Anti-human gas6 antibodies or antigen-binding fragments thereof and uses thereof

Abstract

Disclosed are anti-human GAS6 antibodies, antigen-binding fragments, and uses thereof.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to anti-human GAS6 antibodies or antigen-binding fragments thereof and their applications, and more specifically to anti-human GAS6 antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibody, vectors and cells expressing the antibody, methods for preparing the antibody, pharmaceutical compositions containing the antibody, and the use of the antibody in the preparation of pharmaceuticals. Background Technology

[0002] Growth arrest-specific gene 6 (GAS6) is a 75 kDa soluble glycoprotein composed of three domains: a γ-carboxylated N-terminal GLA domain, an EGF-like domain containing four EGF repeat motifs, and a C-terminal SHBG-like domain comprising two laminin G (LG) domains. γ-carboxylation of the N-terminal GLA domain is essential for complete receptor activation. GAS6 is widely expressed in various tissues, primarily in the lungs, small intestine, bone marrow, and endothelium.

[0003] The downstream receptors of GAS6 are called TAM receptors (TYRO3, AXL, and MERTK), belonging to the receptor tyrosine kinase family. GAS6 binds to its receptor through the LG domain of its C-terminal SHBG domain. GAS6 interacts with TAM receptors in various biological processes. Upon binding, it activates receptor tyrosine kinase activity, playing a crucial role in cell proliferation, survival, adhesion, migration, autophagy, invasion, angiogenesis, platelet aggregation, and NK cell differentiation through downstream signal transduction pathways, including PI3K, ERK, and NF-κB. In many malignant tumors, AXL and its ligand GAS6 are highly expressed and activated, such as in acute myeloid leukemia, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, and ovarian cancer. Furthermore, the GAS6-AXL signaling pathway plays a key role in promoting tumor growth and metastasis, tumor immune escape, and drug tolerance; their expression levels and interactions are closely related to poor clinical prognosis in patients.

[0004] Cancer is one of the leading causes of death worldwide. Recent clinical and commercial successes of anticancer antibodies have sparked great interest in antibody-based therapies. Therefore, further research is needed on anticancer antibodies for treating cancer. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. Therefore, one object of this invention is to provide an anti-human GAS6 antibody or its antigen-binding fragment, a method for its preparation, and its applications.

[0006] Studies have found that the GAS6-AXL signaling pathway plays a crucial role in promoting tumor growth and metastasis, tumor immune escape, and drug tolerance. The expression levels and interactions of these pathways are closely related to poor clinical prognosis in patients. Furthermore, in studies of GAS6 ectopic and orthotopic homologous mouse tumor models, GAS6-deficient mice showed inhibited tumor growth and metastasis compared to wild-type mice. Therefore, blocking this pathway is considered an effective strategy for cancer treatment.

[0007] Currently, numerous pharmaceutical companies and research institutions are conducting research targeting the GAS6-AXL pathway, primarily including the development of small molecule inhibitors, antagonistic therapeutic antibodies, and soluble extracellular receptors for AXL (AXL decoy receptors). These studies aim to inhibit tumor growth and proliferation by blocking GAS6-AXL signaling. For example, Amgen has developed neutralizing antibodies against GAS6, GMAB1 and GMAB2, which inhibit the growth of pancreatic ductal adenocarcinoma (PDAC) tumors by blocking the GAS6-AXL signaling pathway. Kyowa Kirin has also developed a monoclonal antibody against GAS6. Batiraxcept (AVB-500) (AXL decoy receptor), engineered by Aravive Biologics, binds to GAS6 with high affinity, chelates GAS6 in the cytoplasm, and specifically blocks AXL signaling in tumor cells, thereby effectively inhibiting tumor metastasis and disease progression. This drug is currently mainly used for platinum-resistant recurrent epithelial ovarian cancer and clear cell renal cell carcinoma. This decoy receptor can improve the sensitivity of chemotherapeutic drugs in ovarian cancer and serous uterine carcinoma, and a phase 3 recruitment for platinum-resistant recurrent epithelial ovarian cancer (NCT04729608) has been initiated. All of the above demonstrates that inhibiting GAS6 has a positive therapeutic effect on tumors. Anti-GAS6 antibodies have broad clinical application value in the field of tumor treatment.

[0008] According to one aspect of the present invention, an anti-human GAS6 antibody or an antigen-binding fragment thereof is provided. According to an embodiment of the present invention, the anti-human GAS6 antibody or the antigen-binding fragment thereof comprises:

[0009] The heavy chain variable region (VH) includes antigenic determinant regions (CDRs) 1, 2 and 3, and VH CDRs 1, 2 and 3 respectively include the amino acid sequences shown in the selected VH CDRs 1, 2 and 3;

[0010] The light chain variable region (VL) comprises antigen VL CDRs1, 2 and 3, and VL CDRs1, 2 and 3 respectively comprise the amino acid sequences shown in the selected VH CDRs1, 2 and 3;

[0011] 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:

[0012] (1) The amino acid sequences of the selected VH CDRs1, 2 and 3 are shown in SEQ ID NO: 5, 6 and 7 respectively, and the amino acid sequences of the selected VL CDRs1, 2 and 3 are shown in SEQ ID NO: 8, 9 and 10 respectively;

[0013] (2) The amino acid sequences of the selected VH CDRs1, 2 and 3 are shown in SEQ ID NO: 11, 12 and 13, respectively, and the amino acid sequences of the selected VL CDRs1, 2 and 3 are shown in SEQ ID NO: 14, 15 and 16, respectively.

[0014] (3) The amino acid sequences of the selected VH CDRs 1, 2 and 3 are shown in SEQ ID NO: 49, 50 and 51, respectively, and the amino acid sequences of the selected VL CDRs 1, 2 and 3 are shown in SEQ ID NO: 52, 53 and 54, respectively.

[0015] (4) The amino acid sequences of the selected VH CDRs 1, 2 and 3 are shown in SEQ ID NO: 57, 58 and 59, respectively, and the amino acid sequences of the selected VL CDRs 1, 2 and 3 are shown in 60, 61 and 62, respectively.

[0016] (5) The amino acid sequences of the selected VH CDRs 1, 2 and 3 are shown in SEQ ID NO: 63, 64 and 65, respectively, and the amino acid sequences of the selected VL CDRs 1, 2 and 3 are shown in 66, 67 and 68, respectively.

[0017] The anti-human GAS6 antibody or its antigen-binding fragment according to embodiments of the present invention can specifically bind to human GAS6 and mouse GAS6 proteins, thereby inhibiting the GAS6-AXL signaling pathway and inhibiting cell proliferation, thereby inhibiting tumor progression, metastasis and drug resistance.

[0018] According to an embodiment of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2.

[0019] According to an embodiment of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises the amino acid sequence shown in SEQ ID NO: 4.

[0020] According to an embodiment of the present invention, the antibody or its antigen-binding fragment is a monoclonal antibody.

[0021] According to an embodiment of the present invention, the antibody or its antigen-binding fragment is a chimeric antibody.

[0022] According to an embodiment of the present invention, the antibody or its antigen-binding fragment is an antibody fragment that specifically binds to human GAS6 and is selected from Fv, Fab, Fab', scFv and F(ab')2.

[0023] According to another aspect of the present invention, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned anti-human GAS6 antibody or its antigen-binding fragment.

[0024] According to another aspect of the present invention, a vector is provided. According to an embodiment of the present invention, the vector comprises the aforementioned nucleic acid.

[0025] According to another aspect of the invention, the invention provides an isolated cell. According to an embodiment of the invention, the cell comprises the aforementioned carrier.

[0026] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned anti-human GAS6 antibody or its antigen-binding fragment. According to an embodiment of the present invention, the method includes:

[0027] (1) Culturing the cells of claim 9 under suitable conditions; and

[0028] (2) Separate and recover the aforementioned anti-human GAS6 antibody or its antigen-binding fragment.

[0029] 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 GAS6 antibody or its antigen-binding fragment; and a pharmaceutically acceptable carrier.

[0030] According to another aspect of the present invention, an antibody-drug conjugate is provided. According to embodiments of the present invention, the antibody-drug conjugate comprises the aforementioned antibody or its antigen-binding fragment covalently bound to a therapeutic agent.

[0031] According to an embodiment of the present invention, the therapeutic agent is methylaurestatin E (MMAE) or methylaurestatin F (MMAF).

[0032] According to another aspect of the invention, the invention provides the use of the aforementioned anti-human GAS6 antibody or its antigen-binding fragment in the preparation of a medicament for the treatment of cancer.

[0033] According to embodiments of the present invention, the cancers include 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.

[0034] 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. Attached Figure Description

[0035] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0036] Figure 1 The diagram shows the binding results of immune mouse serum and GAS6 protein according to an embodiment of the present invention, wherein A is the ELISA binding result of immune mouse serum and human GAS6 protein; and B is the ELISA binding result of immune mouse serum and mouse GAS6 protein.

[0037] Figure 2 A schematic diagram showing the blocking effect of mouse serum on GAS6-AXL binding according to an embodiment of the present invention is displayed;

[0038] Figure 3 A schematic diagram showing the result of binding of a chimeric antibody to human GAS6 his protein according to an embodiment of the present invention is shown.

[0039] Figure 4 The binding results of the chimeric antibody to the mouse GAS6 His protein according to an embodiment of the present invention are shown;

[0040] Figure 5 The binding results of the chimeric antibody to human Protein S His protein according to an embodiment of the present invention are shown;

[0041] Figure 6 A schematic diagram showing the evaluation results of the chimeric antibody's inhibition of Ba / F3-AXL cell proliferation according to an embodiment of the present invention is displayed. Detailed Implementation

[0042] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] Anti-human GAS6 antibody and its antigen-binding fragment

[0044] This invention provides antibodies that specifically bind to GAS6 and their antigen-binding fragments. Anti-human GAS6 antibodies or their antigen-binding fragments according to embodiments of the present invention can specifically bind to human GAS6 and mouse GAS6 proteins, thereby inhibiting the GAS6-AXL signaling pathway and suppressing cell proliferation, thus inhibiting tumor progression, metastasis, and drug resistance.

[0045] This invention provides several anti-GAS6 antibodies, such as ch2D3C6 and ch2O18G6, including, for example, mouse antibodies and their chimeric counterparts. The CDR sequences (Kabat definition) and heavy chain variable regions and light chain variable regions of some disclosed antibodies are shown in the table below.

[0046] Table 1

[0047]

[0048] This invention also provides chimeric antibodies. These chimeric antibodies have VH and VL domains derived 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 IgG4). These chimeric antibodies are labeled chHvLv-IgG. Some of the chimeric antibodies described in this disclosure are shown in the table below.

[0049] Table 2

[0050]

[0051]

[0052] In some embodiments, the antibody may have a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR1 amino acid sequence; CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR2 amino acid sequence; and CDR3 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH CDR3 amino acid sequence. The light chain variable region includes CDRs 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL. The CDR1 amino acid sequence has or is composed of an amino acid sequence that has at least 80%, 85%, 90%, or 95% identity with the selected VLCDR2 amino acid sequence, the CDR2 region contains or is composed of an amino acid sequence that has at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence, and the CDR3 region contains or is composed of an amino acid sequence that has at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence.

[0053] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable region containing one, two, or three of the following CDRs: SEQ ID NO:5-7, SEQ ID NO:8-10, SEQ ID NO:11-13, or SEQ ID NO:14-16, SEQ ID NO:49-51, SEQ ID NO:52-54, SEQ ID NO:57-59, SEQ ID NO:60-62, SEQ ID NO:63-65, or SEQ ID NO:66-68, having zero, one, or two amino acid insertions, deletions, or substitutions on one, two, or three of the selected CDRs. For example, the antibody or antigen-binding fragments described herein may contain a light chain variable domain, wherein the light chain variable domain contains one, two, or three of the following CDRs: SEQ ID NO:5 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:6 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:7 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0054] This invention also provides antibodies or antigen-binding fragments thereof that bind to 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 the Kabat definition, the Chothia definition, or the 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.

[0055] Nucleic acids, vectors and cells

[0056] Embodiments of the present invention also provide nucleic acids comprising polynucleotides encoding polypeptides, said polypeptides comprising immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains comprise CDRs as shown in Tables 1 and 2, or have sequences as shown in Tables 1 and 2. When the polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to GAS6 (e.g., human GAS6).

[0057] Embodiments of the present invention also provide recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells incorporating the recombinant vectors (i.e., host cells containing polynucleotides and / or vectors containing polynucleotides), and recombinant antibody polypeptides or fragments thereof generated by recombinant technology.

[0058] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is expressed within the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or polyadenylate tails, said regulatory elements being located within the vector or at, near, or flanking, the integration site of the target polynucleotide in the genome of the host cell, such that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0059] Vectors can be introduced into host cells using 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 viruses). Therefore, non-limiting embodiments of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0060] This invention provides host cells transformed using the vector described above. The host cell can be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is *Escherichia coli*. Preferably, the eukaryotic cell is selected from protist 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*.

[0061] Methods for preparing anti-GAS6 antibodies

[0062] Standard techniques for preparing polyclonal and monoclonal antibodies can be used to generate antibodies by using isolated fragments of human GAS6 as an immunogen. Polyclonal antibodies can be generated in animals through multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an immunogenic agent in the species to be immunized. The antigenic peptide or protein can be injected into the animal multiple times.

[0063] Immunogens are typically used to prepare antibodies by immunizing suitable subjects (e.g., humans or transgenic animals expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, recombinantly expressed peptides or chemically synthesized peptides (e.g., fragments of human GAS6). The formulation may further contain adjuvants, such as Freund's complete or incomplete adjuvants, or similar immunostimulants.

[0064] Pharmaceutical compositions, uses and treatments

[0065] This invention also provides pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

[0066] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which increase the shelf life or potency of the antibody.

[0067] In one aspect, the present invention provides the use of the aforementioned anti-human GAS6 antibody or its antigen-binding fragment in the preparation of a medicament for treating cancer. According to embodiments of the invention, the cancers include 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.

[0068] This invention provides one or more antibodies or antigen-binding fragments thereof that can be used for a variety of therapeutic purposes. In one aspect, this disclosure provides methods for treating a subject with cancer, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment can lead to a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0069] In one aspect, this disclosure is characterized by a method comprising administering a therapeutically effective dose of the disclosed antibody or antigen-binding fragment thereof to a subject in need (e.g., a subject who has, or is identified as having, or is diagnosed as having, cancer), the cancer being, for example, breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, neuroendocrine tumors, pancreatic cancer, bladder cancer, and head and neck cancer.

[0070] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0071] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0072] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products, such as those purchased from Sigma.

[0073] Example 1: Animal Immunization

[0074] Female Balb / c mice aged 6-8 weeks were immunized with recombinant human GAS6 hFc protein in groups of five. Specifically, each mouse received a primary immunization via subcutaneous injection of 20 μg of recombinant GAS6 protein (Human GAS6 C-Fc, Novoprotein, Cat#C12W) mixed with Freund's complete adjuvant (CFA). A total of five immunizations were administered. The subsequent four immunizations were each administered with a mixture of 20 μg of recombinant GAS6 protein and Freund's incomplete adjuvant (IFA), with a three-week interval between each immunization. The final two immunizations were administered with 20 μg of recombinant mouse GAS his protein (Sino Biological, Cat#58026-M08H). Starting with the second immunization, orbital blood samples were collected one week after immunization. The obtained serum was used for antigen-specific titer testing and GAS6-AXL binding blockade testing.

[0075] (1) Serum titer antigen binding detection

[0076] Serum ELISA binding assays were performed using recombinant human GAS6 his protein (Novoprotein, Cat#C01W) and mouse GAS6 his protein (SinoBiological, Cat#58026-M08H). Specifically, 1 μg / ml of human GAS6 his protein and 1 μg / ml of mouse GAS6 his protein were coated into 96-well plates, 100 μL / well, and incubated overnight. On the second day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared with 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, serially diluted serum was added, and the plates were incubated at 37°C for 1 hour. After incubation and washing, 1:5000 diluted goat anti-mouse IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, 115-005-008) was added, and the plates were incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The OD450 nm reading was detected using a Spectra M5e instrument.

[0077] The results are as follows Figure 1 As stated, after five immunizations with human and mouse GAS6 proteins, mouse serum showed varying degrees of specific binding to both antigens, with the serum of mouse #4 exhibiting a stronger response to both antigens.

[0078] (2) Serum titer ligand receptor binding blockade detection

[0079] The blocking effect of mouse serum on the binding of human GAS6 and human AXL proteins was assessed using an ELISA method. Specifically, recombinant human AXL his protein (AcroBiosystems, Cat#AXL-H5226) was coated in 96-well plates at 1 μg / ml, 100 μl / well, and incubated overnight. On the second day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared with 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, and 50 μL of serially diluted serum and 50 μL of recombinant human GAS6 hFc protein (Novoprotein, Cat#C12W) at a final concentration of 150 ng / ml were added to each well and incubated at 37°C for 1 hour. A commercially available GAS6 neutralizing antibody (R&D Systems, AB885) at a final concentration of 20 μg / ml was used as a positive control (PC) and Goat IgG (R&D Systems, AB-108-C) at a final concentration of 20 μg / ml was used as a negative control (NC). After incubation and washing, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added, and the mixture was incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured using a Spectra M5e instrument at OD450 nm. In this system, the wells with only GAS6hFc protein added and incubated with HRP antibody showed the highest absorbance (Max), while the wells with only 1% BSA added showed the lowest absorbance (Mini). Wells with absorbance lower than Max corresponded to serum dilutions containing antibodies that inhibit the binding of human GAS6 and human AXL. The inhibition rate (%) = (OD450 nm) / (Mini). Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100.

[0080] ELISA results as follows Figure 2 The results showed that, at low dilution gradients (third dilution, 1:360), mouse serum inhibited the binding of human GAS6hFc and human AXL his proteins by more than 60%, with mouse serum numbered #4 showing the best inhibitory effect.

[0081] Example 2: Screening and Identification of Hybridoma Cells

[0082] (I) Preliminary screening of hybridoma supernatant

[0083] 1) Preliminary binding screening of hybridoma supernatant

[0084] Based on the serum titer detection results of Example 1, two mice, numbered #4 and #5, were selected for booster immunization, and their spleens and lymph nodes were harvested. The resulting cells were ground and electrofused with mouse myeloma cells. After culturing in HAT and HT media for 10-14 days, the hybridoma supernatant was screened. First, the binding of human and mouse GAS6 his proteins was screened using ELISA, similar to the serum titer ELISA method. Human and mouse GAS6 his proteins were coated in 384-well plates at 1 μg / ml, 40 μl / well, and incubated overnight. After washing and blocking, 30 μl of hybridoma supernatant was added and incubated before binding detection. Ultimately, 372 clones were screened for further blocking assays. Among them, 239 clones showed strong binding to both human and mouse GAS6 proteins; 79 clones showed strong binding to human GAS6 and weak binding to mouse GAS6 protein; and 54 clones showed strong binding to human GAS6 but no binding to mouse GAS6.

[0085] 2) Preliminary blocking screening of hybridoma supernatant

[0086] Thirty-two clones selected from the ELISA binding assay were evaluated for blocking assays. The specific method was similar to the serum titer blocking assay: recombinant human AXL his protein was coated into 96-well plates at 1 μg / ml, 100 μl / well, and incubated overnight. After washing and blocking, 50 μl of hybridoma supernatant and 50 μl of a mixture of recombinant human GAS6 hFc protein to a final concentration of 150 ng / ml were added to each well, and the plates were incubated at 37°C for 1 hour. The 50 μl of hybridoma medium and 50 μl of the 150 ng / ml recombinant human GAS6 hFc protein mixture were used as the maximum signal (Max), while the absorbance of wells containing only hybridoma medium was the minimum (Mini). After washing, add a 1:5000 diluted goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098), incubate, wash again, add 100 μL of TMB substrate to each well for color development, and terminate the reaction with an equal volume of 1N HCl. Read the absorbance at OD450 nm. In this system, the hybridoma supernatant corresponding to wells with absorbance lower than Max contains antibodies that inhibit the binding of human GAS6 and human AXL. Inhibition rate (%) = (OD450 nm / (Axonolactone)). Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100. Finally, 71 clones with an inhibition rate greater than 45% were selected.

[0087] (II) Subclonal screening of hybridoma supernatant

[0088] Based on preliminary ELISA binding and blocking assays of the hybridoma supernatant, 53 clones were selected for subcloning. Among them, 36 clones showed strong binding to both human and mouse GAS6, with ligand-receptor binding inhibition rates greater than 50%; 8 clones showed strong binding to human GAS6 and weak binding to mouse GAS6, with ligand-receptor binding inhibition rates greater than 60%; and 9 clones showed strong binding to both human and mouse GAS6, with ligand-receptor binding inhibition rates greater than 45% and less than 50%. The subclone supernatant was then subjected to ELISA binding assays for human and mouse GAS6 proteins and ligand-receptor binding blocking assays (using the same methods as the preliminary screening), ultimately selecting 11 candidate clones that showed strong binding to both human and mouse GAS6 proteins, with ligand-receptor binding inhibition rates greater than 40%.

[0089] Example 3: Production and Identification of Human-Mouse Chimeric Antibodies

[0090] (I) Extraction of candidate cloned genes

[0091] 1) Extraction of total RNA and synthesis of cDNA from hybridoma cells

[0092] After culturing monoclonal hybridoma cells to the logarithmic growth phase, cells were collected (approximately 1E6 cells / clone) and utilized... Total RNA was extracted from hybridoma cells using RNAPlus (MN, Cat#740984.250). Using 1 μg of extracted RNA, [the following steps were performed]: III RT SuperMix for qPCR (+gDNAwiper) (Vazyme, Cat#R323-01) was used for cDNA synthesis.

[0093] 2) Gene amplification of antibody VH and VL sequences and T vector cloning

[0094] Using the synthesized cDNA as a template, specific amplification of the cDNA was performed using primers Primer A + S mix (containing universal heavy and light chain primers Primer A, and primers for the constant regions of human hIgG1, 2a, 2b, and 3, as well as specific primers for human kappa) and Ex Taq enzyme (TaKaRa, Cat#RR902A). The PCR reaction system and cycles are as follows. The PCR products were purified using a gel extraction kit after 1% agarose gel electrophoresis. Gel and PCR Clean-up (MN, Cat#740609.250) were used to recover the target fragment.

[0095]

[0096] The recovered fragment was then cloned into the pMD19-T (TaKaRa, Cat#3271) vector using Solution I (TaKaRa, Cat#6022Q). The cloned plasmid was then transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL) using a heat stimulation method and evenly spread on 2YT solid plates containing ampicillin. The plates were then sent to Genewiz sequencing company for sequencing using the universal primer PMAL-C2X-R (SEQ ID NO: 70).

[0097] (II) Construction, expression and purification of candidate cloned human-mouse chimeric expression vectors

[0098] 1) Construction of expression carrier

[0099] Analyze the antibody VH and VL sequencing sequences, select TA clone plasmids with correct antibody sequencing, and amplify the target fragment using the antibody VH / VL universal primer mix (containing the expression vector signal peptide) under the action of the high-fidelity enzyme PrimeSTAR (TaKaRa, Cat#R045) and primers (PCR procedure as follows). After electrophoresis, the target fragment is recovered using a gel recovery kit (MN, Cat#740609.250).

[0100]

[0101] The recovered fragments were inserted into the corresponding linearized vectors (pTT5_hIgG1.G1m3 / pTT5_hKappa.Km3) using recombinase (Vazyme, Cat#C112-02). The pTT5_hIgG1.G1m3 vector contained the hIgG1 Fc sequence (SEQ ID NO:69), and the pTT5_hKappa.Km3 vector contained the hKappa sequence (SEQ ID NO:74). The recombinant vectors were transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL) and evenly spread on 2YT solid plates containing ampicillin. The plates were sent to Genewiz sequencing company for sequencing using pTT5-F (SEQ ID NO:71) and pTT5-R (SEQ ID NO:72).

[0102] 2) Expression and purification of chimeric antibodies

[0103] Analyze the sequencing results, amplify the plasmids with correct sequencing, and transfect the heavy and light chain plasmids at a ratio of 2:3 using PEI reagent (Polysciences, Cat#24885) (1 μg plasmid: 4 μg PEI) into HEK293 cells at a density of 2E6 / ml. Incubate the transfected cells at 37°C in a 5% CO2 incubator for 5-7 days. Centrifuge the culture supernatant and filter it through a 0.22 μm filter. Purify the supernatant using a Protein G agarose gel column (GE Healthcare Bio-sciences, 17-0618-05). First, equilibrate the column with 1xPBS (pH 7.4). Load the filtered culture supernatant onto the column, wash the column with 1xPBS (pH 7.4), elute the sample with elution solution (50 nM sodium citrate, pH 2.5), and neutralize the eluted sample with 1M Tris-HCl, pH 9.0 solution. The neutralized sample was replaced with 1xPBS (pH 7.4), filtered through a 0.22µm filter for sterilization, and the concentration of the purified antibody was determined using Nanodrop (Thermo Fisher Scientific Inc).

[0104] (III) Identification of specific binding of human-mouse chimeric antibodies

[0105] 1) Evaluation of the specific binding of anti-human GAS6 chimeric antibody to human GAS6 and its cross-binding activity with mouse GAS6

[0106] ELISA binding assays were performed using recombinant human GAS6 his protein (Novoprotein, Cat#C01W) and mouse GAS6 his protein (SinoBiological, Cat#58026-M08H). Specifically, 0.5 μg / ml human GAS6 his protein and 0.5 μg / ml mouse GAS6 his protein were coated into 96-well plates, 100 μL / well, and incubated overnight. The next day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared with 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, and serially diluted purified antibodies were added and incubated at 37°C for 1 hour. The anti-huGas6 antibody (Santa Cruz, sc-376087) and anti-mGas6 Ab (R&D systems, AF986) were positive control antibodies binding human GAS6his protein and mouse GAS6his protein, respectively. After incubation and washing, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were detected using a Spectra M5e instrument at OD 450 nm.

[0107] Table 3. Summary of ELISA results of chimeric antibody binding to human and mouse GAS6 his protein.

[0108] Combined with human GAS6 Combining 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

[0109] ELISA combination results as follows Figure 3 , Figure 4 As shown in Table 1, nine chimeric antibodies can specifically bind to human and mouse GAS6 his proteins. Among them, ch3K16C8, ch5I23B12, ch2O18G6, ch3F23G12, ch11B11D12, ch12H19D4, and ch18I16C6 exhibit comparable binding activity to human and mouse GAS6 his proteins (Table 3).

[0110] 2) Evaluation of cross-binding between human-mouse chimeric antibody and human Protein S

[0111] ELISA binding assays were performed using recombinant human Protein His protein (R&D, Cat#9489-PS). Specifically, 1 μg / ml of human Protein His protein was coated into 96-well plates (100 μL / well) and incubated overnight. The next day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared with 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, and serially diluted purified antibody was added and incubated at 37°C for 1 hour. After incubation and washing, a 1:5000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were detected using a Spectra M5e instrument at OD 450 nm.

[0112] Table 4. Results of ELISA binding of chimeric antibodies to human Protein Si his protein.

[0113] chimeric antibodies ch2O18G6 ch15G5E5 ch18I16C6 EC50(nM) 0.025 0.200 0.008

[0114] ELISA combination results as follows Figure 5 As shown in Table 4, the chimeric antibodies ch2O18G6, ch15G5E5 and ch18I16C6 all exhibit cross-binding activity with human Protein S his protein.

[0115] The above results indicate that antibodies ch2O18G6, ch15G5E5, and ch18I16C6 have specific binding activity to human GAS6 his protein, mouse GAS6 his protein, and human Protein His protein.

[0116] (iv) Identification of blocking effects of human-mouse chimeric antibodies

[0117] The blocking effect of chimeric antibodies on the binding of human GAS6 and human AXL proteins was evaluated using ELISA. Specifically, recombinant human AXL hFc protein (internal synthesis, sequence as shown in SEQ ID NO: 73) was coated into 96-well plates at 1 μg / ml, 100 μl / well, and incubated overnight. The next day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared with 1xPBST), and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, and 50 μl of serially diluted antibody and 50 μl of a mixture of recombinant human GAS6 his protein (Novoprotein, Cat#C01W) at a final concentration of 4 ng / ml were added to each well, and the plates were incubated at 37°C for 1 hour. A Kyowa Kirin GAS6 neutralizing antibody hzKM5321 LV7bHV0 at a final concentration of 10 μg / ml was used as a positive control, and human IgG at a final concentration of 10 μg / ml was used as a negative control. 50 μl of 1% BSA and 50 μl of recombinant human GAS6 at a final concentration of 4 ng / ml were added to each well. The his protein mixture was used as the maximum signal (Max), and the wells with only 1% BSA showed the lowest absorbance (Mini). After incubation and washing, rabbit anti-6xhis HRP antibody (abcam, ab1187) diluted 1:10000 was added, and the mixture was incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The absorbance at OD450 nm was measured using a Spectra M5e instrument. The results showed that the purified human-mouse chimeric antibody exhibited a certain blocking activity against the binding of GAS6-AXL in this system.

[0118] (V) Identification of the proliferation-inhibiting activity of human-mouse chimeric antibodies

[0119] To evaluate the effect of anti-GAS6 antibody on GAS6-dependent cell growth, a cell proliferation inhibition assay was performed using Ba / F3-AXL cells (Nanjing Kebai, Cat#CBP73249). Log-grown Ba / F3-AXL cells were centrifuged, and the culture supernatant was discarded. The centrifuged cells were washed three times with 1x DPBS and resuspended in fresh RPMI 1640 + 10% FBS + 100 ng / ml recombinant human GAS6 his (R&D, Cat#885-GSB) medium at a cell density of 5*10e4 / ml. The resuspended cells were then seeded into 96-well white blood cell culture plates at 100 μL / well, with three replicates, and incubated at 37°C for 4 hours. After incubation, a serially diluted 10x antibody solution was added at 11.1 μL / well, and the cells were incubated at 37°C for another 72 hours. After 72 hours of incubation, 100 μL of Cell Titer Glo assay reagent (Promega, Cat#G7572) was added to each well and incubated for 30 minutes. RLU values ​​were then read. The RLU reading in well 6 containing only 100 ng / ml GAS (without antibody) was the maximum, and the RLU reading in wells containing only culture medium was the minimum. hIgG1 was used as a negative control. Antibody proliferation inhibition activity (%) = 100 - (sample well RLU - minimum RLU) / (maximum RLU - minimum RLU) x 100.

[0120] The results are as follows Figure 6 The results showed that the chimeric antibodies ch2D3C6 and ch2O18G6 inhibited the growth of Ba / F3-AXL in a concentration gradient-dependent manner, with inhibition rates of approximately 30% and 20% respectively at a concentration of 200 nM.

[0121] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0122] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An anti-human GAS6 antibody or its antigen-binding fragment, characterized in that, include: The heavy chain variable region (VH) comprises VH CDRs 1, 2 and 3, and VH CDRs 1, 2 and 3 have amino acid sequences as shown in SEQ ID NO: 11, 12 and 13, respectively; The light chain variable region (VL) comprises VL CDRs1, 2 and 3, and VL CDRs1, 2 and 3 have amino acid sequences as shown in SEQ ID NO: 14, 15 and 16, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The VH includes the amino acid sequence shown in SEQ ID NO: 3, and the VL includes the amino acid sequence shown in SEQ ID NO:

4.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is a chimeric antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is an antibody fragment that specifically binds to human GAS6 and is selected from Fv, Fab, Fab', scFv and F(ab')2.

6. An isolated nucleic acid, characterized in that, The nucleic acid encodes the anti-human GAS6 antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. A carrier, characterized in that, Includes the nucleic acid described in claim 6.

8. An isolated cell, characterized in that, Includes the carrier as described in claim 7.

9. A method for preparing the anti-human GAS6 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, include: (1) The cells of claim 8 are cultured under suitable conditions; as well as (2) Separate and recover the anti-human GAS6 antibody or its antigen-binding fragment as described in any one of claims 1-5.

10. A pharmaceutical composition, characterized in that, include: The anti-human GAS6 antibody or its antigen-binding fragment as described in any one of claims 1-5; as well as Pharmaceutically acceptable carrier.

11. Use of the anti-human GAS6 antibody or its antigen-binding fragment according to any one of claims 1-5 in the preparation of a medicament for treating cancers that highly express GAS6, said cancers being selected from breast cancer, lung cancer, gastric cancer, colon cancer, neuroendocrine tumors, pancreatic cancer, bladder cancer, head and neck cancer, and chronic and acute myeloid leukemia.

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