CN116063507A

CN116063507A - Anti-human GAS6 antibodies or antigen binding fragments thereof and uses thereof

Info

Publication number: CN116063507A
Application number: CN202211078638.8A
Authority: CN
Inventors: 王蕊; 纪佳丽; 刘闯; 桂勋
Original assignee: Maiwei Shanghai Biotechnology Co ltd
Current assignee: Maiwei Shanghai Biotechnology Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-05-05
Also published as: CN115991770A; CN116003599A; CN114591431B; CN114591431A

Abstract

The invention discloses an anti-human GAS6antibody, an antigen binding fragment and application thereof.

Description

Anti-human GAS6 antibodies or antigen binding fragments thereof and uses thereof

The present application is a divisional application of the invention patent application with the application date of 2022, 5, 11 and the application number of 202210506098.2, and the invention name of "anti-human GAS6antibody or antigen binding fragment thereof and application thereof".

Technical Field

The present invention relates to the field of medicine, and in particular, to an anti-human GAS6antibody or an antigen-binding fragment thereof and uses thereof, and more particularly, to an anti-human GAS6antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody, a vector and a cell expressing the antibody, a method of preparing the antibody, a pharmaceutical composition containing the antibody, and uses of the antibody in the preparation of a medicament.

Background

Growth arrest-specific protein 6 (GAS 6) is a 75kDa soluble glycoprotein consisting of 3 domains including a gamma-carboxylated N-terminal Gla domain (gamma-carboxylated glutamic acid domain), a Growth factor-like domain (EGF-like domain), containing 4 EGF repeat motifs and a sex hormone binding globulin-like domain (SHBG-like domain) at the C-terminus, the latter comprising two Laminin (Laminin G, LGdomain). Wherein gamma-carboxylation of the GLA domain at the N-terminus of GAS6 is necessary for complete activation of the receptor. GAS6 is widely expressed in various tissues, mainly in the lung, small intestine, bone marrow and endothelium.

The GAS6 downstream receptors, known as TAM receptors (TYRO 3, AXL and MERTK), belong to the family of receptor tyrosine kinases, GAS6 binds the receptor through LG domain of its C-terminal SHBG domain. GAS6 and TAM receptor are involved in various biological processes, and after binding to the receptor, it activates receptor tyrosine kinase activity, and plays an important role in cell proliferation, survival, adhesion, migration, autophagy, invasion, angiogenesis, platelet aggregation, NK cell differentiation and the like through downstream signal transduction pathways including PI3K, ERK, NF-kB and the like. In many malignant tumors, AXL is highly expressed and activated with its ligand GAS6, such as acute myeloid leukemia, renal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, etc. Furthermore, the GAS6-AXL signaling pathway plays a key role in promoting tumor growth and metastasis, tumor immune escape and drug tolerance, the expression levels and interactions of which are closely related to the clinically poor prognosis of patients.

Cancer is one of the diseases that currently causes the highest mortality rate in humans. Recent clinical and commercial success of anti-cancer antibodies has led to great interest in antibody-based therapies. Thus, anti-cancer antibodies for treating cancer are to be further studied.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to propose an anti-human GAS6antibody or antigen-binding fragment thereof, and a method for preparing and using the same.

The GAS6-AXL signaling pathway has been found to play a key role in promoting tumor growth and metastasis, tumor immune escape and drug tolerance, and the expression level and interaction of the two are closely related to the clinically poor prognosis of patients. Meanwhile, in the research of tumor models of ectopic and in-situ homologous mice of GAS6, compared with wild mice, the tumor growth and metastasis diffusion of the mice with the defects of GAS6 are hindered. Blocking this pathway has therefore been considered as an effective strategy for treating cancer.

At present, a plurality of pharmaceutical companies and scientific research institutions are researching GAS6-AXL pathway, mainly comprising development of small molecule inhibitors and antagonistic therapeutic antibodies and development of AXL soluble extracellular receptors (AXL decoy receptor, decoy receptors), and inhibition of growth, proliferation and the like of tumors by blocking GAS6-AXL signals. As the amben company developed neutralizing antibodies against GAS6, GMAB1 and GMAB2, antibodies inhibited the growth of pancreatic ductal carcinoma (pancreatic ductal adenocarcinoma, PDAC) tumors by blocking the GAS6-AXL signaling pathway. Monoclonal antibodies against GAS6 have also been developed by Kyowa Kirin. The Batiraxcept (AVB-500) (AXL decoy receptor) engineered by Aravive Biologics can specifically block signals of tumor cells AXL by binding GAS6 in cytoplasm through high affinity, thereby effectively inhibiting metastasis of tumors and disease progression. The bait receptor can improve the sensitivity of chemotherapeutics in ovarian cancer and uterine serous cancers, and can initiate the phase 3 recruitment of platinum-resistant recurrent epithelial ovarian cancer (NCT 04729608). The above results indicate that inhibition of GAS6 exerts positive effects on tumor treatment. The anti-GAS 6antibody has wide clinical application value in the field of tumor treatment.

According to one aspect of the invention there is provided an anti-human GAS6antibody or antigen-binding fragment thereof. According to an embodiment of the invention, the anti-human GAS6antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising epitope regions (CDRs) 1,2 and 3, and VH CDRs1, 2 and 3 comprise the amino acid sequences shown in the selected VH CDRs1, 2 and 3, respectively;

a light chain variable region (VL) comprising antigen VL CDRs1, 2 and 3, and VL CDRs1, 2 and 3 comprise the amino acid sequences shown in the selected VH CDRs1, 2 and 3, respectively;

wherein the amino acid sequences of the selected VH CDRs1, 2 and 3 and the amino acid sequences of the selected VL CDRs1, 2 and 3 are selected from one of the following:

(1) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 5. 6 and 7, the amino acid sequences of the selected VL CDRs1, 2 and 3 are set forth in SEQ ID NOs: 8. shown at 9 and 10;

(2) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 11. 12 and 13, the amino acid sequences of the selected VL CDRs1, 2 and 3 are set forth in SEQ ID NOs: 14. 15 and 16;

(3) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 49. 50 and 51, and the amino acid sequences of the selected VL CDRs1, 2 and 3 are set forth in SEQ ID NOs: 52. 53 and 54;

(4) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 57. 58 and 59, and the amino acid sequences of the selected VL CDRs1, 2 and 3 are shown as 60, 61 and 62, respectively;

(5) The amino acid sequences of the selected VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 63. 64 and 65, and the amino acid sequences of the selected VL CDRs1, 2 and 3 are shown at 66, 67 and 68, respectively.

According to the embodiment of the invention, the anti-human GAS6antibody or the antigen binding fragment thereof can specifically bind to human GAS6 and mouse GAS6 proteins, so that the GAS6-AXL signaling pathway can be inhibited, and the cell proliferation can be inhibited, thereby inhibiting the progress, metastasis and drug resistance of tumors.

According to an embodiment of the invention, the VH comprises SEQ ID NO:1, and the VL comprises the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

According to an embodiment of the invention, the VH comprises SEQ ID NO:3, and the VL comprises the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a monoclonal antibody.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a chimeric antibody.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human GAS6 and is selected from Fv, fab, fab ', scFv and F (ab') ₂ 。

According to another aspect of the invention, an isolated nucleic acid is provided. According to an embodiment of the invention, the nucleic acid encodes the aforementioned anti-human GAS6antibody or antigen-binding fragment thereof.

According to another aspect of the invention, a carrier is provided. According to an embodiment of the invention, the vector comprises the aforementioned nucleic acid.

According to another aspect of the invention, an isolated cell is provided. According to an embodiment of the invention, the cell comprises the aforementioned vector.

According to another aspect of the invention there is provided a method of preparing an anti-human GAS6antibody or antigen-binding fragment thereof as hereinbefore described. According to an embodiment of the invention, the method comprises:

(1) Culturing the aforementioned cells under suitable conditions; and

(2) The aforementioned anti-human GAS6antibody or antigen-binding fragment thereof is isolated and recovered.

According to another aspect of the present invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned anti-human GAS6antibody or antigen-binding fragment thereof; and a pharmaceutically acceptable carrier.

According to another aspect of the invention, there is provided an antibody-drug conjugate. According to an embodiment of the invention, the antibody-drug conjugate comprises the aforementioned antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.

According to an embodiment of the invention, the therapeutic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

According to a further aspect of the invention there is provided the use of an anti-human GAS6antibody or antigen binding fragment thereof as hereinbefore described in the manufacture of a medicament for the treatment of cancer.

According to embodiments of the invention, the cancers include breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, colon cancer, neuroendocrine tumors, pancreatic cancer, bladder cancer, head and neck cancer, and chronic and acute myeloid leukemia.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram showing the binding results of immunized mouse serum to GAS6 protein according to one embodiment of the invention, wherein A is the ELISA binding result of immunized mouse serum to human GAS6 protein; b is ELISA combination result of immune mouse serum and mouse GAS6 protein;

FIG. 2 shows a schematic representation of blocking of GAS6-AXL binding by murine serum according to one embodiment of the present invention;

FIG. 3 shows a schematic representation of the results of binding of chimeric antibodies to human GAS6his protein according to one embodiment of the invention;

FIG. 4 shows the binding of chimeric antibodies to murine GAS6His protein according to one embodiment of the present invention;

FIG. 5 shows the binding results of a chimeric antibody according to one embodiment of the present invention to a human Protein S His Protein;

FIG. 6 shows a schematic diagram of the evaluation of inhibition of Ba/F3-AXL cell proliferation by chimeric antibodies according to one embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.

Anti-human GAS6 antibodies and antigen binding fragments thereof

The present invention provides antibodies and antigen binding fragments thereof that specifically bind GAS 6. According to the embodiment of the invention, the anti-human GAS6antibody or the antigen binding fragment thereof can specifically bind to human GAS6 and mouse GAS6 proteins, so that the GAS6-AXL signaling pathway can be inhibited, and the cell proliferation can be inhibited, thereby inhibiting the progress, metastasis and drug resistance of tumors.

Embodiments of the invention provide several anti-GAS 6 antibodies, such as ch2D3C6 and ch2O18G6, including, for example, mouse antibodies, and chimeric antibodies thereof. The CDR sequences (Kabat definition) and the sequences of the heavy chain variable region and the light chain variable region of some of the disclosed antibodies are shown in the following tables.

TABLE 1

The embodiment of the invention also provides a chimeric antibody. These chimeric antibodies have VH and VL from mouse antibodies. However, the constant domains of these chimeric antibodies are derived from human antibodies (e.g., human IgG1, human IgG2, human IgG3, or human IgG 4). These chimeric antibodies were labeled chHvLv-IgG. Some chimeric antibodies described in the present disclosure are shown in the following table.

TABLE 2

In some embodiments, an antibody may have a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) 1,2, 3, wherein CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR1 amino acid sequence, CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR2 amino acid sequence, and CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VH CDR3 amino acid sequence; the light chain variable region comprises CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR3 amino acid sequence.

In some embodiments, an antibody or antigen binding fragment described herein may contain a light chain variable region comprising one, two, or three CDRs selected from the group consisting of: SEQ ID NOS 5-7, 8-10, 11-13 or 14-16, 49-51, 52-54, 57-59, 60-62, 63-65 or 66-68 having zero, one or two amino acid insertions, deletions or substitutions on one, two or three of the selected CDRs. For example, an antibody or antigen binding fragment described herein may contain a light chain variable domain that contains one, two, or three of the following CDRs: SEQ ID NO. 5 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO. 6 with zero, one or two amino acid insertions, deletions or substitutions; SEQ ID NO. 7 with zero, one or two amino acid insertions, deletions or substitutions.

Embodiments of the invention also provide antibodies or antigen-binding fragments thereof that bind GAS6, wherein the antibodies or antigen-binding fragments have VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are determined based on various CDR definitions known in the art, such as Kabat definition, chothia definition, or IMGT definition. The sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are shown in tables 1 and 2.

Nucleic acids, vectors and cells

Embodiments of the invention also provide nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in tables 1 and 2, or has sequences as shown in tables 1 and 2. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds GAS6 (e.g., human GAS 6).

Embodiments of the invention also provide recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and/or the polynucleotide-containing vectors), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is localized for expression in the vector by being operably linked to regulatory elements such as promoters, enhancers and/or polyadenylation tails, located at or near or flanking the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (useful for producing recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

Embodiments of the invention provide host cells transformed with the vectors described above. The host cell may be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is E.coli (Escherichia coli). Preferably, the eukaryotic cell is selected from: primordial biological cells, animal cells, plant cells, and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. A preferred fungal cell is Saccharomyces cerevisiae.

Methods of making anti-GAS 6 antibodies

Isolated fragments of human GAS6 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies may be raised in an animal by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. The animals may be injected with the antigenic peptide or protein multiple times.

Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides (e.g., fragments of human GAS 6). The formulation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

Pharmaceutical composition, use and method of treatment

The present embodiments also provide pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the embodiments of the present invention. Two or more (e.g., two, three, or four) of any of the antibodies or antigen binding fragments described herein can be present in any combination in a pharmaceutical composition. The pharmaceutical composition may be formulated in any manner known in the art.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or efficacy of the antibody.

In one aspect, the invention provides the use of an anti-human GAS6antibody or antigen-binding fragment thereof as described above in the manufacture of a medicament for the treatment of cancer. According to embodiments of the invention, the cancers include breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, colon cancer, neuroendocrine tumors, pancreatic cancer, bladder cancer, head and neck cancer, and chronic and acute myeloid leukemia.

The present invention provides that one or more antibodies or antigen binding fragments thereof may be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, reducing the rate at which tumor volume increases over time in a subject, reducing the risk of metastasis, or reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may stop, slow, delay or inhibit the progression of the cancer. In some embodiments, the treatment may result in a decrease in the number, severity, and/or duration of one or more symptoms of cancer in the subject.

In one aspect, the disclosure features a method comprising administering to a subject in need thereof (e.g., a subject having, or identified as having, or diagnosed with, a cancer, e.g., breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, neuroendocrine tumor, pancreatic cancer, bladder cancer, and head and neck cancer) a therapeutically effective dose of an antibody disclosed herein, or an antigen-binding fragment thereof.

In some embodiments, the compositions and methods disclosed herein are useful for treating a patient at risk of cancer. Patients with cancer may be identified using a variety of methods known in the art.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are conventional products commercially available, for example, from Sigma company.

EXAMPLE 1 animal immunization

Immunization of 6-8 week old female Balb/C mice with recombinant Human GAS6hFc protein, 5 groups, specifically, 20ug of recombinant GAS6 protein (Human GAS 6C-Fc, novoprotein, cat#c12w) in combination with freund's complete adjuvant (CFA) by subcutaneous injection for a total of 5 immunizations, wherein the next four immunizations were each immunization with 20ug of recombinant GAS6 protein in combination with freund's incomplete adjuvant (IFA), followed by 3 weeks of each immunization; and the last two immunizations were performed with 20ug of recombinant murine GAS his protein (Sino Biological, cat# 58026-M08H). From the second immunization, orbital bleeding was performed 1 week after immunization, and the obtained serum was subjected to antigen-specific titer detection and GAS6-AXL binding blocking detection.

(1) Serum titer antigen binding assays

ELISA binding assays of serum were performed with recombinant human GAS6his protein (Novoprotein, cat#C01W) and murine GAS6his protein (Sino Biological, cat# 58026-M08H). Specifically, 1ug/ml human GAS6his protein and 1ug/ml murine GAS6his protein were individually coated in 96-well plates, 100 ul/well, and incubated overnight. The next day the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% bsa (1 xPBST formulation) and incubated for 1 hour at 37 degrees; after incubation, washing three times with 1xPBST, adding serum diluted in gradient and incubating at 37℃for 1 hour; after incubation and washing, goat anti-mouse IgG fcγ fragment specific HRP antibody (Jackson ImmunoResearch, 115-005-008) was added at 1:5000 dilution and incubated at 37 degrees for 1 hour; after PBST washes 100ul TMB substrate was added to each well to detect antibody binding, and the reaction was stopped with an equal volume of 1N HCl and OD450nm readings were detected using a Spectra M5e instrument.

The results are shown in FIG. 1, where the mouse serum has different degrees of specific binding to both antigens after 5 immunizations with human GAS6 protein and murine GAS6 protein, and where the mouse serum numbered #4 responds more strongly to both antigens.

(2) Serum titer ligand receptor binding blocking assay

The blocking effect of murine serum on the binding of human GAS6 protein and human AXL protein was assessed by ELISA. Specifically, recombinant human AXL his protein (AcroBiosystems, cat#axl-H5226) was coated in 96-well plates, 1ug/ml,100 ul/well, and incubated overnight. The next day the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% bsa (1 xPBST formulation) and incubated for 1 hour at 37 degrees; after incubation, the wells were washed three times with 1xPBST, 50ul of serum diluted in gradient and 50ul of a mixture of recombinant human GAS6hFc protein (Novoprotein, cat#C12W) at a final concentration of 150ng/ml were added to each well and incubated for 1 hour at 37 degrees; commercial GAS was used at a final concentration of 20ug/ml6 neutralizing antibodies (R)&D Systems, AB 885) as Positive Control (PC) and final concentration of 20ug/ml of Goat IgG (R)&D Systems, AB-108-C) as Negative Control (NC). After incubation and washing, goat anti-human IgG fcγ fragment specific HRP antibody (Jackson ImmunoResearch, cat#109-035-098) was added at 1:5000 dilution and incubated at 37 degrees for 1 hour; after PBST wash, 100ul of TMB substrate was added to each well to detect antibody binding, and the reaction was stopped with an equal volume of 1N HCl, resulting in detection of OD450nm reads with a Spectra M5e instrument. In this system, the absorbance of wells after incubation with HRP antibody was maximized (Max) with only GAS6hFc protein and minimized (Mini) with only 1% bsa. The lower well-to-Max serum dilutions contained antibodies that inhibited human GAS6 and human AXL binding, inhibition (%) = (OD 450) _Max -OD450 _Sample )/(OD450 _Max -OD450 _Mini )*100。

The ELISA results show that at low dilution gradients (third dilution, 1:360), the mouse serum has an inhibition of more than 60% of the binding of human GAS6hFc and human AXLhis protein, wherein the inhibition of the mouse serum numbered #4 is better.

Example 2 selection and identification of hybridoma cells

Primary screening of hybridoma supernatants

1) Preliminary binding screening of hybridoma supernatants

According to the serum titer detection results of example 1, two mice #4 and #5 were selected for booster immunization, spleen and lymph node were taken, and the obtained cells were electrically fused with murine myeloma cells after grinding, and hybridoma supernatants were screened after 10-14 days of culture in HAT medium and HT medium. First, binding screening of human GAS6his protein and mouse GAS6his protein was performed by ELISA method, and the test method was similar to serum titer ELISA detection method, human GAS6his protein and mouse GAS6his egg were coated in 384-well plate, 1ug/ml,40 ul/well, incubated overnight, washed and blocked, and then 30ul incubation was added to hybridoma supernatant for binding detection. Finally, 372 clones are selected for the screening of the next blocking test, wherein 239 clones have stronger binding with human and mouse GAS6 protein; 79 clones bound strongly to human GAS6 and weakly to murine GAS6 protein; 54 clones bound strongly to human GAS6 and did not bind to murine GAS 6.

2) Preliminary blocking screening of hybridoma supernatants

The 372 clones selected for ELISA binding assay were evaluated for blocking assay. Specific methods similar to the serum titer blocking assay, recombinant human AXL his protein was coated in 96-well plates, 1ug/ml,100 ul/well, and incubated overnight. After washing blocking, 50ul of hybridoma supernatant and 50ul of recombinant human GAS6hFc protein mixture at a final concentration of 150ng/ml were added to each well and incubated for 1 hour at 37 degrees. Wherein 50ul of the hybridoma medium was mixed with 50ul of recombinant human GAS6hFc protein at a final concentration of 150ng/ml as the signal maximum (Max), and the absorbance of the wells was the minimum (Mini) with the hybridoma medium alone. After washing, goat anti-human IgG Fcgamma fragment specific HRP antibody (Jackson Immuno Research, cat#109-035-098) was added at 1:5000 dilution, washed after incubation, 100ul TMB substrate was added to each well for color development, the reaction was stopped with an equal volume of 1N HCl, and OD450nm absorbance was read. In this system, hybridoma supernatants corresponding to wells having absorbance lower than Max contained antibodies that inhibited binding of human GAS6 to human AXL, inhibition (%) = (OD 450) _Max -OD450 _Sample )/(OD450 _Max -OD450 _Mini ) *100. Finally, 71 clones with inhibition rate greater than 45% were selected.

(II) subclone screening of hybridoma supernatants

According to the preliminary ELISA binding and blocking test screening of hybridoma supernatants, 53 clones are selected for subcloning, wherein 36 clones have stronger binding with human GAS6 and murine GAS6, and the ligand receptor binding inhibition rate is more than 50%;8 clones were stronger in binding to human GAS6 and weaker in binding to murine GAS6, with ligand receptor binding inhibition greater than 60%;9 clones bound strongly to both human GAS6 and murine GAS6, with ligand receptor binding inhibition greater than 45% and less than 50%. And finally, 11 candidate clones which have stronger binding with human GAS6 and mouse GAS6 proteins and have ligand receptor binding inhibition rate of more than 40% are selected through ELISA binding tests of the human GAS6 and mouse GAS6 proteins and ligand receptor binding blocking tests (the method is the same as a preliminary screening application method).

EXAMPLE 3 production and identification of human murine chimeric antibodies

Extraction of candidate cloned Gene

1) Extraction of total RNA and cDNA Synthesis in hybridoma cells

After culturing the monoclonal hybridoma cells to the logarithmic growth phase, the cells (about 1E6 cells/clone) were collected using

RNA Plus (MN, cat# 740984.250) extracted total RNA from hybridoma cells. Using 1ug of extracted RNA, by +.>

III RT SuperMix for qPCR (+gDNA wind) (Vazyme, cat#R323-01) cDNA was synthesized.

2) Gene amplification and T vector cloning of antibody VH and VL sequences

The cDNA was specifically amplified using primers Primer A+Smix (comprising heavy and light chain universal Primer A and human hIgG1,2A,2b,3 constant region Primer and human kappa specific Primer) and Ex Taq enzyme (TaKaRa, cat#RR902A) as templates, and the PCR reaction system and cycle were as follows. Kit for recycling PCR product by gel after electrophoresis of 1% agarose gel

Gel and PCR Clean-up (MN, cat# 740609.250) recovering the fragment of interest.

The recovered fragment was then cloned into pMD19-T (TaKaRa, cat # 3271) vector using Solution I (TaKaRa, cat # 6022Q), the cloned plasmid was transformed into competent cells DH 5. Alpha. By heat stimulation (Yestern, cat # FYE607-80 VL) and spread evenly on ampicillin-containing 2YT solid plates, which were sent to the sequencing company Genewiz for sequencing, the sequencing primer being the universal primer PMAL-C2X-R (SEQ ID NO: 70).

Construction, expression and purification of candidate clone human-mouse chimeric expression vector

1) Construction of expression vectors

The antibody VH and VL sequencing sequences were analyzed, TA cloning plasmids with correct antibody sequencing were selected, the antibody VH/VL universal primer mix (containing the expression vector signal peptide) was used to amplify the fragment of interest (PCR procedure as follows) under the action of the high-fidelity enzyme PrimeSTAR (TaKaRa, cat#R045) and primers, and the fragment of interest was recovered after electrophoresis using a gel recovery kit (MN, cat# 740609.250).

The recovered fragment was inserted into a corresponding linearized vector (pTT5_hIgG1.G1 m 3/pTT5_hkappa.Km3) under the action of a recombinase (Vazyme, cat#C112-02), wherein the pTT5_hIgG1.G1m3 vector contains an hIgG1 Fc sequence (SEQ ID NO: 69) and the pTT5_hkappa.Km3 vector contains an hKappa sequence (SEQ ID NO: 74); the recombinant vector was transformed into competent cells DH 5. Alpha. (Yestern, cat# FYE607-80 VL) and spread evenly on ampicillin-containing 2YT solid plates, which were sent to the sequencing company Genewiz for sequencing, with sequencing primers pTT5-F (SEQ ID NO: 71) and pTT5-R (SEQ ID NO: 72).

2) Expression and purification of chimeric antibodies

Sequencing was analyzed, the above correctly sequenced plasmids were amplified and the heavy and light chain plasmids were transfected with PEI reagent (Polysciences, cat # 24885) (1 ug plasmid: 4ug PEI) at a ratio of 2:3 into HEK293 cells at a density of 2E6/ml, and the transfected cells were placed at 37℃and incubated in a 5% CO2 orbital incubator for 5-7 days. The culture supernatant was centrifuged and filtered with a 0.22um filter, and purified using Protein G agarose packed purification columns (GE Healthcare Bio-sciences, 17-0618-05). The purification column was equilibrated with 1xPBS (pH 7.4), the filtered culture supernatant was applied to the purification column, and after washing the purification column with 1xPBS (pH 7.4), the sample was eluted with an elution solution (50 nM sodium citrate, pH 2.5), and the sample was neutralized with 1M Tris-HCl, pH 9.0 solution. The neutralized sample was replaced with 1xPBS (pH 7.4), sterilized by filtration through a 0.22um filter, and the concentration of purified antibody was measured by Nanodrop (Thermo Fisher Scientific Inc) for use.

Specific binding identification of (III) human murine chimeric antibodies

1) Assessment of specific binding to human GAS6 and Cross-binding Activity of anti-human GAS6 chimeric antibodies to murine GAS6

ELISA binding assays for antibodies were performed with recombinant human GAS6his protein (Novoprotein, cat#C01W) and murine GAS6his protein (Sino Biological, cat# 58026-M08H). Specifically, 0.5ug/ml human GAS6his protein and 0.5ug/ml murine GAS6his protein were individually coated in 96-well plates, 100 ul/well, and incubated overnight. The next day the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% bsa (1 xPBST formulation) and incubated for 1 hour at 37 degrees; three washes with 1xPBST after incubation, adding gradient diluted purified antibody and incubating for 1 hour at 37 degrees, wherein anti-huGas6 anti-ibody (Santa cruz, sc-376087) and anti-mGas6 Ab (R & D systems, AF 986) are positive control antibodies that bind human GAS6his protein and murine GAS6his protein, respectively; after incubation and washing, goat anti-human IgG fcγ fragment specific HRP antibody (Jackson ImmunoResearch, cat#109-035-098) was added at 1:5000 dilution and incubated at 37 degrees for 1 hour; after PBST wash, 100ul of TMB substrate was added to each well to detect antibody binding, and the reaction was stopped with an equal volume of 1N HCl, resulting in detection of OD450nm using a Spectra M5e instrument.

TABLE 3 summary of results of ELISA binding of chimeric antibodies to human and murine GAS6his proteins

	Binding human GAS6	Binding to mouse GAS6
			Chimeric antibodies	EC50(nM)	EC50(nM)
ch2D3C6	0.19	1.42
			ch2O18G6	0.21	0.22
ch3F23G12	0.23	0.21
			ch3K16C8	0.15	0.65
ch5I23B12	0.24	0.52
			ch11B11D12	0.18	0.15
ch12H19D4	0.15	0.13
			ch15G5E5	0.16	4.94
ch18I16C6	0.11	0.10
			anti-Gas6 Antibody	0.43	0.32

ELISA binding results as shown in FIGS. 3, 4 and Table 1, 9 chimeric antibodies were able to specifically bind human GAS6his and murine GAS6his proteins. Of these, ch3K16C8, ch5I23B12, ch2O18G6, ch3F23G12, ch11B11D12, ch12H19D4 and ch18I16C6 were comparable to human murine GAS6his protein binding activity (Table 3).

2) Assessment of human murine chimeric antibody Cross-binding to human Protein S

ELISA binding assays of antibodies were performed with recombinant human Protein his Protein (R & D, cat# 9489-PS). Specifically, 1ug/ml human Protein S his Protein was coated in 96-well plates, 100 ul/well, and incubated overnight. The next day the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% bsa (1 xPBST formulation) and incubated for 1 hour at 37 degrees; after incubation, the antibody was washed three times with 1xPBST, and the purified antibody was added in gradient dilution and incubated at 37℃for 1 hour; after incubation and washing, goat anti-human IgG fcγ fragment specific HRP antibody (Jackson ImmunoResearch, cat#109-035-098) was added at 1:5000 dilution and incubated at 37 degrees for 1 hour; after PBST wash, 100ul of TMB substrate was added to each well to detect antibody binding, and the reaction was stopped with an equal volume of 1N HCl, resulting in detection of OD450nm using a Spectra M5e instrument.

Table 4 results of ELISA binding of chimeric antibodies to human Protein S his Protein

The ELISA binding results are shown in FIG. 5 and Table 4, and the chimeric antibodies ch2O18G6, ch15G5E5 and ch18I16C6 all have cross-binding activity with human Protein S his Protein.

The results show that antibodies ch2O18G6, ch15G5E5 and ch18I16C6 have specific binding activity to human GAS6his Protein, murine GAS6his Protein and human Protein S his Protein.

Blocking Identification of (IV) human murine chimeric antibodies

The blocking effect of the chimeric antibodies on the binding of human GAS6 protein and human AXL protein was assessed by ELISA. Specifically, recombinant human AXL hFc protein (internally synthesized, sequence shown as SEQ ID NO:73 coated in 96-well plate, 1ug/ml,100 ul/well, incubated overnight, the coated 96-well plate was washed three times with 1 xBST and then blocked with 1% BSA (1 xBST formulation) the next day, incubated for 1 hour at 37 degrees, three times with 1 xBST after incubation, 50ul of a gradient diluted antibody and 50ul of a final concentration of 4ng/ml recombinant human GAS6his protein (Novoprotein, cat#C01W) were added to each well and incubated for 1 hour at 37 degrees, a final concentration of 10ug/ml Kyowa Kirin GAS6 and antibody hzKM5321 LV bHV0 as positive control and a final concentration of 10ug/ml human as negative control 50ul 1% BSA and 50ul of 4 ul of recombinant human GAS6his protein mixture as maximum value were added to each well, and the detection of 1ul of light absorption protein was stopped by a dilution system, and the detection of 1ul of light absorption protein was carried out by a detection instrument, and the detection system showed that the detection of specific binding to the chimeric antibody was carried out by 1 ug-1 mg/L, 50ul of protein, and 50ul of the antibody was added to the well (1 mg/L, 1% protein) and 50 mg/ml, and 50% protein was incubated in a well plate.

Identification of proliferation inhibitory Activity of (fifth) human murine chimeric antibody

To assess the effect of anti-GAS 6 antibodies on GAS 6-dependent cell growth, a cell proliferation inhibition assay was performed with Ba/F3-AXL cells (tokyo, cat#cbp 73249). Taking logarithmic growth Ba/F3-AXL cells, centrifuging and discarding culture supernatant, washing the centrifuged cells with 1 xDSL three times, and re-suspending the centrifuged cells in fresh RPMI1640+10% FBS+100ng/ml recombinant human GAS6his (R & D, cat # 885-GSB) medium with a cell density of 5 x10 e4/ml; the resuspended cells were then seeded into 96-well white cell culture plates, 100 ul/well cell suspension, 3 replicate wells were set, and placed in a 37 degree cell incubator for 4 hours. After incubation, a gradient of 10 Xantibody was added in a gradient of dilution, 11.1 ul/well, and incubation was continued for 72 hours in a 37℃cell incubator. After 72 hours incubation, 100ul Cell Titer Glo detection reagent (Promega, cat#g7572) was added to each well and left for 30 minutes to read RLU values. The no antibody added had a maximum RLU reading of only 100ng/ml GAS6 wells, a minimum RLU reading of only medium wells, and hIgG1 as a negative control. Antibody proliferation inhibitory activity (%) =100- (sample well RLU-minimum RLU)/(maximum RLU-minimum RLU) x100.

As a result, as shown in FIG. 6, the chimeric antibodies ch2D3C6 and ch2O18G6 inhibited the growth of Ba/F3-AXL in a concentration gradient, with inhibition ratios of about 30% and 20% on proliferation of cells at a concentration of 200nM, respectively.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An anti-human GAS6antibody or antigen-binding fragment thereof, comprising:

a heavy chain variable region (VH) comprising epitope regions (CDRs) 1,2 and 3;

a light chain variable region (VL) comprising antigen VL CDRs1, 2 and 3;

wherein, the amino acid sequences of the VH CDRs1, 2 and 3 are respectively shown in SEQ ID NO: 57. 58 and 59, the amino acid sequences of the VL CDRs1, 2 and 3 are respectively shown in SEQ ID NOs: 60. 61 and 62.

2. The antibody or binding fragment thereof of claim 1, wherein the VH comprises SEQ ID NO:27, and the VL comprises the amino acid sequence set forth in SEQ ID NO:28, and a polypeptide comprising the amino acid sequence shown in seq id no.

3. The antibody or binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof is a monoclonal antibody.

4. The antibody or binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody.

5. An antibody or binding fragment thereof according to any one of claims 1 to 3 wherein the antibody or antigen binding fragment thereof is an antibody fragment that specifically binds human GAS6 and is selected from Fv, fab, fab ', scFv and F (ab') ₂ 。

6. An isolated nucleic acid encoding the anti-human GAS6antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. An isolated cell comprising the vector of claim 7.

9. A method of making the anti-human GAS6antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising:

(1) Culturing the cell of claim 9 under suitable conditions; and

(2) Isolating and recovering the anti-human GAS6antibody or antigen-binding fragment thereof of claims 1-5.

10. A pharmaceutical composition comprising:

the anti-human GAS6antibody or antigen-binding fragment thereof of any one of claims 1-5; and

a pharmaceutically acceptable carrier.

11. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5 covalently bound to a therapeutic agent.

12. The antibody-drug conjugate of claim 11, wherein the therapeutic agent is monomethyl auristatin E or monomethyl auristatin F.

13. Use of an anti-human GAS6antibody or antigen-binding fragment thereof of any one of claims 1-5 or an antibody-drug conjugate of claims 11-12 in the manufacture of a medicament for the treatment of cancer.

14. The use according to claim 13, wherein the cancer comprises breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, neuroendocrine tumors, pancreatic cancer, bladder cancer, head and neck cancer, and chronic and acute myeloid leukemia.