Anti-human AXL antibody, gene and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an anti-human AXL antibody, a gene and application of the anti-human AXL antibody, 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 antibodies in some examples have the binding capacity EC50 value smaller than 2 nM for AXL antigens and have no binding capacity for irrelevant antigens Her2 and BSA, and it is indicated that the screened targeted AXL antibodies have high specificity and high binding activity. Different AXL / CD3 bispecific antibodies prepared on the basis of the anti-human AXL antibody have relatively strong killing ability (IC50: 0.1-0.5 nM) on AXL positive MDA-MB-231 cells, and have very low killing ability on AXL negative SKBR3 cells, so that off-target toxicity of the different AXL / CD3 antibodies is avoided, and the application of the different AXL / CD3 bispecific antibodies is further widened.

Description

Technical Field

[0001] The present invention belongs to the biological field, and specifically relates to an anti-human AXL antibody, a gene and applications thereof. Background Art

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

[0003] AXL activation modes include: ligand-dependent activation and ligand-independent activation. Among them, ligand-dependent activation is the main mode. Growth Arrest Specific Protein 6 (Gas6) is one of the ligands of TAM receptors. Compared with TYRO-3 and MER, it has the highest affinity for AXL. First, AXL and Gas6 form a high-affinity complex of Gas6 / AXL in a ratio of 1:1, which forms a homodimer with another Gas6-AXL complex. Y779, Y821 and Y866 in the intracellular kinase domain of the dimer are phosphorylated, resulting in interaction between the AXL receptor and its downstream signaling molecules. On the other hand, AXL can also be activated through a Gas6-independent mechanism (Nasimian A, et al. Int J Mol Sci. 2023; 24(4): 3830.), which is due to the fact that AXL overexpression induced by multiple factors can lead to AXL extracellular domain aggregation or ligand-independent dimerization, including: human epidermal growth factor receptor 2 (Her2), epidermal growth factor receptor (EGFR), cellular-mesenchymal epithelial transition factor (c-Met), TYRO-3, etc. For example, AXL heterodimerization with Her2 makes it independent of Gas6 activation and promotes drug resistance and tumor progression of 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. As important downstream pathways of the Gas6 / AXL axis, the PI3K / AKT / mTOR, JAK / STAT, NF-κB and RAS / RAF / MEK / ERK signaling pathways are involved in regulating the survival, proliferation, drug resistance, invasion, metastasis and angiogenesis of tumor cells (Lei T, et al. Pharmacol Res. 2023;188:106668.). The activation of AXL can induce a series of immunosuppressive signals such as 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), thereby facilitating the formation of an immunosuppressive tumor microenvironment. At the same time, the activation of this pathway will induce the production of M2 macrophages, promote the infiltration of monocytes and myeloid-derived suppressor cells (MDSC), and reduce the number of DCs, NK cells, and CD4 + and CD8+ T cells infiltrate tumors and promote tumor development (Kim SY, et al. Sci Rep. 2016;6:29673.).

[0004] Since AXL is highly expressed in a variety of tumor tissues such as lung cancer, breast cancer, prostate cancer, and pancreatic cancer, and plays an important role in tumor immune escape and malignant evolution, 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. At present, 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] AXL inhibitors can be divided into two categories according to the different binding sites with AXL. Type I inhibitors can 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 the catalytic activity of AXL and reducing the expression of AXL; while type II inhibitors can bind to parts 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 have the function of inhibiting AXL. However, the off-target effects of the use of multi-target kinase inhibitors may lead to unpredictable toxic side effects. AXL and other tyrosine kinases (such as c-Met) are highly homologous, and the development of selective AXL inhibitors is difficult. In clinical applications, off-target effects can easily lead to serious toxic side effects.

[0006] The ADC drug targeting AXL (AXL-107-MMAE) is used in combination with a MAPK inhibitor to inhibit the growth of drug-resistant melanoma, but due to potential toxicity, the future clinical prospects of AXL-107-MMAE remain to be further observed. The FDA has approved the ADC drug targeting AXL (BA3011) to enter Phase I / II clinical studies, mainly for the treatment of advanced solid tumors. Enapotamab is an ADC-type antibody drug against AXL human IgG1 and monomethyl Auristatin E. It has shown strong anti-tumor activity in preclinical models of non-small cell lung cancer, but in clinical applications, the efficacy, dose and biomarkers have not met the proof-of-concept standards for continued development, and its clinical development has been terminated (Boshuizen J, et al. Cancer Res. 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 Fc fusion protein of an AXL high-affinity mutant, and its affinity for Gas6 protein is higher than that of AXL wild-type protein. In the treatment of ovarian cancer animal models, MYD1 significantly inhibited the invasion and metastasis of tumor cells. However, since AXL has multiple activation modes that are independent of Cas6, the effectiveness of Fc fusion proteins in cancer patients needs further research and verification.

[0008] The research on antibodies targeting AXL is still in the preclinical stage. The mouse hybridoma-derived 3G9, 8B5, and 12A11 antibodies targeting the extracellular domain of human AXL all attenuated the growth of A549 tumor xenografts by inducing downregulation of AXL receptor expression. However, since these monoclonal antibodies have no cross-reactivity with mouse AXL, it is impossible to evaluate the effects of AXL on tumor angiogenesis and tumor stroma in mice (Li Y, et al. Oncogene. 2009;28(39):3442-3455). To solve the above problems, researchers developed a phage-derived monoclonal antibody (YW327.6S2) that can recognize both human and mouse AXL. This antibody can not only downregulate the expression of AXL, but also affect the tumor stroma by regulating the function of tumor-related vascular system and immune cells, thereby inhibiting the growth of tumor cells. Unfortunately, YW327.6S2 monotherapy did not show satisfactory efficacy in preclinical in vivo studies and has not been studied as a clinical candidate (YeX, et al. Oncogene. 2010;29(38):5254-5264.). Other antibodies include 20G7-D9, which provides a promising therapeutic strategy for triple-negative breast cancer with mesenchymal characteristics by inhibiting AXL-dependent epithelial-mesenchymal transition, tumor growth and metastasis (Leconet W, et al. Clin Cancer Res. 2017;23(11):2806-2816.); MAb173 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 different antigen-binding sites. They have become a hot topic for AXL-targeted therapy due to their novel mechanism of action and therapeutic applications. Bis1 is a bispecific antibody that binds AXL and EGFR. When used in combination with the EGFR inhibitor osimertinib, Bis1 significantly delayed the onset of resistance to osimertinib in vivo and sustained inhibition of lung cancer tumor growth (Simoni-Nieves A, et al. Cell Rep Med. 2024;5(9):101703.). However, the efficacy of these structures in preclinical models is relatively low, and inducing effective T cell-mediated lysis of cancer cells usually requires higher drug concentrations, higher effector-target ratios, and additional co-stimulatory signals. Bispecific T cell engagers (BiTEs) are composed of two different single-chain variable fragments (scFvs) derived from anti-CD3 and anti-TAA antibodies, covalently linked by a small linker peptide. In preclinical models, BiTEs have the characteristics of small size, high flexibility, and high affinity between effector cells and target cells. They have strong anti-tumor activity and are superior to traditional mAbs and other forms of bispecific antibodies (GoebelerME, et al. Nat Rev Clin Oncol. 2020;17(7):418-434.). TM The platform constructed pAXL×CD3ε, an effective BiTEs against ovarian cancer, whose low molecular weight facilitates tumor infiltration, ensures the targeting of cancer cells, and recruits and activates T lymphocytes. Due to its small structure, it can be quickly and economically produced in Escherichia coli and yeast systems, providing a theoretical basis for its development as a new drug in the treatment of ovarian cancer (Riillo C, et al. J Transl Med. 2023;21(1):301.). In summary, due to the wide range of biological defects and functional diversity of AXL, the development of BiTEs targeting AXL has attracted widespread attention. With the continuous improvement of AXL-targeted drugs and the in-depth understanding of the potential mechanisms of AXL, inhibiting AXL is expected to provide an effective strategy for the treatment of cancer patients. Summary of the invention

[0010] Although the development of antibodies targeting AXL has shown certain efficacy, drugs with better therapeutic effects and lower toxic side effects are still needed in clinical applications. Based on phage display technology and mouse immunization strategy, the present invention newly discovered multiple single-chain antibodies targeting AXL, and constructed a new AXL×CD3 bispecific antibody with anti-CD3ε antibody SP34, which has strong killing effect on tumor cells expressing AXL and enriches T cells, promoting their infiltration into tumor cells, providing more effective and safer treatment methods for clinical applications.

[0011] The object of the present invention is to overcome at least one deficiency of the prior art and to provide an anti-human AXL antibody, a gene and applications thereof.

[0012] The technical solution adopted by the present invention is: The 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 the following group: Antibody F1-G, CDR1 to CDR3 of the heavy chain variable region are SYVLH, YINPYFDDTKYNEKFKG, YYPGSMDY, and CDR1 to CDR3 of the light chain variable region are RASQDISNYLS, YTSRLQS, QQGKTLPYT, respectively; Antibody F7-B, CDR1 to CDR3 of the heavy chain variable region are SHVLH, YINPYYDDTKYSEKFKG, YYPGAMDY, and CDR1 to CDR3 of the light chain variable region are RASQDISTYLN, YTSRLHS, QQGKTLPYT, respectively; Antibody F7-G, CDR1 to CDR3 of the heavy chain variable region are GYTMH, YIDPFSGDTSYNLKFKG, ELTTVVAYWYFDV, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPFT; Antibody 3-1E, CDR1 to CDR3 of the heavy chain variable region are GYTMH, YIDPFSGDTSYNLKFKG, ELTTVVAYWYFDV, respectively, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPYT, respectively; Antibody 3-3C, CDR1 to CDR3 of the heavy chain variable region are GYSMH, YIDPFSGAINYNLKFKG, ELTTVVAYWYFDV, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPYT, respectively; Antibody 3-3F, CDR1 to CDR3 of the heavy chain variable region are SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of the light chain variable region are RASQDISNYLN, YSSRLHS, and QQGKTLPYT, respectively.

[0013] In some examples of anti-human AXL antibodies, they are single-chain antibodies, chimeric antibodies, humanized antibodies, scFv fused to Fc fragments, homologous or heterologous bivalent or multivalent antibodies.

[0014] 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 variants and modifications thereof.

[0015] In some examples of anti-human AXL antibodies, different functional fragments in homologous or heterologous bivalent or multivalent antibodies are linked by flexible peptides.

[0016] In some examples of anti-human AXL antibodies, heterologous bivalent antibodies include but are not limited to bispecific antibodies that combine CD3 and AXL domains in different combinations based on Triomabs bispecific antibodies, Crossmab bispecific antibodies, DVD-Ig bispecific antibodies, BiTE bispecific antibodies, DART bispecific antibodies, TandAbs bispecific antibodies, etc.

[0017] In some examples of anti-human AXL antibodies, 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 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; 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; 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; 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.

[0018] The above features can be combined arbitrarily as long as they do not conflict with each other.

[0019] The second aspect of the present invention provides: a gene encoding the anti-human AXL antibody according to the first aspect of the present invention.

[0020] The third aspect of the present invention provides: a recombinant expression vector or a recombinant strain, which expresses the anti-human AXL antibody described in the first aspect of the present invention; or has inserted therein the gene described in the second aspect of the present invention.

[0021] The fourth aspect of the present invention provides: the use of the anti-human AXL antibody according to the first aspect of the present invention, wherein the use is selected from: preparing an antibody conjugate targeting human AXL; preparing diagnostic reagents targeting AXL; preparing a drug delivery vector targeting AXL; preparing drugs targeting AXL; An inducer is prepared, wherein the inducing effector cells kill tumor cells expressing AXL.

[0022] In some application examples, the conjugate in the antibody conjugate is a cytotoxic drug.

[0023] In some application examples, the antibody conjugate is used to kill tumor cells expressing AXL.

[0024] In some application examples, the tumor is selected from lung cancer, breast cancer, prostate cancer, and pancreatic cancer that highly express AXL.

[0025] The beneficial effects of the present invention are: The anti-human AXL antibodies of some examples of the present invention have good affinity and high specificity for AXL and can stably bind to AXL.

[0026] Different AXL / CD3 bispecific antibodies prepared based on the anti-human AXL antibody of the present invention have a binding capacity for AXL antigen EC 50 The values ​​were all less than 2 nM, and there was no binding ability to irrelevant antigens Her2 and BSA, indicating that the screened antibodies targeting AXL have high specificity and strong binding activity.

[0027] Different AXL / CD3 bispecific antibodies prepared based on the anti-human AXL antibody of the present invention have strong killing ability against AXL-positive MDA-MB-231 cells (IC 50: 0.1-0.5 nM), while the killing ability for AXL-negative SKBR3 cells is very low, avoiding the off-target toxicity of different AXL / CD3 antibodies and further broadening their application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the result of the binding ability of different monoclonal phages to antigens.

[0029] Figure 2 These are the experimental results of the ability of different AXL / CD3 bispecific antibodies to bind to the AXL antigen.

[0030] Figure 3 These are the experimental results of the ability of different AXL / CD3 bispecific antibodies to mediate T cell killing of MDA-MB-231 cells. DETAILED DESCRIPTION

[0031] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0032] In the present invention, the term "chimeric antigen receptor" (CAR) is the 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 to be one of the most promising means of conquering tumors. CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0033] In the present invention, the term "antibody" has the conventional meaning in the art and is used in the present invention in the broadest sense. In the field of biological sciences, by analyzing the amino acid sequences of different antibody heavy chains and light chains, it is found that the amino acid sequences of the heavy chains and light chains near the N-terminus vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region near the N-terminus of the antibody light chain and heavy chain where the amino acid sequence varies greatly is called the variable region (variable region, V), and the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (constant region, C). The V region of the heavy chain and light chain is referred to as VH and VL, respectively, and the C region of the heavy chain and light chain is referred to as CH and CL, respectively. There is a small part of the amino acid residues in the variable region of the antibody that vary particularly strongly, and the residue composition and arrangement order of these amino acids are more prone to variation, which is called the hypervariable region (HVR); there are three hypervariable regions in the V region of the L chain and the H chain, and this part can form precise complementarity with the antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementarity determining region (CDR). In antibodies, common CDR division rules include Kabat, AbM, Chothia, Contact, and IMGT, which are well known to those skilled in the art. When a website that implements these rules is used, as long as the VH and VL sequences are input and the corresponding rules are selected, CDR sequences according to different rules can be obtained. It should be understood by those skilled in the art that the scope of protection of the present invention covers the combination of CDR sequences obtained by analyzing using different rules. The six CDR regions of an antibody jointly determine the recognition ability and specificity of the antibody to the corresponding antigen. It should be understood by those skilled in the art that when the present invention defines the amino acid sequences of the six CDR regions, the recognition ability and specificity of the antibody to the corresponding antigen are predictable.

[0034] In the present invention, the term "antigen binding site" has the conventional meaning in the art, and refers to the key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0035] In the present invention, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which connects any structure / region of the antibody or chimeric antigen receptor of the present invention together. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other in space, a longer linker can be used.

[0036] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce or transfect a host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication start site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances.

[0037] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention is further explained below in conjunction with experiments.

[0038] Example 1: Preparation of AXL (ECD) antigen 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, and the target plasmid was obtained by homologous recombination.

[0039] Expression of recombinant AXL(ECD)-hIgG1 Fc protein Plasmid acquisition: E. coli carrying the target plasmid was inoculated into 50 mL LB / ampicillin (Amp, 50 μg / mL) medium and cultured overnight at 37°C in a shaking incubator to amplify the plasmid. High-concentration endotoxin-free plasmid was obtained using the OMEGA Endo-Free Plasmid Midi Kit D6915 kit, and after quality inspection by agarose gel electrophoresis, it was stored at -20°C for future use.

[0040] 293 F cell transfection: 50 mL (1.5×10 65 mL Opti-MEM + 50 μg target plasmid was mixed with 125 μL PEI40K and then incubated at room temperature in the dark for 30 min. The density of 293F cells cultured overnight was adjusted to 3.0×10 6 / mL, draw the incubated plasmid, and shake the shake flask while adding dropwise. Be careful because the medium does not contain antibiotics. After the addition is completed, culture on a shaker for 72 hours, centrifuge at 100 g for 5 minutes, collect the cell supernatant, add 50mL of culture medium to resuspend the cells, continue to culture for 48 hours, centrifuge at 100 g for 5 minutes, discard the cell pellet, and retain the cell supernatant.

[0041] Purification of AXL (ECD) protein Centrifuge 100 mL of cell supernatant at 15000 rpm and 4℃ for 30min, collect the supernatant, filter it through a 0.45 µm filter, and place it on ice for later use. Take 3 mL of Protein G in the chromatography column, replace its solution with Binding buffer, and press the resin surface with a gasket. Use a peristaltic pump to control the flow rate to 0.5 mL / min, so that the cell supernatant passes through the ProteinG column at a uniform speed. Use Binding buffer to wash away proteins that are not bound to Protein G. During elution, add neutralization buffer to the EP tube, add Elution buffer to the column, collect one tube for every 1 mL, and measure the concentration with nanodrop until the protein concentration cannot be measured. According to the Thrombin instruction manual of Solarbio, dialyze the AXL (ECD) -hIgG1 Fc protein and replace it with a buffer solution under enzyme cleavage conditions. Take the dialyzed protein solution, add appropriate Thrombin, and rotate at room temperature for 16h. SDS-PAGE is used to detect the enzyme cleavage effect. The protein sample after enzyme digestion was slowly and evenly passed through the Protein G column to remove the IgG-Fc tag, and eluted according to the above steps. The protein was detected by SDS-PAGE, and the purity of AXL (ECD) protein was greater than 90%.

[0042] Example 2: Immunization of mice using AXL (ECD) antigen AXL (ECD) antigen and adjuvant were mixed to form an emulsion, which was injected subcutaneously at multiple points in each mouse at a dose of 100 μg / time. Immunization was performed once every two weeks for a total of 4 times. The first and second times were complete Freund's adjuvants, and the third and fourth times were incomplete Freund's adjuvants. Blood was collected from the orbital venous plexus of the mice one week after each immunization, and the serum titer was determined (ELISA determination). The spleen could be removed if the titer was higher than 1: 10000.

[0043] Example 3: Extraction and separation of mouse B cells The mice immunized with AXL (ECD) antigen were taken, and their serum titers were measured. They were then killed and disinfected, and the spleens were removed by laparotomy in a sterile operating table. Splenocytes were obtained by grinding and centrifugation. Mouse B cells were sorted according to the instructions of MojoSort™ Mouse Pan B Cell Isolation Kit II. The purity of mouse B cells was 95% by flow cytometry.

[0044] Example 4: Extraction of total RNA from B cells of immunized mice and synthesis of cDNA According to the instructions of TIANGEN RNAprep Pure Cell / Bacteria Kit, total RNA of B cells of immunized mice was extracted, the concentration was determined and verified by agarose gel electrophoresis. ® Ⅱ OneStep RT-PCR Kit Instructions and related primers were used to synthesize the anti-AXL scFv VH and VL regions. The VH and VL fragments were assembled into scFv (VH-VL) by overlap PCR. The product was detected by agarose gel electrophoresis.

[0045] Example 5: Construction and diversity evaluation of Anti-AXL scFv phage library Preparation of pCGMT3 phagemid vector / scFv restriction fragment

[0046] The mixture was placed in a 1.5 mL EP tube and incubated in a water bath at 50°C for 4 h. The product was recovered by agarose gel electrophoresis.

[0047] Ligation of pCGMT3 phagemid vector and scFv The pCGMT3 phagemid vector and the scFv fragment after restriction digestion were ligated at a molar ratio of 3:1 using T4 DNA ligase (NEB) according to the following reaction system. The reaction conditions were 16°C overnight.

[0048]

[0049] The obtained ligation product was purified using OMEGA Cycle-pure kit and the DNA was stored at -20°C.

[0050] Add the DNA product to 100 μL of freshly prepared XL1-Blue electroporation competent cells, avoid blowing, and transfer to a pre-cooled electroporation cup after ice bathing for 0.5 h. The voltage is set to 2.5 KV. After the electroporation, immediately rinse the electroporation cup with 2YT medium preheated at 37°C, mix all the washed bacterial solution into a centrifuge tube, and recover for 1.5 h at 37°C and 200 rpm. Take 10 μL of the recovered XL1-Blue cells for gradient dilution and spread on a 2YT solid culture dish containing ampicillin (Amp) and tetracycline (Tet), and culture at 37°C overnight to determine the storage capacity. Spread all the remaining recovered cells on a 2YT solid culture dish and incubate at 37°C overnight. The next day, use 2YT: 50% glycerol = 1: 1 (v / v) solution, use a coating rod to gently scrape the colonies on the solid culture dish and collect them in a 15 mL centrifuge tube, and use a spectrophotometer to measure the OD600 value of the bacterial solution. All DNA products were electroporated into XL1-Blue cells according to the above method. At this time, the total library capacity was >10 8 , this is the anti-AXL scFv phage library.

[0051] Evaluation of phage library capacity and diversity Take 20 μL of anti-AXL scFv bacterial library in 80 μL 2YT solution, 37°C, 200 rpm, and resuscitate for 1.5 h. Evenly spread it on a freshly prepared 2YT plate containing Amp and Tet, and incubate it at 37°C overnight. On the second day, count the single colonies on the plate, and randomly pick 24 single colonies from the plate for single colony PCR to identify whether they are positive colonies, and then perform sequencing verification.

[0052] Amplification of phage libraries Take 1 mL of anti-AXL scFv bacterial library in 200 mL SB medium (200 μg / mL Amp, 5 μg / mL Tet, 2% glucose) and culture at 37°C and 220 rpm. During this period, the OD600 value was detected several times. The OD600 value of the bacterial solution was shaken to 0.5 in about 2-3 hours. According to the MOI (number of phages: number of bacteria) = 20: 1, VCSM13 helper phage was added, and the culture was incubated in a 37° incubator for 30 min, and then cultured at 220 rpm for 1 hour. The bacterial solution was centrifuged at 3900 rpm for 15 min at room temperature, and the supernatant was discarded to remove glucose and uninfected helper phage. The bacteria were resuspended in 200 mL SB medium (200 μg / mL Amp, 5 μg / mL Tet, 15 μg / mL kanamycin) and shaken at 200 rpm at 30°C overnight to amplify the phage. Take the overnight shaken bacterial solution, centrifuge at 3900rpm, 4°C for 15 min, and collect the supernatant. Add 50 mL of PEG8000 / 2.5MNaCl (5×) solution and the supernatant and mix gently, place on ice for 4 h to precipitate the phage. Centrifuge at 3900rpm, 4°C for 30 min, discard the supernatant, and invert the centrifuge tube on a clean paper to dry. Add 80 mL of PBS to resuspend the phage, add 20 mL of PEG8000 / 2.5M NaCl (5×) solution again, mix gently, and place on ice to precipitate the phage for 2 h. Centrifuge at 3900 rpm, 4°C for 30 min, discard the supernatant, and invert the centrifuge tube on a clean paper to dry. Add 2 mL of PBS: 50% glycerol = 1: 1 (v / v) solution, divide and store at -20℃, this is the first round of phage library input.

[0053] Example 6: Screening of phage library based on immunotube-immobilized antigen First round of screening Take out the screening antigen from the -80℃ freezer and place on ice to thaw; coat the AXL (ECD) antigen (positive screening protein) and hIgG1 Fc protein (negative screening protein) in the immunotube (50 µg / tube, coating solution is CBS, pH9.6, 2 mL / tube), and rotate slowly at 4°C overnight. Discard the liquid in the overnight coated immunotube, add 3 mL of PBS solution to seal and rotate at room temperature for 5 min, repeat 3 times to wash the protein that is not bound to the immunotube; add 2 mL of 3% BSA-PBST solution, rotate and block at room temperature for 2 h; discard the blocking solution, add 3 mL of 0.05% PBST (1×PBS plus 0.05% Tween20, the same below) buffer, rotate at room temperature for 5 min, and wash the immunotube 3 times; discard the washing solution in the negative screening protein immunotube, add 1 mL PBS (3% BSA) and 1 mL of the first round input phage, incubate at room temperature for 1 h, and then transfer to the positive screening protein immunotube and rotate and incubate at room temperature for 1 h. Discard the liquid in the positive screening protein immunotube, add 3 mL of 0.05% PBST buffer, seal and rotate at room temperature for 5 min / time, and wash the immunotube 10 times in total; discard the liquid in the immunotube, add 1 mL of Trypsin (0.25 mg / mL) solution, rotate at room temperature for 30 minutes, add 10 μL of 10% AEBSF to terminate elution, and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube, which is the output of the first round of phage screening eluate.

[0054] First round of phage eluate titer testing After the XL1-Blue strain stored in the low-temperature refrigerator was revived, a single colony was streaked on a plate containing the corresponding antibiotic (Tet), cultured overnight at 37°C, and a single colony was picked from the plate for amplification. The bacteria were cultured at 37°C and 240 rpm until OD600 = 0.5; Take 10 μL of the first round of phage elution and dilute it 10 times with SB medium in a 96-well U-shaped plate. That is, take 10 μL of the phage library and add it to 90 μL of medium. After blowing, take 10 μL from it and add it to 90 μL of new medium. Repeat this process for a total of 10 dilutions to 10. -10 ; Take 10 μL from each dilution well of the gradient and add it to 90 μL XL1-Blue bacterial solution with OD600=0.5, mix well and incubate at 37℃ for 30min; then take 10 μL from each gradient of XL1-Blue bacterial solution and add it dropwise to SB solid medium (Amp, Tet), and invert and culture overnight at 37℃. The next day, record the number of single colonies and calculate the titer of the phage library. At the same time, determine the first round of input titer according to the above method and compare it with the first round of output titer.

[0055] Amplification of the first round of phage eluates Take 800 μL of the first round of phage eluate and transfer it to 10 mL XL1-Blue (5 μg / mL Tet) bacterial solution with OD600=0.5, incubate at 37°C for 30 min, and continue to culture at 220 rpm for 30 min for infection; evenly spread all the bacterial solution on a 15 cm medium plate (200 μg / mL Amp and 5 μg / mL Tet), and culture at 37°C overnight; add 2YT: 50% glycerol = 1: 1 (v / v) solution to the overnight cultured plate, gently scrape the colonies on the plate into a 15 mL centrifuge tube with a coating rod, and use a spectrophotometer to measure the OD600 value of the bacterial solution, which is the first round of bacterial library. According to the method of phage library amplification in Example 5, the first round of bacterial library was amplified and phage was purified to obtain the second round of phage library input.

[0056] The second and subsequent rounds of screening were similar to the first round of screening. As the number of screening rounds increased, the screening conditions became more stringent, and ultimately phage clones with high affinity for the AXL (ECD) antigen were obtained.

[0057] Example 7: Monoclonal ELISA detection Take 10 μL of each round of elution output, transfect into XL1-Blue strain with OD600=0.5, incubate at 37℃, 200rpm for 1 h, spread on solid culture medium containing corresponding resistance, and culture at 37℃ overnight. Randomly pick single clone colonies from the culture medium plate after overnight culture and place them in a sterile 96-well cell culture plate. Add 180 μL SB culture medium (200μg / mL Amp and 5μg / mL Tet) to each well. Incubate the culture medium at 37℃, 240 rpm, shake for 3-5 h to make the bacterial solution in each well OD600=0.5. Take 100 μL from each well to a new 96-well plate. Add glycerol to the remaining bacterial solution and store it at -20℃. Add VCSM13 helper phage with MOI=20 to the new 96-well plate, incubate at 37℃ for 30 min, add Kan with a final concentration of 50 μg / mL, and culture overnight at 30℃, 200 rpm. Centrifuge the 96-well culture plate after overnight culture at 4℃, 3900 rpm for 10 min, and store the supernatant at 4℃ for later use. 100 μL / well of 10 μg / mL antigen was coated on the ELISA plate, and Her2(ECD)-hIgG1 Fc and BSA were coated in parallel as negative controls at 4°C overnight. The overnight coating liquid was discarded, and 250 μL / well PBS buffer was added. The ELISA plate was shaken at room temperature for 5 min / time and washed 3 times. 200 μL of blocking solution (3% BSA) was added to each well and blocked for 2 h at room temperature. The blocking solution was discarded, and 250 μL / well PBST buffer was added. The plate was washed 3 times at room temperature for 5 min each time. The diluted supernatant (120 μL 3% BSA was added to 80 μL of the supernatant after centrifugation) was added to each well and incubated at room temperature for 2 h. The ELISA plate was washed in the same way as above. 100 μL of Anti-M13 Antibody, Mouse Monoclonal diluted 1:8000 with blocking solution was added to each well and incubated at room temperature for 1 h. After washing, 100 μL of After incubation in dark, add 100 μL 2M H2SO4 to each well to terminate the reaction. Read the OD450 value with a microplate reader, record and save. To ensure the accuracy of the experimental results, the clones initially identified as positive were subjected to secondary ELISA verification using the same method as above. Figure 1 As shown, the ability of different monoclonal phages to bind to antigens was detected by ELISA. The AXL antigen had a higher absorbance value, while the absorbance values ​​for other antigens Her2 and BSA were lower, proving that positive phage clones targeting the AXL antigen were screened.

[0058] Example 8: Sequencing and sequence analysis of positive clones Based on the results of two ELISA tests, positive monoclones were selected, and the positive clones were cultured at 37°C and 240 rpm. Part of the culture was sequenced and the remaining culture was stored. The sequenced sequences were compared using software, and the antibody sequences were translated into amino acids. The sequencing results of the screened antibodies are as follows: 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 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; 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; 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; 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.

[0059] The CDR sequences of each antibody determined based on the Kabat numbering scheme are shown in Table 1.

[0060] Table 1. CDR analysis results of different AXL antibodies

[0061] Example 9: Construction of AXL / CD3 bispecific antibody Construction of AXL / CD3 bispecific antibody plasmid The anti-AXL scFv fragment obtained by sequencing was constructed at the C-terminus of the CD3(SP34) VH-CH1 region by the PCR method, and the two were connected by (G4S)3Linker. The anti-CD3(SP34) VH-CH1-(G4S)3Linker-anti-AXL scFv fragment was constructed into the eukaryotic expression plasmid pCAGGS vector by homologous recombination. The anti-CD3(SP34) VL-CL region was inserted into the pCAGGS vector by homologous recombination. The anti-CD3(SP34) sequence is public, and the specific amino acid sequence is as follows: SP34-VL-CL: QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPDHLFRGLIGGTNKRAPGTPARFSGSLLGDKAALTISGAQPEDEAEYYCALWYSNLWVFGGG TKLTVLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:35).

[0062] SP34-VH-CH1:EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:36).

[0063] Expression of AXL / CD3 bispecific antibody 50 mL of 293 F cells (1.5 × 10 6 The cells were inoculated into a cell shaker at 37°C, 5% CO2, 101 rpm, and cultured overnight. The next day, the 293 F cell density was adjusted to 3.0×10 6 / mL. Add 25 μg of the heavy chain and light chain endotoxin-free plasmids constructed above to 5 mL Opti-MEM transfection reagent, add 125 μL PEI40K solution at the same time, slowly invert up and down to mix, and incubate at room temperature in the dark for 30 min. Pipette the incubated plasmid and add it drop by drop to the 293F cells with adjusted cell density. After culturing for 72 h at 37°C, 5% CO2, 101 rpm, centrifuge at 100 g for 5 min, collect the supernatant, add new culture medium to re-cell, continue to culture for 48 h, and centrifuge to remove the cell precipitate to obtain the supernatant. The supernatant obtained twice was centrifuged at 4°C, 15000 rpm for 30 min, filtered through a 0.45 μm filter membrane, and passed through a ProteinG column as in the antigen purification method in Example 1 to obtain the AXL / CD3 bispecific antibody, and the concentration was determined and verified by SDS-PAGE.

[0064] Example 10: Antigen Binding Validation of AXL / CD3 Bispecific Antibodies AXL (ECD), Her2 (ECD)-hIgG1 Fc (irrelevant antigen containing negative sieve protein), and BSA (negative control) were coated onto ELISA plates, 1 μg protein per well, overnight at 4 °C. The liquid in the wells was discarded, and 250 μL PBST was added to the plates and washed 3 times at room temperature, 5 min each time. 200 μL PBST solution containing 2% BSA was added to each well and blocked for 1 h at room temperature. The blocking solution was discarded and washed as described above. AXL / CD3 bispecific antibody was diluted with 2% BSA PBST solution, with a starting concentration of 200 nM and 8 dilutions. 100 μL of the diluted bispecific antibody solution was added to the ELISA plate per well and incubated at room temperature for 1 h. The bispecific antibody solution was discarded and washed. 100 μL of secondary antibody (HRP: anti-mouse) diluted 1:5000 with 2% BSA was added to each well and incubated at room temperature for 1 h. After washing, 100 μL of TMB colorimetric solution was added to each well and reacted at room temperature for 3-5 min. 100 μL of 2 M H2SO4 solution was added to terminate the reaction and the absorbance was measured at 450 nm. The results are shown in Table 2 and Figure 2 As shown, the binding ability of different AXL / CD3 clones to immobilized antigen was detected by ELISA. 50 The values ​​were all less than 2 nM, and there was no binding ability to irrelevant antigens Her2 and BSA, indicating that the screened antibodies targeting AXL have high specificity and strong binding activity.

[0065] Table 2. Comparison of the binding ability of AXL / CD3 bispecific antibodies to AXL antigen

[0066] Example 11: In vitro tumor killing activity of different AXL / CD3 bispecific antibodies PBMCs were extracted from healthy volunteers and activated with CD3 / CD28 antibodies, followed by T cell expansion with 300 IU / mL IL-2. MDA-MB-231 (AXL) breast cancer cells were revived 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 cells were adjusted to a cell density of 2×10 6 The cell density of breast cancer cells MDA-MB-231 and SKBR3 was adjusted to 2×10 5 / mL. T cells were mixed with MDA-MB-231 and SKBR3 cells at a ratio of 1:1 (v / v), at which time E:T=10:1, and 100 μL of the mixed cells were added to each well of a 96-well plate. At the same time, different AXL / CD3 bispecific antibodies were diluted 10 times with cell culture medium (initial concentration was 10 nM), 10 μL per well. The experiment set up a positive control (cell lysate added to tumor cells) and a negative control (tumor cells and T cells), placed in a cell culture incubator at 37°C, 5% CO2, cultured for 24 hours, and centrifuged at 3000 rpm for 5 minutes. The supernatant was taken for lactate dehydrogenase (LDH) detection, and the OD490 reading in the well was detected by a multifunctional microplate reader, and data analysis was performed. The results are shown in Tables 3 and Figure 3 As shown in Figure 2, by detecting the release of LDH in cells, different AXL / CD3 antibodies have a strong killing ability against AXL-positive MDA-MB-231 cells (IC 50 : 0.1-0.5 nM), while the killing ability for AXL-negative SKBR3 cells is very low, avoiding the off-target toxicity of different AXL / CD3 antibodies and further broadening their application.

[0067] Table 3. Comparison of the killing ability of AXL / CD3 bispecific antibody-mediated T cells against MDA-MB-231 cells

[0068] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. Any deduction or change made by any person familiar with the technology without departing from the concept disclosed by the present invention is 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 of the heavy chain variable region and the light chain variable region are selected from the following group: Antibody F1-G, CDR1 to CDR3 of the heavy chain variable region are SYVLH, YINPYFDDTKYNEKFKG, YYPGSMDY, and CDR1 to CDR3 of the light chain variable region are RASQDISNYLS, YTSRLQS, QQGKTLPYT, respectively; Antibody F7-B, CDR1 to CDR3 of the heavy chain variable region are SHVLH, YINPYYDDTKYSEKFKG, YYPGAMDY, and CDR1 to CDR3 of the light chain variable region are RASQDISTYLN, YTSRLHS, QQGKTLPYT, respectively; Antibody F7-G, CDR1 to CDR3 of the heavy chain variable region are GYTMH, YIDPFSGDTSYNLKFKG, ELTTVVAYWYFDV, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPFT; Antibody 3-1E, CDR1 to CDR3 of the heavy chain variable region are GYTMH, YIDPFSGDTSYNLKFKG, ELTTVVAYWYFDV, respectively, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPYT, respectively; Antibody 3-3C, CDR1 to CDR3 of the heavy chain variable region are GYSMH, YIDPFSGAINYNLKFKG, ELTTVVAYWYFDV, and CDR1 to CDR3 of the light chain variable region are RSSQSIVHSNGNTYLE, KVSNRFS, FQGSHVPYT, respectively; Antibody 3-3F, CDR1 to CDR3 of the heavy chain variable region are SYVLH, YINPYFDDTKYNEKFKG, and YYPGSMDY, respectively, and CDR1 to CDR3 of the light chain variable region are RASQDISNYLN, YSSRLHS, and QQGKTLPYT, respectively.

2. The anti-human AXL antibody according to claim 1, characterized in that It is a single-chain antibody, a chimeric antibody, a humanized antibody, a scFv fused to an Fc fragment, a homologous or heterologous bivalent or multivalent antibody.

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 and modifications.

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 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; 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; 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; 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. A gene encoding the anti-human AXL antibody according to any one of claims 1 to 4.

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

7. The use of the anti-human AXL antibody according to any one of claims 1 to 4, wherein the use is selected from: preparing an antibody conjugate targeting human AXL; preparing diagnostic reagents targeting AXL; preparing a drug delivery vector targeting AXL; preparing drugs targeting AXL; An inducer is prepared, wherein the inducing effector cells kill tumor cells expressing AXL.

8. The use according to claim 7, characterized in that: The conjugate in the antibody conjugate is a cytotoxic drug.

9. The use according to claim 7, characterized in that: The antibody conjugate is used to kill tumor cells expressing AXL.

10. The use according to claim 9, characterized in that: The tumor is selected from lung cancer, breast cancer, prostate cancer, and pancreatic cancer that highly express AXL.

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