Anti-human axl antibodies, genes, and uses thereof

Abstract

The application belongs to the technical field of biology, discloses an anti-human AXL antibody, a gene and application thereof, and amino acid sequences of a heavy chain variable region and a light chain variable region of the anti-human AXL antibody are respectively shown as SEQ ID NO: 23-34. The anti-human AXL antibody of some examples of the application has a binding capacity EC 50 all less than 2 nM, and has no binding capacity for irrelevant antigens Her2 and BSA, which indicates that the screened antibody targeting AXL has high specificity and strong binding activity. Different AXL / CD3 bispecific antibodies prepared based on the anti-human AXL antibody of the application have strong killing capacity (IC 50 : 0.1-0.5 nM) on AXL positive MDA-MB-231 cells, and have low killing capacity on AXL negative SKBR3 cells, which avoids off-target toxicity of different AXL / CD3 antibodies and further widens the application.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to an anti-human AXL antibody, gene, and its applications. Background Technology

[0002] AXL (also known as ARK, UFO, and TYRO-7) belongs to the receptor tyrosine kinase TAM family, along with TYRO-3 and MER. AXL derives from the Greek word "Anexelekto," meaning "uncontrolled," and was first isolated from chronic myeloid leukemia cells in 1988. It originates from chromosome 19q13.2. AXL Gene encoding. Structurally, the AXL protein is mainly composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain of the AXL protein contains two immunoglobulin-like domains (Ig) and two fibronectin type III (FNIII) domains. The Ig domain is the region that binds to endogenous ligands, while the FNIII domain plays a regulatory role in the binding of the AXL protein to its ligands. The intracellular region of the AXL protein is a tyrosine kinase domain, which has kinase activity and can participate in the transmission of various signals in normal cells and tumor cells (Tang Y, et al. J ExpClin Cancer Res.2023;42(1):148.).

[0003] AXL activation occurs through both ligand-dependent and ligand-independent activation, with ligand-dependent activation being the predominant pathway. Growth Arrest Specific Protein 6 (Gas6), one of the ligands for the TAM receptor, exhibits the highest affinity for AXL compared to TYRO-3 and MER. First, AXL and Gas6 form a high-affinity Gas6 / AXL complex in a 1:1 ratio. This complex then forms a homodimer with another Gas6-AXL complex. Phosphorylation of Y779, Y821, and Y866 in the intracellular kinase domain of this dimer enables interactions between the AXL receptor and its downstream signaling molecules. On the other hand, AXL can also be activated via a Gas6-independent mechanism (Nasimian A, et al. Int J Mol Sci. 2023;24(4):3830.). This is because AXL overexpression induced by various factors can lead to the aggregation or ligand-independent dimerization of AXL extracellular domains, including human epidermal growth factor receptor 2 (Her2), epidermal growth factor receptor (EGFR), cellular-mesenchymal epithelial transition factor (c-Met), and TYRO-3. For example, heterodimerization of AXL with Her2 enables its activation in a Gas6-independent manner and promotes drug resistance and tumor progression in Her2-positive breast cancer cells. In addition, the stable interaction between AXL and EGFR can also activate AXL in a Gas6-independent manner and promote the activation of downstream signaling pathways. The PI3K / AKT / mTOR, JAK / STAT, NF-κB, and RAS / RAF / MEK / ERK signaling pathways, as important downstream pathways of the Gas6 / AXL axis, participate in regulating tumor cell survival, proliferation, drug resistance, invasion and metastasis, and angiogenesis (Lei T, et al. Pharmacol Res. 2023;188:106668.). Activation of AXL can induce a series of immunosuppressive signals, including downregulation of MHC-I expression on the surface of tumor cells, overexpression of PD-L1, and promotion of the secretion of immunosuppressive cytokines (CCL3-5, G-CSF, IL-3, IL-4, IL-6, TGF-β, and TNF-α), thus contributing to the formation of an immunosuppressive tumor microenvironment. Simultaneously, activation of this pathway induces the production of M2 macrophages, promotes the infiltration of monocytes and myeloid-derived suppressor cells (MDSCs), and reduces the number of dendritic cells (DCs), NK cells, and CD4+ cells. + and CD8+ T cell infiltration in tumors promotes tumor development (Kim SY, et al. Sci Rep. 2016;6:29673.).

[0004] Because AXL is highly expressed in various tumor tissues such as lung cancer, breast cancer, prostate cancer, and pancreatic cancer, and plays an important role in tumor immune escape and malignant progression, AXL is an attractive target for cancer treatment. There are two main strategies for targeting AXL: one is to target its intracellular kinase domain to inhibit its activation, and the other is to target its extracellular region to prevent AXL from interacting with its ligand Gas6 or other receptors. Currently, drugs targeting AXL mainly include small molecule kinase inhibitors, AXL nucleic acid aptamers, antibody-drug conjugates (ADCs), and Gas6 / AXL-Fc fusion proteins. (Miao YR, et al. Nat Rev Drug Discov. 2024;23(3):201-217.)

[0005] Based on their binding sites to AXL, AXL inhibitors can be divided into two categories. Type I inhibitors bind to the aspartate-phenylalanine-glycine (DFG) motif of the AXL kinase domain. For example, BGB324 and TP-0903 are specific and highly selective inhibitors of AXL, blocking its catalytic activity and reducing its expression. Type II inhibitors, on the other hand, can bind to sites other than DFG (Sun ZG, et al. Curr Top Med Chem. 2019;19(15):1338-1349.). For example, FDA-approved cabozantinib and foretinib are multi-target small molecule tyrosine kinase inhibitors that inhibit AXL. However, the off-target effects of multi-target kinase inhibitors can lead to unpredictable toxic side effects. AXL and other tyrosine kinases (such as c-Met) are highly homologous, making the development of selective AXL inhibitors difficult. In clinical applications, off-target effects can easily lead to serious toxic side effects.

[0006] An AXL-targeting ADC drug (AXL-107-MMAE) used in combination with a MAPK inhibitor inhibits the growth of drug-resistant melanoma. However, due to potential toxicity, the future clinical prospects of AXL-107-MMAE still require further observation. The FDA has approved an AXL-targeting ADC drug (BA3011) for Phase I / II clinical trials, primarily for the treatment of advanced solid tumors. Enapotamab is an anti-AXL human IgG1 and monomethyl Auristatin E ADC antibody drug that showed strong anti-tumor activity in a preclinical model of non-small cell lung cancer. However, its efficacy, dosage, and biomarkers did not meet the proof-of-concept criteria for continued development in clinical applications, leading to the termination of its clinical development (Boshuizen J, et al. CancerRes. 2021;81(7):1775-1787.).

[0007] With the gradual development of therapeutic Fc fusion proteins in tumor treatment, high-affinity fusion proteins targeting AXL have become a research hotspot. MYD1 is an AXL high-affinity mutant Fc fusion protein, exhibiting a higher affinity for Gas6 protein than the wild-type AXL protein. In animal models of ovarian cancer, MYD1 significantly inhibited tumor cell invasion and metastasis. However, due to the existence of multiple Cas6-independent activation mechanisms of AXL, the effectiveness of Fc fusion proteins in cancer patients requires further investigation and validation.

[0008] Research on antibodies targeting AXL is still in the preclinical stage. Mouse hybridoma-derived antibodies targeting the extracellular domain of human AXL, such as 3G9, 8B5, and 12A11, have all attenuated the growth of A549 tumor xenografts by inducing downregulation of AXL receptor expression. However, because these monoclonal antibodies do not cross-react with mouse AXL, the effects of AXL on tumor angiogenesis and the tumor stroma in mice cannot be assessed (Li Y, et al. Oncogene. 2009;28(39):3442-3455). To address this issue, researchers developed a phage-derived monoclonal antibody (YW327.6S2) capable of recognizing both human and mouse AXL. This antibody not only downregulates AXL expression but also inhibits tumor cell growth by modulating the tumor stroma through the regulation of tumor-associated vascular system and immune cell function. Unfortunately, YW327.6S2 monotherapy did not show satisfactory efficacy in preclinical in vivo studies and has not been investigated as a clinical candidate (YeX, et al. Oncogene. 2010;29(38):5254-5264.). Other antibodies include 20G7-D9, which provides a promising treatment strategy for triple-negative breast cancer with stromal features by inhibiting AXL-dependent epithelial-mesenchymal transition, tumor growth, and metastasis (Leconet W, et al. Clin Cancer Res. 2017;23(11):2806-2816.); and MAb173, which inhibits Kaposi's sarcoma growth, increases tumor cell apoptosis, and significantly reduces AXL protein levels in tumors (Liu R, et al. Blood. 2010;116(2):297-305.).

[0009] Bispecific antibodies (bsAbs) are antibody-based molecules with two distinct antigen-binding sites. Due to their novel mechanisms of action and therapeutic applications, they have become a research hotspot for targeted AXL therapy. Bis1 is a bispecific antibody that binds to both AXL and EGFR. When used in combination with the EGFR inhibitor osimertinib, Bis1 significantly delayed the onset of resistance to osimertinib and continuously inhibited the growth of lung cancer tumors in vivo (Simoni-Nieves A, et al. Cell Rep Med. 2024;5(9):101703.). However, these structures showed relatively low efficacy in preclinical models, and inducing effective T cell-mediated cancer cell lysis usually requires high drug concentrations, high effector-to-target ratios, and additional co-stimulatory signals. Bispecific T cell engagers (BiTEs) consist of two distinct single-chain variable fragments (scFvs) derived from anti-CD3 and anti-TAA antibodies, covalently linked by small linker peptides. In preclinical models, BiTEs exhibit small size, high flexibility, and high affinity between effector and target cells, demonstrating strong antitumor activity superior to traditional mAbs and other forms of bispecific antibodies (Goebeler ME, et al. Nat Rev Clin Oncol. 2020;17(7):418-434.). Based on Pronectin TM The platform constructed pAXL×CD3ε, a potent BiTE for ovarian cancer. Its low molecular weight facilitates tumor invasion, ensuring targeted cancer cell therapy, while simultaneously recruiting and activating T lymphocytes. Its small size allows for rapid and economical production in E. coli and yeast systems, providing a theoretical basis for its development as a novel drug in ovarian cancer treatment (Riillo C, et al. J Transl Med. 2023;21(1):301.). In summary, due to the extensive biological defects and functional diversity of AXL, the development of BiTEs targeting AXL has attracted widespread attention. With the continuous improvement of AXL-targeting drugs and a deeper understanding of the potential mechanisms of AXL, inhibiting AXL holds promise for providing an effective strategy for the treatment of cancer patients. Summary of the Invention

[0010] Although the development of antibodies targeting AXL has shown some efficacy, drugs with better therapeutic effects and lower toxicity are still needed in clinical applications. This invention, based on phage display technology and utilizing a mouse immunization strategy, has newly discovered several single-chain antibodies targeting AXL. These antibodies are then combined with the anti-CD3ε antibody SP34 to construct a novel AXL×CD3 bispecific antibody. This antibody exhibits strong killing effect on AXL-expressing tumor cells while enriching T cells and promoting their infiltration into tumor cells, providing a more effective and safer treatment method for clinical applications.

[0011] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an anti-human AXL antibody, gene and its application.

[0012] The technical solution adopted in this invention is:

[0013] A first aspect of the present invention provides: an anti-human AXL antibody, comprising a heavy chain variable region and a light chain variable region, wherein the CDRs of the heavy chain variable region and the light chain variable region are selected from a group consisting of:

[0014] Antibody F1-G has CDR1 to CDR3 of heavy chain variable region as SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISNYLS, YTSRLQS, and QQGKTLPYT, respectively.

[0015] Antibody F7-B has CDR1 to CDR3 of heavy chain variable region as SHVLH, YINPYYDDTKYSEKFKG, and YYPGAMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISTYLN, YTSRLHS, and QQGKTLPYT, respectively.

[0016] Antibody F7-G has CDR1 to CDR3 of heavy chain variable region as GYTMH, YIDPFSGDTSYNLKFKG, and ELTTVVAYWYFDV, respectively, and CDR1 to CDR3 of light chain variable region as RSSQSIVHSNGNTYLE, KVSNRFS, and FQGSHVPFT, respectively.

[0017] Antibody 3-1E has CDR1 to CDR3 of heavy chain variable region as GYTMH, YIDPFSGDTSYNLKFKG, and ELTTVVAYWYFDV, respectively, and CDR1 to CDR3 of light chain variable region as RSSQSIVHSNGNTYLE, KVSNRFS, and FQGSHVPYT, respectively.

[0018] Antibody 3-3C has CDR1 to CDR3 of heavy chain variable region as GYSMH, YIDPFSGAINYNLKFKG, and ELTTVVAYWYFDV, respectively, and CDR1 to CDR3 of light chain variable region as RSSQSIVHSNGNTYLE, KVSNRFS, and FQGSHVPYT, respectively.

[0019] Antibody 3-3F has CDR1 to CDR3 of heavy chain variable region as SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISNYLN, YSSRLHS, and QQGKTLPYT, respectively.

[0020] In some instances, anti-human AXL antibodies are single-chain antibodies, chimeric antibodies, humanized antibodies, scFvs fused with Fc fragments, or homologous or heterologous bivalent or multivalent antibodies.

[0021] In some examples of anti-human AXL antibodies, the Fc fragment is selected from the Fc fragment of human IgG1, IgG2, IgG3 or IgG4 or its variants or modifications.

[0022] In some examples of anti-human AXL antibodies, flexible peptides are used to link different functional fragments in homologous or heterologous bivalent or multivalent antibodies.

[0023] In some examples of anti-human AXL antibodies, heterobivalent antibodies include, but are not limited to, those based on Triomabs bispecific antibodies, Crossmab bispecific antibodies, DVD-Ig bispecific antibodies, BiTE bispecific antibodies, DART bispecific antibodies, TandAbs bispecific antibodies, etc., which combine the CD3 and AXL domains in different combinations to form various forms of bispecific antibodies.

[0024] In some examples of anti-human AXL antibodies, the antibody is selected from one of the following antibodies:

[0025] Antibody F1-G: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24;

[0026] Antibody F7-B: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26;

[0027] Antibody F7-G: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28;

[0028] Antibody 3-1E: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30;

[0029] Antibody 3-3C: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32;

[0030] Antibody 3-3F: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:34.

[0031] The above features can be combined arbitrarily as long as they do not conflict with each other.

[0032] A second aspect of the invention provides: a gene encoding the anti-human AXL antibody described in the first aspect of the invention.

[0033] A third aspect of the invention provides: a recombinant expression vector or recombinant bacterial strain expressing the anti-human AXL antibody as described in the first aspect of the invention; or having inserted the gene as described in the second aspect of the invention.

[0034] A fourth aspect of the present invention provides: the application of the anti-human AXL antibody described in the first aspect of the present invention, wherein the application is selected from:

[0035] Preparation of antibody-drug conjugates targeting human AXL;

[0036] Preparation of diagnostic reagents targeting AXL;

[0037] Preparation of drug delivery vehicles targeting AXL;

[0038] To prepare drugs targeting AXL;

[0039] Prepare an inducer, which induces effector cells to kill tumor cells expressing AXL.

[0040] In some applications, the conjugate in the antibody-drug conjugate is a cytotoxic drug.

[0041] In some applications, the antibody conjugate is used to kill tumor cells expressing AXL.

[0042] In some application examples, the tumor is selected from lung cancer, breast cancer, prostate cancer, and pancreatic cancer that highly express AXL.

[0043] The beneficial effects of this invention are:

[0044] The anti-human AXL antibodies of some examples of the present invention have good affinity for AXL, high specificity, and can stably bind to AXL.

[0045] Based on the anti-human AXL antibody prepared according to the present invention, different AXL / CD3 bispecific antibodies have varying binding capacity (ECG) for AXL antigen. 50 The values ​​were all less than 2 nM, and there was no binding ability for unrelated antigens Her2 and BSA, indicating that the selected antibodies targeting AXL have high specificity and strong binding activity.

[0046] Different AXL / CD3 bispecific antibodies prepared based on the anti-human AXL antibody of this invention exhibit strong killing ability (IC50) against AXL-positive MDA-MB-231 cells. 50 (0.1-0.5 nM), while having very low killing ability against AXL-negative SKBR3 cells, thus avoiding the off-target toxicity of different AXL / CD3 antibodies and further broadening their application. Attached Figure Description

[0047] Figure 1 This is the result of the binding ability of different monoclonal bacteriophages to antigens.

[0048] Figure 2 These are experimental results showing the binding ability of different AXL / CD3 bispecific antibodies to the AXL antigen.

[0049] Figure 3 These are experimental results regarding the ability of different AXL / CD3 bispecific antibodies to mediate the killing effect of T cells on MDA-MB-231 cells. Detailed Implementation

[0050] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0051] In this invention, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0052] In this invention, the term "antibody" has its conventional meaning in the art and is used in its broadest sense. In the field of bioscience, analysis of the amino acid sequences of different antibody heavy and light chains reveals that the amino acid sequences near the N-terminus of both heavy and light chains vary considerably, while the amino acid sequences in other parts remain relatively constant. Therefore, the regions in the antibody light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the antibody variable regions, a small number of amino acid residues exhibit particularly strong variations. These regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVRs). There are three hypervariable regions in the V regions of both the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDRs). In antibodies, common CDR (Cellular Recognition Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When using websites that apply these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of this invention covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine its recognition ability and specificity against the corresponding antigen. Those skilled in the art should understand that when this invention defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity against the corresponding antigen are predictable.

[0053] In this invention, the term "antigen binding site" has the conventional meaning in the art, referring to a key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0054] In this invention, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of the antibody or chimeric antigen receptor of this invention. Linking sequences may consist of different amino acid residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. Longer linking sequences may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0055] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into it, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0056] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention are further explained below in conjunction with experiments.

[0057] Example 1: Preparation of AXL (ECD) antigen

[0058] Construction of pCAGGS-AXL(ECD)-hIgG1 Fc-His Tag plasmid: pCAGGS vector was obtained by double digestion with NheI and NotI; AXL(ECD)-hIgG1 Fc-His Tag fragment was synthesized by the company's gene company and the target plasmid was obtained by homologous recombination.

[0059] Expression of recombinant AXL(ECD)-hIgG1 Fc protein

[0060] Plasmid acquisition: E. coli containing the target plasmid was inoculated into 50 mL LB / ampicillin (Amp, 50 μg / mL) medium and cultured overnight at 37°C with a shaker to amplify the plasmid. A high concentration of endotoxin-free plasmid was obtained using the OMEGA Endo-Free Plasmid Midi Kit D6915, and after quality control by agarose gel electrophoresis, it was stored at -20°C for later use.

[0061] 293F cell transfection: 50 mL of healthy 293F cells (1.5 × 10⁻⁶ cells) were transfected. 6 Cells (number of cells / mL) were seeded in shake flasks and cultured overnight on a shaker at 101 rpm in 5% CO2. 5 mL of Opti-MEM + 50 μg of the target plasmid were mixed, followed by 125 μL of PEI40K, which was then slowly inverted to mix. The cells were incubated at room temperature in the dark for 30 min. The density of the overnight 293F cells was adjusted to 3.0 × 10⁻⁶ cells / mL. 6 Add the incubated plasmid at a rate of 1 / mL, while simultaneously shaking the flask. Handle with care as the culture medium contains no antibiotics. After addition, incubate on a shaker for 72 h, then centrifuge at 100 g for 5 min, collect the cell supernatant, add 50 mL of culture medium to resuspend the cells, continue culturing for 48 h, then centrifuge at 100 g for 5 min, discard the cell pellet, and retain the cell supernatant.

[0062] Purification of AXL(ECD) protein

[0063] Centrifuge 100 mL of cell supernatant at 15000 rpm for 30 min at 4°C. Collect the supernatant and filter it through a 0.45 µm filter membrane. Store on ice. Add 3 mL of Protein G to the chromatography column, replace the solution with binding buffer, and press the resin surface with a pad. Use a peristaltic pump to control the flow rate at 0.5 mL / min to ensure the cell supernatant passes through the Protein G column at a uniform rate. Wash away unbound Protein G protein with binding buffer. During elution, add neutralization buffer to the EP tube and Elution buffer to the column. Collect 1 mL of protein per tube and measure the concentration with a nanodropper until the protein concentration is undetectable. Dialyze AXL(ECD)-hIgG1 Fc protein according to the Solarbio Thrombin instructions to replace it with the enzyme digestion buffer. Take the dialyzed protein solution, add the appropriate amount of Thrombin, and rotate at room temperature for 16 h. Analyze the enzyme digestion effect using SDS-PAGE. The enzyme-digested protein sample was slowly and uniformly passed through a Protein G column to remove the IgG-Fc tag, and eluted according to the above steps. The protein was then detected by SDS-PAGE, and the purity of AXL(ECD) protein was greater than 90%.

[0064] Example 2: Immunization of mice using AXL (ECD) antigen

[0065] The AXL (ECD) antigen was mixed with adjuvant to prepare an emulsion, which was then injected subcutaneously at multiple sites into each mouse at a dose of 100 μg per injection. Immunization was performed every two weeks for a total of four injections. The first and second injections used complete Freund's adjuvant, while the third and fourth injections used incomplete Freund's adjuvant. One week after each immunization, blood was collected from the orbital venous plexus of the mice for serum titer determination (ELISA). Spleens were harvested when the titer was higher than 1:10000.

[0066] Example 3: Extraction and isolation of mouse B cells

[0067] Mice immunized with AXL (ECD) antigen were sacrificed and sterilized after serum titer was measured. The spleen was removed via abdominal surgery in a sterile environment, and spleen cells were obtained by grinding and centrifugation. Mouse B cells were sorted according to the instructions of the MojoSort™ Mouse Pan B Cell Isolation Kit II. Flow cytometry analysis showed the mouse B cell purity to be 95%.

[0068] Example 4: Extraction of total RNA and synthesis of cDNA from B cells of immunized mice

[0069] Total RNA was extracted from immunized mouse B cells according to the instructions for use of the TIANGEN RNAprep Pure Cell / Bacteria Kit, and the concentration was determined and verified by agarose gel electrophoresis. According to the Vazyme HiScript... ® II. Using the OneStep RT-PCR Kit instruction manual and relevant primers, synthesize the anti-AXL scFv VH and VL regions. Assemble the VH and VL fragments into scFv (VH-VL) using overlap PCR. Detect the products using agarose gel electrophoresis.

[0070] Example 5: Construction and diversity evaluation of Anti-AXL scFv phage library

[0071] Preparation of pCGMT3 phage vector / scFv restriction fragment

[0072]

[0073] Place the product into a 1.5 mL EP tube and incubate in a water bath at 50°C for 4 h. Recover the product by agarose gel electrophoresis.

[0074] ligation of pCGMT3 phage vector and scFv

[0075] The digested pCGMT3 phage vector and scFv fragment were ligated using T4 DNA ligase (NEB) at a molar ratio of 3:1 in the following reaction system, and the reaction was carried out overnight at 16°C.

[0076]

[0077] The obtained ligation products were purified using the OMEGA Cycle-pure kit, and the DNA was stored at -20°C.

[0078] Add 100 μL of freshly prepared XL1-Blue electroporation competent cells to 100 μL of the DNA product, avoiding pipetting, and incubate on ice for 0.5 h before transferring to a pre-chilled electroporation cuvette. The voltage was set to 2.5 kV. After electroporation, immediately rinse the cuvette with 2YT medium preheated to 37°C. Mix all the rinsed bacterial cultures into centrifuge tubes and incubate at 37°C, 200 rpm for 1.5 h. Take 10 μL of the incubated XL1-Blue cells, serially dilute them, and spread them onto 2YT solid culture dishes containing ampicillin (Amp) and tetracycline (Tet). Incubate overnight at 37°C to determine the library volume. Spread the remaining incubated cells onto 2YT solid culture dishes and incubate overnight at 37°C. The next day, use a 2YT: 50% glycerol = 1:1 (v / v) solution to gently scrape the colonies from the solid culture dishes with a spreader and collect them into 15 mL centrifuge tubes. Measure the OD600 value of the bacterial culture using a spectrophotometer. All DNA products were electroporated into XL1-Blue cells using the method described above. At this point, the total library volume was >10. 8 This is an anti-AXL scFv phage library.

[0079] Evaluation of phage library capacity diversity

[0080] 20 μL of anti-AXL scFv bacterial library was added to 80 μL of 2YT solution and incubated at 37°C and 200 rpm for 1.5 h. The solution was then evenly spread onto freshly prepared 2YT plates containing Amp and Tet, and incubated overnight at 37°C. The next day, single colonies were counted on the plates, and 24 single colonies were randomly selected for single-colony PCR to identify whether they were positive colonies, followed by sequencing verification.

[0081] Amplification of phage libraries

[0082] Take 1 mL of anti-AXL scFv bacterial library and incubate it in 200 mL of SB medium (200 μg / mL Amp, 5 μg / mL LTet, 2% glucose) at 37°C and 220 rpm. Check the OD600 value multiple times during this period. After approximately 2-3 hours, shake the bacterial suspension until the OD600 reaches 0.5. Add VCSM13 helper phage at an MOI (phage count: bacteria count) of 20:1, incubate statically at 37°C for 30 min, then incubate with shaking at 220 rpm for 1 h. Centrifuge the bacterial suspension at 3900 rpm for 15 min at room temperature, discard the supernatant, and remove glucose and uninfected helper phage. Resuspend the bacteria in 200 mL of SB medium (200 μg / mL Amp, 5 μg / mL LTet, 15 μg / mL kanamycin) and incubate overnight at 30°C with shaking at 200 rpm to amplify the phage. Take the bacterial culture after overnight shaking, centrifuge at 3900 rpm, 4°C for 15 min, and collect the supernatant. Add 50 mL of PEG8000 / 2.5M NaCl (5×) solution and gently mix with the supernatant. Place on ice for 4 h to allow the phage to settle. Centrifuge at 3900 rpm, 4°C for 30 min, discard the supernatant, and invert the centrifuge tube onto clean paper to air dry. Resuspend the phage in 80 mL of PBS, add 20 mL of PEG8000 / 2.5M NaCl (5×) solution again, gently mix, and place on ice to allow the phage to settle for 2 h. Centrifuge at 3900 rpm, 4°C for 30 min, discard the supernatant, and invert the centrifuge tube onto clean paper to air dry. Add 2 mL of PBS:50% glycerol = 1:1 (v / v) solution, aliquot, and store at -20°C. This is the first round of phage library input.

[0083] Example 6: Phage library screening based on immunotube immobilized antigens

[0084] First round of screening

[0085] Remove the screening antigen from the -80°C freezer and thaw on ice. Coat the immunotubes with AXL(ECD) antigen (positive screening protein) and hIgG1 Fc protein (negative screening protein) (50 µg / tube, coating buffer: CBS, pH 9.6, 2 mL / tube) and incubate overnight at 4°C with gentle rotation. Discard the liquid in the overnight coated immunotubes, add 3 mL of PBS solution, seal, and rotate at room temperature for 5 min. Repeat 3 times to wash away any unbound proteins. Add 2 mL of 3% BSA-PBST solution and incubate at room temperature for 2 h with rotation. Discard the blocking solution, add 3 mL of 0.05% PBST (1×PBS plus 0.05% Tween 20, the same below) buffer, and incubate at room temperature for 5 min. Wash the immunotubes 3 times. Discard the washing solution in the negative screening protein immunotubes, add 1 mL of PBS (3% BSA) and 1 mL of the first round of input phage, incubate at room temperature with rotation for 1 h, and then transfer to the positive screening protein immunotubes and incubate at room temperature with rotation for 1 h. Discard the liquid in the positive screening protein immunotherapy tube, add 3 mL of 0.05% PBST buffer, seal the tube, and rotate at room temperature for 5 min / time, washing the immunotherapy tube a total of 10 times; discard the liquid in the immunotherapy tube, add 1 mL of Trypsin (0.25 mg / mL) solution, rotate at room temperature for 30 min, then add 10 μL of 10% AEBSF to stop elution, and transfer the solution in the immunotherapy tube to a new 1.5 mL centrifuge tube, which is the first round of phage screening elution output.

[0086] First round of phage eluent titer testing

[0087] After reviving the XL1-Blue strain stored in a low-temperature freezer, single colonies were streaked on a plate containing the corresponding antibiotic (Tet), incubated overnight at 37°C, and single colonies were picked from the plate for amplification. The culture was then incubated at 37°C and 240 rpm until OD600=0.5.

[0088] Take 10 μL of the first round of phage elution buffer and perform 10-fold serial dilutions in a 96-well U-plate using SB medium. Specifically, add 10 μL of the phage library to 90 μL of medium, mix well, then add 10 μL of that mixture to another 90 μL of fresh medium, and so on, for a total of 10 dilutions to 10⁻¹⁰. -1010 μL of each dilution well was added to 90 μL of XL1-Blue bacterial culture with an OD600 of 0.5, mixed well, and incubated at 37°C for 30 min. Then, 10 μL of each XL1-Blue bacterial culture was added dropwise to SB solid medium (Amp, Tet) and incubated overnight at 37°C with the culture inverted position. The number of single colonies was recorded the following day, and the phage library titer was calculated. Simultaneously, the first-round input titer was determined using the same method and compared with the first-round output titer.

[0089] Amplification of the first round of phage elution buffer

[0090] 800 μL of the first-round phage eluent was transferred to 10 mL of XL1-Blue (5 μg / mL Tet) bacterial culture with OD600=0.5. The culture was incubated statically at 37°C for 30 min, followed by a further 30 min incubation at 220 rpm for infection. The entire bacterial culture was then evenly spread onto 15 cm agar plates (200 μg / mL Amp and 5 μg / mL Tet) and incubated overnight at 37°C. A 2YT:50% glycerol solution (1:1 v / v) was added to the overnight plates, and colonies were gently scraped from the plates into 15 mL centrifuge tubes using a spreader. The OD600 value of the bacterial culture was measured using a spectrophotometer; this constituted the first-round bacterial library. The first-round bacterial library was amplified and purified according to the phage library amplification method described in Example 5 to obtain the second-round phage library input.

[0091] The second and subsequent rounds of screening were similar to the first round. As the number of screening rounds increased, the screening conditions became more stringent, ultimately yielding phage clones with high affinity for the AXL (ECD) antigen.

[0092] Example 7: Monoclonal ELISA Detection

[0093] Take 10 μL of each eluent output and transfect it into XL1-Blue strain with OD600=0.5. Incubate at 37℃ and 200rpm for 1 h, then spread it on solid medium containing the corresponding resistance and incubate overnight at 37℃. Single colonies were randomly picked from the overnight culture plates and placed into sterile 96-well cell culture plates. 180 μL of SB medium (200 µg / mL Amp and 5 µg / mL Tet) was added to each well, and the plates were incubated at 37°C and 240 rpm for 3–5 h with shaking until the OD600 of each well reached 0.5. 100 μL of the culture from each well was transferred to a new 96-well plate, and the remaining culture was stored in glycerol at -20°C. VCSM13 helper phage with an MOI of 20 was added to the new 96-well plate, and the plates were incubated at 37°C for 30 min. Kan was then added to a final concentration of 50 µg / mL, and the plates were incubated overnight at 30°C and 200 rpm. The overnight culture plates were centrifuged at 4°C and 3900 rpm for 10 min, and the supernatant was stored at 4°C for later use.

[0094] Coat each well with 100 μL of 10 μg / mL antigen (10 μg / mL), and simultaneously coat Her2(ECD)-hIgG1 Fc and BSA as negative controls. Incubate overnight at 4°C. Discard the overnight coating liquid, add 250 μL / well of PBS buffer, and wash 3 times by shaking the ELISA plate at room temperature for 5 min each time. Add 200 μL of blocking buffer (3% BSA) to each well and block at room temperature for 2 h. Discard the blocking buffer, add 250 μL / well of PBST buffer, and wash 3 times at room temperature for 5 min each time. Add the diluted supernatant (120 μL of 3% BSA to 80 μL of supernatant after centrifugation) to each well and incubate at room temperature for 2 h. Wash the ELISA plate using the same method as above. Add 100 μL of Anti-M13 Antibody diluted 1:8000 with blocking buffer to each well and incubate at room temperature for 1 h. After washing, add 100 μL of... After incubating with TMB single-component chromogenic solution in the dark, add 100 μL of 2M H2SO4 to each well to stop the reaction. Read the OD450 value using a microplate reader, record and save the results. To ensure the accuracy of the experimental results, clones initially identified as positive were subjected to a second ELISA verification using the same method. Results are as follows: Figure 1 As shown, the binding ability of different monoclonal phages to antigens was detected by ELISA. The phages showed high absorbance values ​​for the AXL antigen, while the absorbance values ​​for other antigens Her2 and BSA were low, which proves that positive phage clones targeting the AXL antigen were obtained through screening.

[0095] Example 8: Sequencing and Sequence Analysis of Positive Clones

[0096] Positive single clones were selected based on two ELISA test results. The bacterial culture of the positive clones was amplified and cultured at 37°C and 240 rpm. A portion of the bacterial culture was sequenced, and the remaining culture was stored. The sequenced sequences were aligned using software, and the antibody sequences were translated into amino acids. The sequencing results of the selected antibodies are as follows:

[0097] Antibody F1-G: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24;

[0098] Antibody F7-B: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26;

[0099] Antibody F7-G: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28;

[0100] Antibody 3-1E: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:30;

[0101] Antibody 3-3C: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32;

[0102] Antibody 3-3F: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:34.

[0103] The CDR sequences of each antibody, determined based on the Kabat numbering scheme, are shown in Table 1.

[0104] Table 1. CDR analysis results of different AXL antibodies

[0105]

[0106] Example 9: Construction of AXL / CD3 bispecific antibody

[0107] Construction of AXL / CD3 bispecific antibody plasmid

[0108] The anti-AXL scFv fragment obtained by sequencing was constructed at the C-terminus of the CD3(SP34) VH-CH1 region using PCR. The two fragments were then linked via a (G4S)3-Linker, and the anti-CD3(SP34) VH-CH1-(G4S)3Linker-anti-AXL scFv fragment was constructed into the eukaryotic expression plasmid pCAGGS via homologous recombination. The anti-CD3(SP34) VL-CL region was inserted into the pCAGGS vector via homologous recombination. The anti-CD3(SP34) sequence is publicly available; the specific amino acid sequence is as follows:

[0109] SP34-VL-CL: QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPDHLFRGLIGGTNKRAPGTPARFSGSLLGDKAALTISGAQPEDEAEYYCALWYSNLWVFGGG TKLTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:35).

[0110] SP34-VH-CH1:EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:36).

[0111] Expression of AXL / CD3 bispecific antibody

[0112] 50 mL of 293F cells (1.5 × 10⁻⁶) were added. 6 Cells (number of cells / mL) were seeded in shake flasks and cultured overnight at 37°C, 5% CO2, 101 rpm. The next day, the 293F cell density was adjusted to 3.0 × 10⁻⁶ cells / mL. 6Cells / mL. Add 25 μg each of the constructed heavy and light chain endotoxin-free plasmids to 5 mL of Opti-MEM transfection reagent, along with 125 μL of PEI40K solution. Mix thoroughly by slow inversion and incubate at room temperature in the dark for 30 min. Add the incubated plasmids dropwise to 293F cells with adjusted cell density. Incubate at 37°C, 5% CO2, 101 rpm for 72 h. Centrifuge at 100 g for 5 min, collect the supernatant, add fresh culture medium, and continue culturing for 48 h. Centrifuge to remove cell pellet and obtain the supernatant. Centrifuge the supernatant obtained twice at 4°C, 15000 rpm for 30 min, filter through a 0.45 μm filter membrane, and purify the antigen using the same method as in Example 1. Pass the supernatant through a Protein G column to obtain AXL / CD3 bispecific antibody. Determine the concentration and verify by SDS-PAGE.

[0113] Example 10: Validation of antigen binding of AXL / CD3 bispecific antibody

[0114] AXL (ECD), Her2 (ECD)-hIgG1 Fc (an irrelevant antigen containing negative screening proteins), and BSA (negative control) were coated onto ELISA plates, 1 μg protein per well, and incubated overnight at 4 °C. The liquid in the plates was discarded, and the plates were washed three times with 250 μL of PBST at room temperature, vortexing for 5 min each time. 200 μL of PBST solution containing 2% BSA was added to each well, and the plates were blocked for 1 h at room temperature. The blocking solution was discarded, and the plates were washed as described above. The AXL / CD3 bispecific antibody was diluted with 2% BSA PBST solution to an initial concentration of 200 nM, with eight dilutions. 100 μL of the diluted antibody solution was added to each well of the ELISA plate, and the plates were incubated for 1 h at room temperature. The antibody solution was discarded, and the plates were washed. 100 μL of secondary antibody (HRP: anti-mouse) diluted 1:5000 with 2% BSA was added to each well, and the plates were incubated for 1 h at room temperature. After washing, add 100 μL of TMB chromogenic solution to each well and react at room temperature for 3–5 min. Stop the reaction by adding 100 μL of 2 M H₂SO₄ solution and immediately detect the absorbance at 450 nm. The results are shown in Table 2 and [Table data would be inserted here]. Figure 2 As shown, the binding ability of different AXL / CD3 clones to immobilized antigens was detected by ELISA. The binding ability of AXL antigen was measured by EC50. 50 The values ​​were all less than 2 nM, and there was no binding ability for unrelated antigens Her2 and BSA, indicating that the selected antibodies targeting AXL have high specificity and strong binding activity.

[0115] Table 2. Comparison of the binding ability of AXL / CD3 bispecific antibodies to AXL antigen.

[0116]

[0117] Example 11: In vitro tumor-killing activity of different AXL / CD3 bispecific antibodies

[0118] PBMCs were extracted from healthy volunteers, activated with CD3 / CD28 antibody, and then T cells were expanded with 300 IU / mL IL-2. MDA-MB-231(AXL) breast cancer cells were resuscitated and cultured in DMEM medium containing 10% fetal bovine serum. + ) and SKBR3 (AXL - Before the experiment, T cells were resuspended in RPMI-1640 medium without IL-2 and cultured overnight. The next day, the T cell density was adjusted to 2 × 10⁶ cells / year. 6 The density of breast cancer cells MDA-MB-231 and SKBR3 was adjusted to 2 × 10⁶ cells / mL. 5 T cells were mixed with MDA-MB-231 and SKBR3 cells at a 1:1 (v / v) ratio, with an E:T ratio of 10:1. 100 μL of the mixed cells were added to each well of a 96-well plate. Simultaneously, different AXL / CD3 bispecific antibodies were diluted 10-fold with cell culture medium (initial concentration 10 nM), with 10 μL added to each well. Positive controls (cell lysate with added tumor cells) and negative controls (tumor cells and T cells) were included. The plates were incubated at 37°C with 5% CO2 for 24 h. After centrifugation at 3000 rpm for 5 min, the supernatant was collected for lactate dehydrogenase (LDH) detection. OD490 readings in the wells were measured using a multi-mode microplate reader, and the data were analyzed. The results are shown in Table 3. Figure 3 As shown, by detecting the release of LDH in cells, different AXL / CD3 antibodies have a strong killing ability (IC50) against AXL-positive MDA-MB-231 cells. 50 (0.1-0.5 nM), while having very low killing ability against AXL-negative SKBR3 cells, thus avoiding the off-target toxicity of different AXL / CD3 antibodies and further broadening their application.

[0119] Table 3. Comparison of the killing ability of AXL / CD3 bispecific antibody-mediated T cells against MDA-MB-231 cells.

[0120]

[0121] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. Any deductions or modifications made by those skilled in the art without departing from the concept disclosed in the present invention are within the protection scope of the present invention.

Claims

1. An anti-human AXL antibody, comprising a heavy chain variable region and a light chain variable region, characterized in that: The CDRs for the heavy chain variable region and the light chain variable region are selected from one of the following groups: Antibody F1-G has CDR1 to CDR3 of heavy chain variable region as SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISNYLS, YTSRLQS, and QQGKTLPYT, respectively. Antibody F7-B has CDR1 to CDR3 of heavy chain variable region as SHVLH, YINPYYDDTKYSEKFKG, and YYPGAMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISTYLN, YTSRLHS, and QQGKTLPYT, respectively. Antibody 3-3F has CDR1 to CDR3 of heavy chain variable region as SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of light chain variable region as RASQDISNYLN, YSSRLHS, and QQGKTLPYT, respectively.

2. The anti-human AXL antibody according to claim 1, characterized in that, These include single-chain antibodies, chimeric antibodies, humanized antibodies, scFvs fused with Fc fragments, and homologous or heterologous multivalent antibodies.

3. The anti-human AXL antibody according to claim 1, characterized in that, The Fc fragment is selected from the Fc segment of human IgG1, IgG2, IgG3 or IgG4 or its variants or modifiers.

4. The anti-human AXL antibody according to claim 1, characterized in that, The antibody is selected from one of the following antibodies: Antibody F1-G: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24; Antibody F7-B: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:26; Antibody 3-3F: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

34.

5. The gene encoding the anti-human AXL antibody as described in any one of claims 1 to 4.

6. A recombinant expression vector or recombinant bacterial strain, characterized in that, It expresses the anti-human AXL antibody as described in any one of claims 1 to 4; or has the gene as described in claim 5 inserted.

7. The application of the anti-human AXL antibody according to any one of claims 1 to 4, wherein the application is selected from: Preparation of diagnostic reagents targeting AXL; To develop drugs that target AXL for the treatment of breast cancer.

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