invention can be coupled with a targeted AXL antibody by a simple chemical method, compared with the traditional coupling mode, the distribution of DAR values of an AXL antibody drug conjugate obtained by applying the linker is very narrow, so that the uniformity of a generated product is high, the single-distribution component (DAR is 4) of the obtained linker accounts for more than 80%, and the in-vitro tumor cell proliferation inhibition activity of the linker is improved or maintained in the aspects of the patent medicine properties such as the biological activity, the safety and the like of the traditional mcVC-PAB cross-linking.

5. The disulfide chain bridging based on the maleimide has better stability, the substituent introduced into the Ar' position can regulate the reaction speed of the ring-opening hydrolysis of the maleimide and slow down the ring-closing secondary hydrolysis reaction after the ring-opening of the maleimide, the thiol exchange and the ring-closing secondary hydrolysis reaction after the ring-opening are not easy to occur in vivo, and the stability of the AXL antibody-drug conjugate in vitro and in vivo is further enhanced.

6. Compared with the AXL07-vc-MMAE in the prior art, the preferred antibody and the antibody-drug conjugate have more excellent in vitro and in vivo anti-tumor treatment effects.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The cell lines were either conventional commercially available or purchased from ATCC, and the plasmids were all commercially available.

Example 1 discovery and preparation of monoclonal antibodies targeting human AXL

Step one, preparation of hybridoma cells:

the extracellular domain of the human AXL protein (AXL-ECD) is first prepared as an antigen. Reference to NCBI: the 33 rd site to the 449 th site of the amino acid NP-068713.2, adopting gene cloning technology and a mammalian vector expression system to obtain the antigen of a C-terminal polyhistidine-tagged, wherein the specific amino acid sequence is as follows (SEQ ID NO: 36):

the human AXL extracellular domain protein expressed and prepared in HEK293T cells is adopted to immunize Balb/c mice, the dosage is 50 mug/mouse, so as to prepare immune spleen cells; murine myeloma cells (SP2/0) and feeder cells were prepared in time for fusion.

After the preparation of the three cells, the immune spleen cells and SP2/0 cells were fused by PEG-mediated fusion, PEG was removed, resuspended in HAT complete medium containing feeder cells, inoculated into a 96-well plate for culture, and positive well screening was performed by ELISA/FACS method. And finally, cloning and culturing the cells in the positive holes by a limiting dilution method, screening the cells which have high titer and good form and are in monoclonal growth by ELSIA or FASCS, and continuing to perform subclone screening until the positive cloning rate is all 100% after three continuous screening, namely performing expanded culture and library construction on the cell strain.

Step two, purification of the targeted human AXL murine monoclonal antibody:

after the hybridoma cells selected in step (i) were subjected to extensive culture in a roller bottle for 14 days, a cell culture supernatant was collected, filtered through a 0.22 μ M filter, and the obtained culture supernatant was applied to a Protein a resin column equilibrated in advance at a constant rate, and the column was equilibrated with 0.1M Tris-HCl (PH 8.0, containing 1.5M NaCl). Then, the equilibrium column was eluted with 0.1M sodium citrate buffer, and the eluate was collected and quantified and subjected to SDS-PAGE electrophoresis, SEC-HPLC and endotoxin detection. Subpackaging the obtained purified antibody and freezing at-80 ℃ for later use.

Step three, determination of biological activity and specificity of the targeted human AXL mouse-derived monoclonal antibody:

and through repeated screening, the biological activity and the target specificity of the 6 selected hybridoma monoclonal antibodies are determined. As shown in FIG. 1A, when the supernatant of the culture solution of the monoclonal cell was detected by a flow cytometric fluorescence sorter (FACS), 6 monoclonal antibodies were able to specifically bind to MDA-MB-231 cells (AXL-P) with high expression of human AXL, but had no significant binding activity to MDA-MB-453 cells (AXL-N) with low expression of AXL. As shown in FIG. 1B, the purified antibody samples were used for subtype detection, and mAb 001-mAb 005 were identified as IgG1/k and mAb006 was IgG 2B/k.

Step (iv) using the purified antibody sample, performing gradient dilution and ELISA detection, wherein mAb 001-mAb 006 have excellent binding affinity to AXL-ECD as shown in Table-1, wherein EC of mAb001, mAb002, mAb005 and mAb006 ₅₀ All were < 0.1 nM.

TABLE-1: ELISA Activity of Targeted human AXL original murine antibody

Example 2 sequencing of antibodies, identification of Complementarity Determining Regions (CDRs)

Based on excellent specificity and affinity, mAb001, mAb002, mAb005 and mAb006 are preferably selected for antibody sequencing identification. Primers were designed to amplify the heavy (VH) and light (VL) variable region fragments (see fig. 2) by conventional PCR techniques, cloned into vectors, and sequenced. The following heavy chain variable region (VH), light chain variable region (VL) amino acid sequences, Complementarity Determining Region (CDR) information were obtained using conventional sequencing and analysis by Kabat database (http:// www.bioinf.org.uk), and the CDR-1/2/3 amino acid sequence is shown underlined as "__". The CDR sequences of mAb006c and mAb005c were highly similar to each other as noted after gene sequencing and are not separately listed.

SEQ ID No.:7 mAb002 heavy chain variable region (VH) amino acid sequence

SEQ ID No.: 15 mAb005 heavy chain variable region (VH) amino acid sequence

SEQ ID No.: 23 mAb001 heavy chain variable region (VH) amino acid sequence

SEQ ID No.:8 mAb002 light chain variable region (VL) amino acid sequence

SEQ ID No.: 16 mAb005 light chain variable region (VL) amino acid sequence

SEQ ID No.: variable region (VL) amino acid sequence of 24 mAb001 light chain

EXAMPLE 3 preparation of human-murine chimeric antibody

3 groups of variable region sequences (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) were cloned into a vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region by gene recombination technique, sequenced, and then transfected and mammalian expression system (FreeStyle) ^TM 293T cells) the constructed chimeric antibody was expressed and purified (see fig. 3), and the obtained human-mouse chimeric antibody, the number, heavy chain and light chain composition are shown in table-2. The humanized antibody AXL07 disclosed in the invention patent application CN201580045131.4 was prepared in the same manner as a control.

TABLE-2: preparation of human-mouse chimeric antibody

Example 4 ELISA assay of the affinity of chimeric antibodies to human AXL protein

The extracellular domain of AXL protein (AXL-ECD) was diluted to 1. mu.g/mL with a coating solution, and ELISA plates were coated at 100. mu.L/well at 4 ℃ overnight. Washing off redundant antigen, blocking for 2h at room temperature by using 1% BSA, then adding each monoclonal antibody diluted by 3 times in a gradient manner, and incubating for 1h at room temperature, wherein each monoclonal antibody is 100L/hole; unbound antibody was washed off, a suitable concentration of horseradish peroxidase-labeled anti-mouse secondary antibody was added, 100. mu.L/well, and incubated at room temperature for 0.5 h. Unbound secondary antibody was washed off, TMB developing solution was added for reaction for about 15min, 1N HCl was added at 50. mu.L/well to terminate the developing reaction, and then absorbance was measured at 450nm and the data was analyzed.

The detection results are shown in FIG. 4, mAb001c, mAb002c, mAb005c and mAb006c have strong affinity to AXL-ECD, and specific EC ₅₀ Values as shown in table-3, mAb002c had slightly higher affinity for AXL-ECD than the control antibody AXL 107.

TABLE-3: ELISA assay Activity of chimeric antibodies

Example 5 AXL protein is highly expressed in various tumor cells

Aiming at a plurality of different molecular typing breast cell strains (MDA-MB-231, Hs578T and MDA-MB-453), lung cancer cell strains (NCI-H1299, Calu-1 and NCI-H460) and pancreatic cancer cell strains (SW1990, Capan-2, Panc-1 and Canpan-1), total cell protein is prepared and accurately quantified, and the expression level of the AXL protein is detected by an immunoblot test (Western blot). The results are shown in fig. 5, and AXL protein is expressed in abnormal activation in most of breast cancer, lung cancer and pancreatic cancer cell lines tested.

Example 6 human tumor and Normal tissue Gene expression database analysis

The expression of AXLmRNA levels in tumor Cell populations (e.g., breast Cancer, lung Cancer, brain glioma, melanoma) relative to human normal tissues was analyzed by downloading gene expression information from the CCLE (Cancer Cell line encyclopedia) database, the G-Tex (human normal tissue) database, and the 51-line human breast Cancer Cell line database (New RM et al, Cancer Cell 2006; 10: 515-27). The present example also analyzed the expression levels of axlmna in breast cancers of different molecular classifications (e.g., luminal versus basal), lung cancers of different degrees of malignancy (e.g., epithelial versus interstitial).

The results are shown in fig. 6, comparing the CCLE database and the G-Tex database, the average AXL mRNA expression level of highly aggressive breast cancer, lung cancer, glioma and melanoma cell lines is significantly higher than that of normal tissues. The antibody taking the AXL as the target point has obvious effects in the application of diagnosing, preventing and treating triple negative breast cancer, lung cancer and glioma.

As shown in fig. 7, the mean axlmna expression level of the Basal-type (Basal-type) breast cancer cell line with high invasion and high metastasis was significantly higher than that of the Luminal type (Luminal-type) breast cancer cell line, and was statistically significant. Since basal breast cancer is the main source of clinical triple negative breast cancer, the antibody targeting AXL of the present invention will have more significant effects in the application of diagnosis, prevention and treatment of triple negative breast cancer.

The results are shown in fig. 8, which shows that the mean AXL mRNA expression level of the high metastatic/interstitial (EMT-high) lung cancer cell line is significantly increased compared to the low metastatic/epithelial (EMT-low) lung cancer cell line, and has statistical significance. As most of the highly metastatic lung cancer is clinically drug-resistant and has poor prognosis, the antibody taking the AXL as the target point has more remarkable effects in the application of diagnosing and treating the highly metastatic, drug-resistant and advanced lung cancer.

Example 7 FACS detection of specific binding of the AXL protein on the surface of tumor cells to the chimeric antibody

The combination of the chimeric antibody mAb002c on the cell surface AXL is determined by using AXL-high expression non-small cell lung cancer cells NCI-1299 and LCLC-103H, Calu-1, high expression triple negative breast cancer cells MDA-MB-231 and Hs578T, and AXL-low expression breast cancer cell MDA-MB-453 as a target cell. Using 3x10 ⁵ The individual tumor cells were mixed with the antibody (final concentration 5. mu.g/mL), incubated at 4 ℃ for 1 hour, washed with PBSCells were washed twice to remove unbound primary antibody, then 200 μ L (2 μ g/mL) PE-labeled secondary antibody was incubated at 4 ℃ for 30min, PBS washed twice to remove unbound secondary antibody, and finally cells were resuspended in 200 μ L PBS, and the Binding affinity (Binding affinity) of the test antibody to cell surface AXL, or the total Binding fluorescence intensity (MFI) at the same antibody concentration for different tumor cells was determined by flow cytometry FACSCalibur.

As shown in FIG. 9, the chimeric antibody mAb002c specifically recognized and bound AXL-highly expressed tumor cells, and the fluorescence intensity sequence of the binding rate was NCI-H1299, LCLC-103H, MDA-MB-231 and Hs578T, while the antibody mAb MDA-MB-453 shows weak binding fluorescence intensity to AXL-less expressed tumor cells. Comparing the binding rates (MFI) of NCI-H1299 and LCLC-103H to the antibody with the binding rates of MDA-MB-453 to the antibody, it can be seen that the binding rates of mAb002c differ by 127-fold and 91-fold, respectively.

Example 8 determination of the binding affinity of the chimeric antibody to AXL on the surface of tumor cells

AXL-highly expressed triple negative breast cancer cell MDA-MB-231 was used as a target cell, 100. mu.L of test antibody diluted from 200nM to 0.091nM in a 3-fold gradient was used as a primary antibody, and the primary antibody was separately added to 1X10 suspended in 100. mu.L of RPMI-1640 serum-free medium ⁵ The MDA-MB-231 was mixed well and incubated at 4 ℃ for 1h, the cells were washed twice with PBS to remove unbound primary antibody, the target cells were incubated with 200. mu.L, 2. mu.g/mL, PE-labeled secondary antibody for 30min at 4 ℃, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200. mu.L of PBS and the Binding affinity (Binding affinity) of the test antibody to cell surface AXL was determined by flow cytometry FACSCalibur.

The test results are shown in FIG. 10 and Table 4, and mAb001c, mAb002c, mAb005c and mAb006c have strong affinity to MDA-MB-231 cells, wherein mAb002c has significantly higher affinity to MDA-MB-231 cells than control antibody AXL 107.

TABLE-4: binding Activity of chimeric antibodies to MDA-MB-231 cells

Using AXL-highly expressed lung cancer NCI-H1299 as a target cell, 100. mu.L of a test substance diluted in a 3-fold gradient was used as a primary antibody against 1X10 suspended in 100. mu.L of RPMI-1640 serum-free medium ⁵ The NCI-H1299 cells were mixed well and, as above, resuspended in 200. mu.L PBS and the Binding affinity (Binding affinity) of the test antibody for cell surface AXL was determined by flow cytometry, FACSAria II.

The test results are shown in FIG. 11 and Table 5, and mAb001c, mAb002c, mAb005c and mAb006c have strong affinity to NCI-H1299 cells, wherein mAb002c has significantly higher affinity to NCI-H1299 cells than the control antibody AXL 107.

TABLE-5: binding Activity of chimeric antibodies on NCI-H1299 cells

Example 9 preparation of humanized antibody

The humanized template that best matches the mAb002 non-CDR regions was retrieved and selected in the Germline database, the CDR regions of the antibody were then grafted onto the selected humanized template, the CDR regions of the human template were replaced, and the antibody was recombined with the IgG1 constant region, while the embedded residues, residues that directly interact with the CDR regions, and residues that had important effects on the conformation of VL and VH were back mutated based on the three-dimensional structure of the murine antibody.

Specifically, the humanization implementation of mAb002c yielded 3 variable regions of the humanized heavy chain (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27), and 8 variable regions of the humanized light chain (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.：25 mAb002_VHg0

SEQ ID NO.：26 mAb002_VHg1

SEQ ID NO.：27 mAb002_VHg2

SEQ ID NO.：28 mAb002_VKg0

SEQ ID NO.：29 mAb002_VKg1

SEQ ID NO.：30 mAb002_VKg2

SEQ ID NO.：31 mAb002_VKg3

SEQ ID NO.：32 mAb002_VKg4

SEQ ID NO.：33 mAb002_VKg5

SEQ ID NO.：34 mAb002_VKg6

SEQ ID NO.：35 mAb002_VKg7

Cloning the designed humanized variable region sequence into a vector containing human IgG1 heavy chain constant region and Kappa chain constant region by gene recombination technology, sequencing, and using transfection technology and mammalian expression system (FreeStyle) ^TM 293 cells) the constructed humanized antibody expression vector. The final mAb002c group obtained 24 humanized antibodies by expressing these humanized heavy and light chains in combination, respectively, and the corresponding heavy and light chain combinations for each antibody are shown in Table-6.

TABLE-6: preparation of humanized antibody

Example 10 binding affinity of humanized antibodies to AXL-ECD

The 24 humanized antibodies in Table-6 were diluted in a gradient and their affinity for the AXL-ECD protein was determined by ELISA, and the experimental procedure was as described in example 4.

The experimental results are shown in FIG. 12 and Table-7, and the humanized antibodies Hu002c-1 to Hu002c-24 all have strong binding affinity to AXL-ECD protein, and EC ₅₀ The values were 0.043nM to 0.082 nM.

TABLE-7: ELISA test Activity of humanized antibodies

Example 11 binding affinity of humanized antibody to tumor cell AXL

The 24 humanized antibodies in Table-6 were diluted in a gradient and their affinity for AXL on the cell surface of MDA-MB-231 was determined by flow cytometry, the experimental procedure being as described in example 8.

Experimental results As shown in FIG. 13 and Table-8, the humanized antibody has high binding affinity activity to AXL on the cell surface of MDA-MB-231, and EC ₅₀ The values were 0.073nM to 0.17nM, indicating a higher affinity than the control AXL107(0.43 nM).

TABLE-8: binding Activity of humanized antibodies to MDA-MB-231 cells

The 4 humanized antibodies in table-6 were diluted in a gradient and their affinity for LCLC-103H cell surface AXL was determined by flow cytometry, the experimental procedure was according to example 8.

As shown in FIG. 14, the results of the experiments showed that the humanized antibodies Hu002-1, Hu002-2, Hu002-4 and Hu002-5 have high binding affinity activity to AXL on the surface of LCLC-103H cells, and EC has been shown ₅₀ The values were 0.28nM, 0.37nM, 0.49nM, 0.36nM, respectively, indicating a higher affinity than the control AXL107(0.80 nM).

Example 12 binding of humanized antibodies to tumor cells results in endocytosis to intracellular lysosomes

Adopting an AXL-high-expression mammary gland cell strain MDA-MB-231 as a target cell, paving 50% density MDA-MB-231, culturing the MDA-MB-231 in a laser confocal culture dish for 16h, adding 5 mu g/mL (diluted in 1640 culture medium containing 10% fetal bovine serum) of an AXL humanized antibody Hu002-2, and respectively incubating at 37 ℃ for 4 h or 4 ℃ for 1h (serving as a control); PBS was washed 3 times to remove the antibody not bound to the cells, fixed with 4% paraformaldehyde (diluted in PBS) for 30min at room temperature; PBS wash 3 times, using 0.4% Triton X-100 (diluted in PBS) room temperature in the permeabilization of cells for 10 min; after 3 times of PBS washing, the cells were incubated for 1 hour at 37 ℃ using LAMP-2 (rabbit anti-human) antibody to label the lysosome position of the cells; washing away the unbound antibody by PBS, and incubating the R-PE labeled goat anti-human and Alexa 488 secondary antibody for 30min at 37 ℃; unbound secondary antibody was washed with PBS, stained with DAPI for 10min to locate nuclei, after which endocytosis of the antibody was detected using a Fluorescence microscope (Leica, 20 ×).

As a result, Hu002-2 was rapidly and extensively endocytosed into lysosomes by MDA-MB-231 cells, as shown in FIG. 15. The result shows that the antibody is suitable for preparing an antibody-drug conjugate (ADC), and the AXL-ADC has good ADC drug characteristics and can be used for broad-spectrum and high-specificity treatment of the AXL-positive tumor targeted drug.

Example 13 binding of humanized antibodies to tumor cells results in a reduction in protein expression levels of tumor AXL

Adopting an AXL-high-expression lung cancer cell LCLC-103H as a target cell, and paving the target cell in a 12-hole plate according to 16% confluence; 16 hours after attachment, the medium was changed to serum-free medium containing PBS, subtype control hIgG1, Hu002-2 of 2. mu.g/mL, or antibody-drug conjugate (ADC) Hu002-2-BL20-MMAE (prepared in example 15 below), respectively, in each of two wells; respectively collecting protein samples which are incubated for 24 hours and 48 hours; the change in the amount of AXL protein expression in cells was detected using immunoblotting.

The results are shown in fig. 16, where there was no significant change in AXL expression in the cell as a whole after PBS or hIgG1 administration, whereas AXL expression was significantly down-regulated in LCLC-103H cells after Hu002-2 or its ADC administration, probably due to degradation in lysosomes after the antibody or ADC binds to the cell and is endocytosed.

Example 14 preparation of AXL107-vc-MMAE, AXL107-BL20-MMAE

The humanized antibody AXL107 stock solution targeting AXL was added with PBS/EDTA (pH 7.4) buffer to a concentration of 20mg/ml, then reduced with 2.6eq of TCEP at 25 ℃ for 2 hours, taken out and placed on ice to cool, 6.0eq of mc-VC-PAB-MMAE (purchased from shanghai hao chem., pre-dissolved in DMA) was added without purification, reacted at 0 ℃ for 1 hour, and the reaction was stopped by adding cysteine. Removing excessive small molecules by using G25 desalting column, replacing with 20mM citric acid-sodium citrate/6% sucrose, pH 6.6 buffer, sterilizing with 0.22 μm pore size filter device, and storing at-80 deg.C to obtain antibody conjugate named AXL 107-vc-MMAE.

As shown in FIGS. 17, 19 and 20, the HIC and mass spectra (FIGS. 17 and 20) of the humanized antibody AXL107 and the antibody conjugate AXL107-vc-MMAE both showed that the antibody conjugate AXL107 was formed after the conjugation reaction of the humanized antibody AXL107, the molecular weight of the conjugate coincided with the expected value, and the average DAR value was about 4.0.

The AXL107 stock solution was replaced with 50mM sodium dihydrogenphosphate-disodium hydrogenphosphate (NaH2PO4-Na2HPO4)/150mM sodium chloride (NaCl)/2mM ethylenediaminetetraacetic acid (EDTA) at pH 7.0 to give a concentration of 10mg/mL, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a molar excess ratio of 10 times, and the reaction mixture was stirred at 25 ℃ for 4 hours. Excess TCEP was removed by means of a G25 desalting column, and then a suitable amount of Diethylacetamide (DMA) was added to the reduced antibody collected, followed by addition of 6-fold excess molar ratio of compound Ic-4(10mg/ml pre-dissolved in DMA) to ensure that the volume of DMA in the reaction system was not more than 10%, and the coupling was carried out by stirring at 20 ℃ for 2.0 hours. The coupling reaction mixture was purified by filtration through a desalting column using Tris-HCl/sucrose gel pH 7.5, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.22 micron pore size filter unit and stored at-80 ℃ and the resulting antibody conjugate was designated AXL107-BL 20-MMAE.

The results are shown in fig. 18, 19 and 21, and the mass spectrum (fig. 19) of the humanized antibody AXL107 and the HIC and mass spectrum (fig. 18 and 21) of the antibody conjugate AXL107-BL20-MMAE both indicate that the antibody conjugate AXL107-BL20-MMAE is formed after the antibody AXL107 is subjected to the conjugation reaction, the molecular weight of the conjugate is consistent with the expected value, and the DAR is about 4.0.

Example 15 preparation of mAb002c-vc-MMAE, mAb002c-BL20-MMAE, humanized antibody series Hu002-BL20-MMAE

To a stock solution of the chimeric antibody mAb002c targeting AXL was added PBS/EDTA (pH 7.4) buffer at 20mg/ml, followed by 2.6eq of TCEP at 25 ℃ for 2 hours, taken out and cooled on ice, and 6.0eq of mc-VC-PAB-MMAE (purchased from shanghai hao chem., pre-dissolved in DMA) was added without purification, reacted at 0 ℃ for 1 hour, and then terminated by addition of cysteine. Excess small molecules were removed using a G25 desalting column and replaced into 20mM citric acid-sodium citrate/6% sucrose, pH 6.6 buffer, sterilized by a 0.22 micron pore size filter unit, -stored at 80 ℃ and the resulting antibody conjugate was designated mAb002 c-vc-MMAE.

As shown in FIGS. 22, 24 and 25, the mass spectrum of antibody mAb002c (FIG. 24) and the HIC and mass spectrum of its antibody conjugate mAb002c-vc-MMAE (FIG. 22 and FIG. 25) both showed that antibody mAb002c formed the antibody conjugate mAb002c-vc-MMAE after conjugation, the molecular weight of the conjugate was consistent with the expected value, and the average DAR value was about 4.0.

The mAb002c stock solution was replaced with 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (NaH2PO4-Na2HPO4)/150mM sodium chloride (NaCl)/2mM ethylenediaminetetraacetic acid (EDTA) at pH 7.0 to give a concentration of 10mg/mL, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a molar excess ratio of 10 times, and the reaction solution was stirred at 25 ℃ for 4 hours. Excess TCEP was removed by means of a G25 desalting column, and then a suitable amount of Diethylacetamide (DMA) was added to the collected reduced antibody, followed by addition of 6-fold excess molar ratio of compound Ic-4(10mg/ml pre-dissolved in DMA) to ensure that the volume ratio of DMA in the reaction system did not exceed 10%, and the coupling was carried out by stirring at 20 ℃ for 2.0 hours. The coupling reaction mixture was purified by filtration through a desalting column using Tris-HCl/sucrose gel pH 7.5, and peak samples were collected according to UV280 UV absorbance. The antibody conjugate was then sterilized by filtration through a 0.22 micron pore size filter unit and stored at-80 ℃ and was designated mAb002c-BL 20-MMAE.

As shown in FIGS. 23, 24 and 26, the mass spectrum of the antibody mAb002c (FIG. 24) and the HIC and mass spectrum of its antibody conjugate mAb002c-BL20-MMAE (FIGS. 23 and 26) both showed that the antibody mAb002c formed the antibody conjugate mAb002c-BL20-MMAE after conjugation reaction, the molecular weight of the conjugate coincided with the expected value, and the DAR was about 4.0.

The preparation method of the humanized serial antibody Hu002-BL20-MMAE is the same as the preparation method of the mAb002c-BL 20-MMAE. Taking the humanized antibody Hu002-2-BL20-MMAE as an example, the results are shown in FIGS. 27, 28 and 29, and the mass spectrum (FIG. 52) of the antibody Hu002-2 and the HIC and mass spectrum (FIGS. 27 and 28) of the antibody conjugate Hu002-2-BL20-MMAE show that the antibody Hu002-2 forms the antibody conjugate Hu002-2-BL20-MMAE after the coupling reaction, the molecular weight of the conjugate is consistent with the expected value, and the DAR is about 4.0.

Example 16 in vitro anti-tumor Activity of AXL chimeric antibody-drug conjugate (AXL-ADC) against AXL highly expressed triple negative breast, lung and brain glioma cells

The cell lines used in this example were purchased from the American Type Culture Collection (ATCC) or cell bank of national academy of sciences 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, U87 MG.

Cell proliferation assay: inoculating the cells in logarithmic growth phase into a 96-well cell culture plate at a density of 600-2 and 500 cells per well (according to the growth rate of different cells), culturing at 37 ℃ and 5% CO2 for about 5-12 hours, adding AXL-ADCs at different concentrations, setting 3 multiple wells and corresponding solvent control and blank control wells for each drug concentration, pouring out the culture solution after 4-6 days of action, adding MTS reaction solution (purchased from Promega, cat # G3581) and 100 muL per well, reacting to the expected color depth at 37 ℃, and then measuring the cell viability (OD490nm) of each group by a multifunctional enzyme labeling instrument (BioTek Synergy II) and calculating the cell viability according to the following formula: survival rate was (OD dose-OD blank)/(OD control-OD blank) × 100%. Each proliferation assay set was independently repeated 3-4 times. The data were analyzed by GraphPad Prism 5 software and the IC of the drug on different cell lines was calculated separately ₅₀ The value is obtained.

The experimental result shows that the AXL antibody-drug conjugate mAb002c-vc-MMAE and mAb002c-BL20-MMAE have high AXL-targeting specificityThe sexual cytotoxic activity is that no obvious proliferation inhibition effect is exerted on MDA-MB-453 cells with low AXL-expression (figure 30), and strong proliferation inhibition activity is exerted on MDA-MB-231 (figure 31), Hs578T (figure 32), lung cancer Calu-1 (figure 33), LCLC-103H (figure 34) and glioma U87MG (figure 35) cells with high AXL-expression, and IC is ₅₀ The values were 0.03nM to 0.07nM (Table-9). In addition, the inhibitory activity of the antibody-drug conjugate mAb002c-vc-MMAE and mAb002c-BL20-MMAE on AXL-high expression tumor cells is obviously stronger than that of the contrast antibody-drug conjugate AXL107-vc-MMAE and AXL107-BL 20-MMAE.

TABLE-9: in vitro anti-tumor Activity of chimeric antibody-drug conjugates

Example 17 in vitro anti-tumor Activity of humanized AXL-ADCs

Similarly, referring to the detection method of example 16, the AXL humanized antibody-drug conjugate Hu002-1/2/4/5/7/16-BL20-MMAE of the present invention has very high AXL-targeting specific cytotoxic activity, i.e. has no obvious proliferation inhibition effect on AXL-low expressing MDA-MB-453 cells, while it has very strong proliferation inhibition activity on AXL-high expressing triple negative breast cancer MDA-MB-231 (fig. 36), Hs578T (fig. 37), glioma U87MG (fig. 38) and lung cancer LCLC-103H (fig. 39) cells, IC ₅₀ The values were 0.013nM to 0.05nM (Table-10).

TABLE-10: in vitro anti-tumor Activity of humanized AXL-ADC

Example 18 in vivo anti-tumor Activity of AXL-ADC

Respectively mixing 200 μ L of the extract containing 5 × 10 ⁶ U87MG, LCLC-103H cell suspension was inoculated subcutaneously into the back of female immunodeficient mice (Balb/c nude, 6-8 weeks old). When the volume of the tumor reaches 100-300 mm ³ And can observe obvious tumor growth according to the tumor volumeRandomly grouping the size and the weight of the nude mice (n is 6-8), and respectively adopting doses of 25mg/kg, 3mg/kg, 1mg/kg and 0.5mg/kg for intravenous administration once per week for 2 weeks; meanwhile, hIgG1-BL20-MMAE is 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. After the experiment is finished, counting and analyzing experimental data, drawing a tumor growth curve and a weight change curve of a nude mouse, taking out the subcutaneous transplanted tumor by an operation, and weighing, wherein the tumor volume (V) is calculated by the following formula: v ═ lxw ² And/2, wherein L, W represents the length and width of the tumor respectively.

The chimeric antibody mAb002c and 4 preferred humanized antibodies Hu002-1, Hu002-2, Hu002-4 and Hu002-5 were respectively coupled with vc-MMAE or BL20-MMAE, and the in vivo antitumor activity was evaluated by comparing the prior art AXL 107-vc-MMAE. Preparation of humanized AXL-ADC reference example 15.

As shown in FIG. 40, mAb002c-BL20-MMAE or mAb002c-vc-MMAE at a dose of 5mg/kg in the U87MG tumor model showed very significant and similar anti-tumor effects during the administration period, but the mAb002c-vc-MMAE group showed tumor recovery growth after 3 weeks of drug withdrawal, and the anti-tumor effect was significantly lower than that of mAb002c-BL20-MMAE group, indicating that BL20-MMAE linker was more advantageous in vivo.

As shown in fig. 41 and 42, in the U87MG tumor model, 4 preferred humanized AXL-ADCs at a dose of 3mg/kg and AXL107-vc-MMAE were all observed to have excellent antitumor activity when tested for pharmacodynamic effects in vivo; and preferred AXL-ADCs of the invention are capable of delivering a more significant in vivo tumor treatment effect than AXL 107-vc-MMAE.

The results of the three in vivo efficacy tests described above with U87MG were summarized and counted (Table-11).

TABLE-11: in vivo antitumor Activity of AXL-ADC (U87MG model)

As shown in FIGS. 43 and 44, in the LCLC-103H tumor model, the 4 preferred AXL-ADCs of the present invention showed dose-related therapeutic effects at both the administered doses of 3mg/kg and 1mg/kg, and resulted in significant tumor regression, indicating that LCLC-103H tumors are highly sensitive to treatment with AXL-ADC.

As shown in FIG. 45, in the repeated experiments of LCLC-103H tumor, Hu002-1-BL20-MMAE, Hu002-2-BL20-MMAE and Hu002-5-BL20-MMAE administered at a dose of 1mg/kg all resulted in significant tumor regression, and among them Hu002-2-BL20-MMAE had the most significant effect on tumor regression.

The results of the three LCLC-03H in vivo efficacy tests described above were summarized and counted (Table-12).

TABLE-12: in vivo antitumor Activity of AXL-ADC (LCLC-103H model)

As shown in FIG. 46 and Table-13, in another repeated experiment of LCLC-103H, Hu002-2-BL20-MMAE and Hu002-5-BL20-MMAE administered at a dose of 1mg/kg were again confirmed to induce tumor regression, and the effect was significantly better than that of AXL107-vc-MMAE at the same dose; meanwhile, 0.5mg/kg Hu002-2-BL20-MMAE also has a certain tumor inhibition effect.

TABLE-13: in vivo anti-tumor Activity of AXL-ADC (LCLC-103H model)

Example 19 regressive Activity of humanized AXL-ADC on Large-volume tumors

Based on the high sensitivity of LCLC-103H lung cancer to AXL-ADC, this example studies the activity of AXL-ADC on tumors of very large volume; when the growth volume of the tumor reaches 1000- ³ Treatment is administered and the inhibition of tumor growth or regression of the tumor is observed, as is the test method described in example 18.

The results are shown in FIG. 47, in which LCLC-103H tumors grew in vivo to a volume of 800mm ³ The single administration of 5mg/kg Hu002-2-BL20-MMAE at day 34 of treatmentBy the time the tumor has completely regressed.

The results are shown in FIG. 48, and similarly LCLC-103H tumors grew in vivo to a volume of 1800mm ³ When 10mg/kg dose of Hu002-2-BL20-MMAE is administered once, the tumor regression rate reaches more than 90 percent on the 20 th day of treatment.

Example 20 determination of binding Activity of humanized AXL antibodies on mouse AXL and cynomolgus monkey AXL

1. The protein sequence of monkey full-length AXL (Genebank ID: XP-014979499.1; 894 amino acids) and the protein sequence of mouse full-length AXL (Genebank ID: NP-033491.2; 888 amino acids) were used. The specific amino acid sequences are respectively represented by SEQ ID No.: 39. SEQ ID No.: 40 is listed;

2. constructing the total length gene sequence of artificially synthesized cynomolgus monkey and mouse into a mammal expression vector pcDNA3.1 to prepare positive expression vector plasmid;

3. selecting HEK293T cells, plating them in 50% density in petri dishes; after overnight incubation at 37 ℃ the confluency was about 80%, 2. mu.g of the monkey/mouse-AXL vector plasmid prepared above was transiently transfected using Liposome Lipo2000 (Invitrogen); proteins were harvested 24 hours after transfection for immunoblot (Western blot) analysis, while cells were harvested for FACS detection of Hu002-2 binding activity to monkey/mouse AXL on the surface of HEK293T cells. The FACS detection assay method is described in example 8.

The results are shown in fig. 49, the AXL humanized antibody Hu002-2 has similar effect to the control antibody AXL107, has poor binding ability to mouse AXL on the HEK-293T surface, and still does not reach a binding saturation platform at the highest concentration (200nM) of the antibody.

The results are shown in fig. 50, and demonstrate the transient transfection efficiency of monkey AXL and the binding ability of humanized antibody Hu002-2 to it by immunoblotting (fig. 50A); meanwhile, through FACS detection, Hu002-2 has excellent binding affinity to the surface of HEK-293T cell cynomolgus monkey AXL, EC ₅₀ At 0.135nM (fig. 50B), this result supports the use of cynomolgus monkeys in preclinical study models to evaluate toxicity, pharmacokinetics, and toxicity of the Hu002 series of antibodies.

Comparison of the antibodies, antibody-drug conjugates described in example 21 with the prior art

The heavy chain and light chain variable regions of AXL107 antibody disclosed in patent application No. CN201580045131 are artificially synthesized and cloned into a vector containing human IgG1 heavy chain constant region and Kappa chain constant region, and the sequence is determined without errors in FreeStyle ^TM AXL107 was obtained by expression and purification of 293T cell line (example 3), which was then prepared as an antibody-drug conjugate of AXL107 (example 14). AXL107, AXL107-vc-MMAE, AXL107-BL20-MMAE were added to the study of the present invention as reference drugs.

AXL 107-heavy chain variable region (VH) SEQ ID No.: 37

AXL 107-light chain variable region (VL) SEQ ID No.: 38

The activity results of the comparative tests are summarized below:

1. as a result, as shown in tables-3, 4, 5, 7 and 8, mAb005c of the present invention had a target affinity comparable to that of AXL107, whereas mAb002c and the humanized Hu002 series of antibodies had a higher affinity for AXL, and a higher tumor inhibitory activity was expected;

2. the results are shown in tables-9 and-10, and based on higher tumor cell affinity, the AXL-ADC prepared by mAb002c-vc-MMAE, mAb002c-BL20-MMAE and corresponding humanized Hu002 antibody all show stronger AXL-targeting specific in vitro anti-tumor effect;

3. the results are shown in Table-11 (FIG. 41) and Table-13 (FIG. 46), consistent with the in vitro results, a plurality of preferred humanized AXL-ADCs of the present invention have excellent antitumor effects in vivo, with activities superior to those of the prior art AXL 107-vc-MMAE;

4. the results are shown in FIG. 23 and FIG. 26, mAb002c-BL20-MMAE has higher material uniformity than AXL107-vc-MMAE (FIG. 17 and FIG. 20) in the prior art, and the proportion of single distribution component (DAR4) reaches more than 90%;

5. further in vivo tumor pharmacodynamic tests directly compared the therapeutic effects of BL20-MMAE and vc-MMA linker, the results showed that BL20-MMAE was more excellent than vc-MMAE (FIG. 40, Table-11).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> university of Compound Dan

Shanghai Medicine Inst., Chinese Academy of Sciences

<120> AXL-targeted antibody, antibody-drug conjugate, preparation method and application thereof

<130> P2019-2058

<150> CN201810464287.1

<151> 2018-05-15

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Ser Gly Tyr Trp Ser

1 5

<210> 18

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Tyr Met Thr Tyr Ser Gly Ala Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 19

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Gly Asn Ser Tyr Phe Phe Asp Tyr

1 5

<210> 20

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala

1 5 10

<210> 21

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ala Ala Thr Asn Leu Ala Asp

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gln His Phe Trp Gly Thr Pro Leu Thr

1 5

<210> 23

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Ser Met

35 40 45

Gly Tyr Met Thr Tyr Ser Gly Ala Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Asn Ser Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Leu Lys Gln Gly Lys Ser Pro His Leu Leu Val

35 40 45

Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ile Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 25

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asp Phe

20 25 30

Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 26

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asp Phe

20 25 30

Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 27

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asp Phe

20 25 30

Tyr Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 28

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 29

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ser Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 31

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Ser Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Ser Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 35

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Gln Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Gly Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Ser Leu Ile Tyr

35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 429

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Gln Ala Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn Ile Thr Gly

1 5 10 15

Ala Arg Gly Leu Thr Gly Thr Leu Arg Cys Gln Leu Gln Val Gln Gly

20 25 30

Glu Pro Pro Glu Val His Trp Leu Arg Asp Gly Gln Ile Leu Glu Leu

35 40 45

Ala Asp Ser Thr Gln Thr Gln Val Pro Leu Gly Glu Asp Glu Gln Asp

50 55 60

Asp Trp Ile Val Val Ser Gln Leu Arg Ile Thr Ser Leu Gln Leu Ser

65 70 75 80

Asp Thr Gly Gln Tyr Gln Cys Leu Val Phe Leu Gly His Gln Thr Phe

85 90 95

Val Ser Gln Pro Gly Tyr Val Gly Leu Glu Gly Leu Pro Tyr Phe Leu

100 105 110

Glu Glu Pro Glu Asp Arg Thr Val Ala Ala Asn Thr Pro Phe Asn Leu

115 120 125

Ser Cys Gln Ala Gln Gly Pro Pro Glu Pro Val Asp Leu Leu Trp Leu

130 135 140

Gln Asp Ala Val Pro Leu Ala Thr Ala Pro Gly His Gly Pro Gln Arg

145 150 155 160

Ser Leu His Val Pro Gly Leu Asn Lys Thr Ser Ser Phe Ser Cys Glu

165 170 175

Ala His Asn Ala Lys Gly Val Thr Thr Ser Arg Thr Ala Thr Ile Thr

180 185 190

Val Leu Pro Gln Gln Pro Arg Asn Leu His Leu Val Ser Arg Gln Pro

195 200 205

Thr Glu Leu Glu Val Ala Trp Thr Pro Gly Leu Ser Gly Ile Tyr Pro

210 215 220

Leu Thr His Cys Thr Leu Gln Ala Val Leu Ser Asp Asp Gly Met Gly

225 230 235 240

Ile Gln Ala Gly Glu Pro Asp Pro Pro Glu Glu Pro Leu Thr Ser Gln

245 250 255

Ala Ser Val Pro Pro His Gln Leu Arg Leu Gly Ser Leu His Pro His

260 265 270

Thr Pro Tyr His Ile Arg Val Ala Cys Thr Ser Ser Gln Gly Pro Ser

275 280 285

Ser Trp Thr His Trp Leu Pro Val Glu Thr Pro Glu Gly Val Pro Leu

290 295 300

Gly Pro Pro Glu Asn Ile Ser Ala Thr Arg Asn Gly Ser Gln Ala Phe

305 310 315 320

Val His Trp Gln Glu Pro Arg Ala Pro Leu Gln Gly Thr Leu Leu Gly

325 330 335

Tyr Arg Leu Ala Tyr Gln Gly Gln Asp Thr Pro Glu Val Leu Met Asp

340 345 350

Ile Gly Leu Arg Gln Glu Val Thr Leu Glu Leu Gln Gly Asp Gly Ser

355 360 365

Val Ser Asn Leu Thr Val Cys Val Ala Ala Tyr Thr Ala Ala Gly Asp

370 375 380

Gly Pro Trp Ser Leu Pro Val Pro Leu Glu Ala Trp Arg Pro Gly Gln

385 390 395 400

Ala Gln Pro Val His Gln Leu Val Lys Glu Pro Ser Thr Pro Ala Phe

405 410 415

Ser Trp Pro His His His His His His His His His His

420 425

<210> 37

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Thr Ser Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ile Trp Ile Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val

<210> 38

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

Claims (12)

1. An antibody targeting AXL, wherein the antibody has a heavy chain variable region and a light chain variable region,

the heavy chain variable region comprises the following three Complementarity Determining Regions (CDRs):

CDR1 shown in SEQ ID NO. 1,

CDR2 shown in SEQ ID NO. 2,

CDR3 shown in SEQ ID NO. 3; and

the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

SEQ ID NO. 4 shows CDR1',

CDR2' shown in SEQ ID NO. 5,

the CDR3' shown in SEQ ID NO. 6;

or

The heavy chain variable region comprises the following three Complementarity Determining Regions (CDRs):

CDR1 shown in SEQ ID NO.9,

SEQ ID NO. 10 shows the CDR2,

a CDR3 shown in SEQ ID No. 11; and

the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

SEQ ID NO. 12 shows the CDR1',

CDR2' shown in SEQ ID NO. 13,

CDR3' as shown in SEQ ID NO. 14.

2. The antibody of claim 1, wherein the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID No. 7, 15, or 25-27.

3. The antibody of claim 1, wherein the light chain variable region has an amino acid sequence as set forth in any one of SEQ ID No. 8, 16, or 28-35.

4. A recombinant protein, said recombinant protein having:

(i) the antibody of claim 1; and

(ii) optionally a tag sequence to assist expression and/or purification.

5. A CAR construct comprising a scFV fragment of the monoclonal antibody antigen binding region that specifically binds to AXL, wherein the scFV fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO. 1,

CDR2 shown in SEQ ID NO. 2,

CDR3 shown in SEQ ID NO. 3; and

the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

CDR1' shown in SEQ ID NO. 4,

CDR2' shown in SEQ ID NO. 5,

CDR3' as shown in SEQ ID No. 6;

or

The heavy chain variable region comprises the following three Complementarity Determining Regions (CDRs):

CDR1 shown in SEQ ID NO.9,

SEQ ID NO. 10 shows the CDR2,

a CDR3 shown in SEQ ID NO. 11; and

the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

SEQ ID NO. 12 shows the CDR1',

CDR2' shown in SEQ ID NO. 13,

CDR3' as shown in SEQ ID NO. 14.

6. A recombinant immune cell expressing an exogenous CAR construct of claim 5.

7. An antibody drug conjugate, comprising:

(a) an antibody moiety selected from the group consisting of: the antibody of claim 1, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof.

8. The antibody drug conjugate of claim 7, wherein said antibody moiety is conjugated to said conjugate moiety via a chemical bond or a linker.

9. The antibody drug conjugate of claim 8, wherein the linker is selected from the group consisting of: 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid imidate, maleimidocaproyl, 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl and a disubstituted maleimide linker.

10. The antibody drug conjugate of claim 7, wherein the conjugate moiety (D) is a cytotoxic drug and the cytotoxic drug is: microtubule-targeted drugs and/or DNA-targeted drugs and/or topoisomerase inhibitors.

11. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 4, the immune cell of claim 6, the antibody drug conjugate of claim 7, or a combination thereof, wherein the active ingredient is used to (a) prepare a detection reagent, a detection panel, or a kit; and/or (b) preparing a medicament for preventing and/or treating AXL-related diseases.

12. The use of claim 11, wherein the medicament is for the treatment or prevention of AXL-high expressing tumors, tumor migration, or tumor resistance.