Anti-human MerTK antibody and application thereof

Abstract

The invention discloses an anti-human MerTK antibody and an application of the anti-human MerTK antibody. The anti-human MerTK antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The invention also discloses nucleic acid for coding the antibody, a recombinant expression vector, a transformant, a preparation method of the antibody, a chimeric antigen receptor, an antibody drug conjugate, a pharmaceutical composition or a kit containing the antibody, and applications of the nucleic acid, the recombinant expression vector, the transformant, the chimeric antigen receptor, the antibody drug conjugate, the pharmaceutical composition or the kit in preparation of drugs for preventing and / or treating tumors. The antibody disclosed by the invention is high in affinity, can specifically inhibit the interaction of the ligand Gas6-MerTK and the ligand protein S-MerTK at the same time, and does not inhibit the interaction of the ligand Tulp1-MerTK. And potential toxicity to the retina can be avoided when inhibition activity is exerted in vivo. In addition, the anti-human MerTK antibody provided by the invention can specifically and effectively inhibit phosphorylation of AKT in a Gas6-MerTK signal, and shows certain tumor inhibition activity in a mouse in-vivo model test.

Description

[0001] This application claims priority to Chinese patent application No. 2024103455407, filed on March 25, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of antibody drugs, and specifically relates to an anti-human MerTK antibody and an application thereof. Background Art

[0003] The occurrence and development of tumors depend on multiple mechanisms, one of which is immune escape to avoid recognition and elimination by the immune system.

[0004] MerTK (myeloid-epithelial-reproductive tyrosine kinase) is a phagocytic receptor expressed on macrophages. It belongs to the TAM receptor tyrosine kinase family (MerTK, Axl, Tyro3) and consists of two IgC2-like globulin domains, two type III fibronectin domains, and a tyrosine kinase domain. During apoptosis, phosphatidylserine (PtdSer) is exposed on the cell surface. MerTK, through the help of bridge molecules such as Gas6 or Protein S, recognizes PtdSer, mediating the rapid phagocytosis and clearance of apoptotic cells by macrophages, a process known as macrophage efferocytosis. Timely and efficient clearance of apoptotic cells prevents the release of damage-associated molecular patterns (DAMPs) into the tissue microenvironment, ensuring so-called "immune silencing," which is crucial for maintaining normal tissue homeostasis.

[0005] MerTK is overexpressed in a variety of liquid tumors (B / T lymphocytic leukemias) and solid cancers (gastric cancer, NSCLC, colon cancer, prostate cancer, melanoma, and glioblastoma). It is not expressed in normal mouse and human T and B lymphocytes or normal bone marrow cells. On the one hand, MerTK expression in various tumor cells promotes tumor survival, proliferation, and migration through the PI3K / AKT and Raf / MEK signaling pathways and by regulating anti-apoptotic proteins such as Bcl-X1 and Bcl-2. Therefore, blocking MerTK signaling has potential therapeutic benefits for tumors. On the other hand, tumors often undergo cell death (i.e., apoptosis, necrosis, and pyroptosis) during their development and progression, and this is further increased during cytotoxic therapy. Similarly, tumors utilize the complete efferocytosis system of tumor-associated macrophages (TAMs) to eliminate apoptotic tumor cells, influencing the immune phenotype within the tumor microenvironment (TME). This leads to M2-like macrophage polarization, production of cytokines (such as IL-10, IL-13, and TGF-β), recruitment of FOXP3+ regulatory T cells, and the creation of a tumor-tolerant environment that prevents tumor recognition by the immune system, thereby weakening the immune response against the tumor, promoting tumor growth, and tolerance to immune checkpoint blockade (Kumar et al., 2017). When efferocytosis is impaired, secondary necrosis continues, leading to the release of proinflammatory damage-associated molecular patterns (DAMPs). Proinflammatory DAMPs drive the polarization of M1-like macrophages, the production of proinflammatory cytokines (such as TNF, IFN, and IL-12), and the recruitment of cytotoxic cells (such as CD8+ T cells and natural killer cells), thereby killing tumors. Currently, antibodies that block MerTK signaling for tumor treatment have been developed. For example, patent applications WO2020076799A1, WO2016106221A1, WO2020214995A1, and WO202363026A1 all report an anti-MerTK antibody. However, despite the clear mechanism and research direction, due to many uncontrollable factors in the antibody drug development process, anti-human MerTK antibodies still need further research as clinical drugs. Summary of the Invention

[0006] This invention provides a series of novel anti-MerTK antibodies with specific binding and high affinity. These antibodies can specifically inhibit both the Gas6-MerTK and Protein S-MerTK interactions, while not inhibiting the Tulp1-MerTK interaction. Theoretically, when these antibodies exert their inhibitory activity in vivo, they should avoid potential retinal toxicity.

[0007] Specifically, one of the technical solutions provided by the present invention is: an anti-human MerTK antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 14, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 15, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 16; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 17, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 18, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 19;

[0008] Or, the heavy chain variable region includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 30, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 60; the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 33 or SEQ ID NO: 61, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35. Specifically, according to some embodiments of the present invention, the heavy chain variable region includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 30, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 32; the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 33, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35; or, the heavy chain variable region includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 30, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 60; the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 61, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35.

[0009] The term "antibody" herein is used in the broadest sense, encompassing monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), and full-length antibodies (antibodies with a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds) and antigen-binding fragments thereof (also referred to as antigen-binding portion, which is a part of the full-length antibody that exhibits the desired antigen-binding activity, such as Fab, Fab', F(ab')2 or Fv, etc.). In one embodiment, the anti-human MerTK antibody is selected from the following:

[0010] (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13;

[0011] Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;

[0012] Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;

[0013] Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29;

[0014] Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39;

[0015] Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43;

[0016] (2) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13;

[0017] Or, the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23;

[0018] Or, the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25; the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23;

[0019] or, the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29;

[0020] or, the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37; the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 39;

[0021] Or, the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 41; and the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43.

[0022] In some embodiments, the anti-human MerTK antibody is selected from one or more of the following:

[0023] (1) Full-length antibody, Fab, F(ab)2, Fv;

[0024] (2) Monoclonal antibodies or polyclonal antibodies;

[0025] (3) Bispecific antibodies or multispecific antibodies.

[0026] In certain embodiments of any of the aforementioned anti-human MerTK antibodies, the antibody is a full-length antibody, which further includes at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE constant regions and their related variants.

[0027] In one embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 10; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 12;

[0028] Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 20; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22;

[0029] Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 24; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22;

[0030] Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 26; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 28;

[0031] Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 38;

[0032] Alternatively, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 40; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 42.

[0033] In some of the above embodiments, the amino acid sequences of the listed CDRs are determined according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (world wide web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity determining regions defined by any of the above-mentioned known schemes, or the complementarity determining regions defined by any other definition rules based on the antibodies of the present invention.

[0034] The second technical solution provided by the present invention is: a nucleic acid molecule encoding any anti-human MerTK antibody described in one of the technical solutions.

[0035] The third technical solution provided by the present invention is: a recombinant expression vector comprising the nucleic acid molecule described in the second technical solution;

[0036] In some embodiments, the recombinant expression vector is any one or a combination of at least two of a viral vector, a plasmid, a DNA fragment, or an RNA;

[0037] In some preferred embodiments, the recombinant expression vector is a plasmid.

[0038] The fourth technical solution provided by the present invention is: a transformant comprising the recombinant expression vector described in the third technical solution in a host cell;

[0039] In some preferred embodiments, the host cell is a mammalian cell.

[0040] The fifth technical solution provided by the present invention is: a method for preparing an anti-human MerTK antibody, which comprises culturing the transformant as described in the fourth technical solution, and obtaining the anti-human MerTK antibody from the culture.

[0041] The sixth technical solution provided by the present invention is: a chimeric antigen receptor, which comprises any anti-human MerTK antibody described in one of the technical solutions.

[0042] The seventh technical solution provided by the present invention is: a genetically modified cell, which includes the chimeric antigen receptor described in the sixth technical solution; in some preferred embodiments, the host of the genetically modified cell is a T cell or a NK cell.

[0043] The eighth technical solution provided by the present invention is: an antibody-drug conjugate, which comprises a cytotoxic agent or a label, and any anti-human MerTK antibody described in one of the technical solutions.

[0044] A ninth technical solution provided by the present invention is: a pharmaceutical composition or a drug kit containing the same, comprising any anti-human MerTK antibody described in one of the technical solutions, the genetically modified cell described in the seventh technical solution, or the antibody-drug conjugate described in the eighth technical solution, and at least one of a pharmaceutically acceptable carrier, excipient, or diluent;

[0045] In some embodiments, the pharmaceutical composition or a kit containing the same further contains one or more of the group consisting of a hormone agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

[0046] The tenth technical solution provided by the present invention is: use of any antibody described in the first technical solution, or the genetically modified cell described in the seventh technical solution, or the antibody-drug conjugate described in the eighth technical solution, or the pharmaceutical composition described in the ninth technical solution, or a kit containing the same, in the preparation of a drug for preventing and / or treating tumors, wherein the tumor is a MerTK-overexpressing tumor;

[0047] In some embodiments, the tumor is one of the following: B or T lymphocytic leukemia, gastric cancer, NSCLC, colon cancer, prostate cancer, melanoma, glioblastoma.

[0048] The eleventh technical solution provided by the present invention is: use of any antibody described in the first technical solution, or the genetically modified cell described in the seventh technical solution, or the antibody-drug conjugate described in the eighth technical solution, or the pharmaceutical composition described in the ninth technical solution, or a drug kit containing the same, in the treatment of tumors, wherein the tumor is a MerTK-overexpressing tumor;

[0049] In some embodiments, the tumor is one of the following: B or T lymphocytic leukemia, gastric cancer, NSCLC, colon cancer, prostate cancer, melanoma, glioblastoma.

[0050] The twelfth technical solution provided by the present invention is: using any antibody described in the first technical solution, or the genetically modified cell described in the seventh technical solution, or the antibody-drug conjugate described in the eighth technical solution, or the pharmaceutical composition described in the ninth technical solution, or a drug kit containing the same, to treat a tumor, wherein the tumor is a MerTK-overexpressing tumor;

[0051] In some embodiments, the tumor is one of the following: B or T lymphocytic leukemia, gastric cancer, NSCLC, colon cancer, prostate cancer, melanoma, glioblastoma.

[0052] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0053] The reagents and raw materials used in the present invention are commercially available.

[0054] To better understand the present invention, some terms are first defined. Other definitions are listed throughout the detailed description.

[0055] As used herein, the terms "full-length antibody," "complete antibody," and "conventional antibody" are used interchangeably to refer to glycoproteins comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain in a full-length antibody consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is composed of a single domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ heavy chains. Mammalian light chains are classified as λ or κ light chains. Immunoglobulins comprising α, δ, ε, γ, and μ heavy chains are immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions (and, for IgE and IgM, the fourth constant region) of two heavy chains, bound together by disulfide bonds (interchain bonds) in the hinge. Heavy chains γ, α, and δ have a constant region composed of three tandem (in a row) Ig domains and a hinge region for added flexibility; heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y consists of the variable region of a single heavy chain bound to a single light chain and the first constant region (CH1). The "Fc" region is the two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and hydrophobic interactions within the CH3 domain.

[0056] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain involved in antigen binding. VH and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region of the variable domain primarily responsible for antigen binding; "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0057] In the present invention, "Fab" consists of one light chain and the CH1 and VH domains of one heavy chain. "Fab'" contains one light chain and a portion encompassing the VH and CH1 domains, as well as the region between the CH1 and CH2 domains. Interchain disulfide bonds can form between the two heavy chains of two Fab' fragments, forming a F(ab')2 molecule. A F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0058] In the present invention, the scFv (single-chain antibody fragment) VH and VL domains are connected by a linker (also known as a linker) to form a polypeptide chain. The VL and VH domains are paired via a linker that enables them to be produced as a single polypeptide chain to form a monovalent molecule [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating G4S amino acid sequence or variants thereof. For example, linkers having the amino acid sequences (G4S)4 or (G4S)3 may be used, although variants thereof may also be used.

[0059] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with two or more epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has two or more epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; and antibodies having two or more single variable domains, each binding to a different target or a different epitope of the same target.

[0060] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody. An epitope can be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by antigen folding (i.e., through tertiary folding of proteinaceous antigens). Conformational epitopes differ from linear epitopes in that antibody binding to a conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning and peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463).

[0061] The term "specific binding" means that the antibody binds to an antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes. -7 M or smaller (e.g., approximately 1×10 -8 M or smaller, approximately 1×10 - 9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 In some embodiments, an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of 1 M or less. In some embodiments, the KD of the antibody for binding to the antigen is 10% or 1% of the KD of the antibody for binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures, such as by BIACORE® surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous), such as humans or monkeys, e.g., cynomolgus (Macaca fascicularis), chimpanzee (Pan troglodytes), or marmoset (Callithrix jacchus).

[0062] The term "affinity" refers to the overall strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art (including those described herein). The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, while the term "kdis" or "kd" as used herein refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using well-established methods in the art. Methods for determining antibody KD include measuring surface plasmon resonance using biosensing systems such as the ELISA system, or measuring affinity in solution by solution equilibrium titration (SET).

[0063] The term "EC50," also known as half-maximal effect concentration, refers to the concentration of an antibody that elicits 50% of the maximal effect.

[0064] The term "IC50," also known as the inhibitory concentration (IC50), refers to the concentration of a drug or inhibitor required to inhibit a given biological process or a component of that process (e.g., an enzyme, receptor, or cell) by half. In competitive ELISAs, it is an important indicator of competitive inhibition.

[0065] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-additive mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can instruct the expression of the gene to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Typically, expression vectors useful in recombinant DNA technology typically exist in the form of a plasmid. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0066] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0067] The term "polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may comprise one or more polypeptides.

[0068] The term "host cell" refers to a cell in which a vector can be propagated and its DNA can be expressed, and the cell can be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the subject host cell. It should be understood that not all progeny are identical to the parent cell, as mutations may occur during the replication process, and such progeny are included.

[0069] The term "purify" and its grammatical variations are used to refer to the complete or partial removal of at least one impurity from a mixture comprising a protein and one or more impurities, reducing the content of the impurity in the composition, thereby increasing the level of purification of the protein in the composition.

[0070] The terms "anti-human MerTK antibody" and "antibody that specifically binds to human MerTK" refer to antibodies that can bind to human MerTK with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting human MerTK. In certain embodiments, the antibody that binds to human MerTK has a dissociation constant (KD) of < about 1 µM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -12 M, for example 10 -9 M to 10 -10 In certain embodiments, the anti-human MerTK antibody binds to an epitope that is conserved among MerTKs from different species.

[0071] The antibodies of the present invention include monoclonal antibodies (abbreviated as mAb or Ab), which refer to antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage clonal cell lines.

[0072] The positive progress effect of the present invention is:

[0073] The present invention provides a series of novel anti-MerTK antibodies with specific binding and high affinity. These antibodies can specifically inhibit both the Gas6-MerTK and Protein S-MerTK interactions, while not inhibiting the Tulp1-MerTK interaction. Theoretically, when these antibodies exert their inhibitory activity in vivo, they should avoid potential retinal toxicity. Purified human MerTK antibodies specifically and effectively inhibit AKT phosphorylation in the Gas6-MerTK signaling pathway and demonstrate modest tumor-suppressing activity in mouse models. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 ELISA assay for binding of immune serum to recombinant human MerTK ECD his protein and recombinant mouse MerTK ECD his protein.

[0075] Figure 2 The immune serum was combined with recombinant CHO human MerTK overexpressing cells (clone number: 1-T-53) and CHO null cells by FACS.

[0076] Figure 3 The results show the binding of rabbit anti-human MerTK antibody to recombinant human MerTK overexpressing cells and CHO null cells. Figure 3 A, 3C, and 3E are the binding curves of antibodies and overexpressing cells. Figure 3 B, 3D, and 3F are the binding curves of antibodies to empty cells.

[0077] Figure 4 To evaluate the blocking activity of rabbit anti-MerTK antibody on GAS6-MerTK binding.

[0078] Figure 5 Comparison of 8F7 and 3E6 rabbit and humanized antibody sequences. The CDR regions of the VH and VL sequences are shown in Kabat format.

[0079] Figure 6 Binding of humanized antibodies to recombinant overexpressing cells and tumor cells. Figure 6 A, 6C, and 6E are the binding results of 8F7 humanized antibody with recombinant human high-expressing MerTK cells, low-expressing MerTK cells, and MerTK-overexpressing tumor cells, respectively. Figure 6 B, 6D, and 6F are the binding results of 3E6 humanized antibody with recombinant human high-expressing MerTK cells, low-expressing MerTK cells, and MerTK-overexpressing tumor cells, respectively.

[0080] Figure 7 The results of humanized antibodies blocking the binding of ligand to MerTK. Figure 7A and 7B are the blocking activities of 8F7 humanized antibody on the binding of two ligands to receptors. Figure 7 Tables C and D show the blocking activity of the 3E6 humanized antibody against two ligand-receptor binding events. Table 7 summarizes the blocking activity of the humanized antibodies against ligand-receptor binding. Figure 7 E is the effect of humanized antibody 8F7H3L1 LALA treatment on the binding of ligand TULP1-MerTK.

[0081] Figure 8 Inhibitory activity of the humanized antibody against AKT phosphorylation downstream of MerTK.

[0082] Figure 9 The humanized antibody binds to different domain proteins of MerTK and other proteins in the family. Figure 9 A represents the binding of antibodies to MerTK proteins with different structural domains. Figure 9 B and Figure 9 C shows the binding of antibodies to Axl and Tyro3 proteins, respectively.

[0083] Figure 10 Evaluation of the tumor growth inhibitory activity of MerTK humanized antibodies. DETAILED DESCRIPTION

[0084] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0085] Example 1 Expression of MerTK in tumor cells

[0086] MerTK is expressed in various liquid and solid tumors. Flow cytometry was used to assess MerTK expression in various tumor cells. Specifically, exponentially growing tumor cells were harvested, adjusted to a cell density of 2E6 cells / ml, and plated into 96-well plates at 100 μl / well. After centrifugation (2000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 100 μl of either 5 μg / ml or 1 μg / ml antibody, respectively. The plates were incubated at 4°C for 1 hour. Following incubation, the plates were centrifuged as above and washed three times with PBS. Then, 100 μl / well of either a 1:1000 dilution of goat anti-mouse F(ab')2 fragment-specific APC antibody (Jackson ImmunoResearch, 115-136-146) or goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the cells were resuspended in 80 μl of PBS and analyzed by flow cytometry using the iQue Screener PLUS. The results were analyzed using GraphPad Prism. A commercial anti-human MerTK antibody (Biolegend, 367602) was used for HT29, A549, MDA-MB-468, and BT-474 cells. For other cell types, the MerTK antibody 8N42 (sequence from BMS PCT patent application WO2020076799A1, VH sequence as shown in SEQ ID NO:237, VL sequence as shown in SEQ ID NO:238) was used. The results are shown in Table 1. Tumor cells exhibited varying levels of MerTK expression, with Colo-699, HepG2, BT474, and RT4 cells showing higher MerTK expression.

[0087] Table 1 MerTK expression in different tumor cells

[0088] cell describe MFI-5μg / mlMerTK Ab MFI-1μg / mlMerTK Ab MFI- 5μg / mlIsotype Ab MFI-1μg / mlIsotype Ab 5μg / ml MerTK / Isotype ratio 5μg / ml MerTK / Isotype ratio HT29 human colon cancer cells 47612 21209 3900 3740 12.2 5.7 A549 Human alveolar adenocarcinoma basal epithelial cells 50447 28346 2985 3070 16.9 9.2 MDA-MB-468 human breast cancer cells 28941 13369 4592 4183 6.3 3.2 RT4 Bladder cancer papilloma cells 126593 104371 4045 4005 31.3 26.1 HepG2 Human hepatocellular carcinoma cells 221085 221618 5114 5008 43.2 44.3 BT-474 human breast cancer cells 134523 113796 5038 4717 26.7 24.1 K562 human chronic myeloid leukemia cells 20742 14761 3110 2996 6.7 4.9 RPMI8226 human myeloma cells 39891 22644 4465 2993 8.9 7.6 Capan-2 Human pancreatic cancer cells 20742 11097 2436 2927 8.5 3.8 BEWO human placental choriocarcinoma cells 41694 28270 3892 1677 10.7 16.9 A431 Human skin cancer cells 14500 7224 3409 3204 4.3 2.3 OVCAR3 human ovarian adenocarcinoma cells 63963 44107 3634 3174 17.6 13.9 Colo-699 Human lung adenocarcinoma cells 218291 190359 4361 2864 50.1 66.5 SKOV3 human ovarian cancer cells 32328 13102 4318 4397 7.5 3.0

[0089] Example 2 Animal Immunization

[0090] Two New Zealand white rabbits (E18978, E18979) were immunized with recombinant human MerTK mFc protein. Specifically, 400 μg of recombinant human MerTK mFc protein (SEQ ID NO: 1) mixed with complete Freund's adjuvant (CFA) was injected subcutaneously at multiple sites for the first immunization. Four immunizations were performed, with the next three immunizations each using 200 μg of recombinant human MerTK mFc protein mixed with incomplete Freund's adjuvant (IFA). The four immunizations were performed one, two, and three weeks apart, respectively. Serum was collected one week after the third and fourth immunizations, and the obtained sera were tested for antigen-specific titers.

[0091] (1) Serum titer protein binding test

[0092] Serum ELISA binding assays were performed using recombinant human MerTK ECD his protein (SEQ ID NO: 2) and recombinant mouse MerTK ECD his protein (mouse MerTK sequence: Q60805 Met 1-F498, SEQ ID NO: 3). Specifically, 1 μg / mL of human MerTK ECD his protein and 1 μg / mL of mouse MerTK ECD his protein were coated in 384-well plates, 25 μl / well, and incubated overnight at 4°C. The next day, the coated 384-well plates were washed five times with 1xPBST and then blocked with 1% BSA (prepared in 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed five times with 1xPBST and serially diluted serum (starting at 1:1000, 3-fold dilution, 8 steps) was added and incubated at 37°C for 1 hour. After incubation and washing, 1:5000 diluted goat anti-rabbit IgG (H+L) HRP antibody (Jackson ImmunoResearch, 111-035-045) was added at 25 μl / well and incubated at 37°C for 1 hour. After washing with PBST, 25 μl TMB substrate (TMB, Thermo Fisher, CAT: 34029) was added to each well to detect antibody binding. The plates were developed in the dark for 3 min at room temperature and 10 μl / well of stop solution (2 M H2SO4) was added. The OD450n\630nm plate was read, and the background at 450nm was subtracted from the 630nm background. The results are as follows Figure 1 As shown, Figure 1 A and 1B are the binding of two rabbit pre-immune sera (PB), three-immune sera (TB1) and four-immune sera (TB2) to human MerTK ECD his protein, respectively. The immunized sera all showed specific binding. Figure 1Figures C and D show the binding of pre-immune sera (PB), serum after three immunizations (TB1), and serum after four immunizations (TB2) from two rabbits to the mouse MerTK ECD his protein, respectively. All immunized sera showed specific binding. The results showed that after four immunizations with human MerTK ECD mFc protein, the sera from both rabbits showed varying degrees of specific binding to both antigens, with the serum from rabbit number E18979 showing a stronger response to both antigens.

[0093] (II) Combination Detection of Serum Titer and Recombinant Overexpressing Cells

[0094] Cell-based binding assays were performed with rabbit serum using recombinant CHO cells overexpressing human MerTK (the human MerTK sequence used is Q12866 Met1-Met999, SEQ ID NO: 4). Specifically, the original serum was diluted 100-fold as the first dilution, followed by six 3-fold dilutions. Recombinant CHO cells overexpressing human MerTK (clone 1-T-53) and CHO null cells were harvested from the exponentially growing phase, adjusted to a cell density of 2E6 cells / ml, and plated into 96-well plates at 100 μl per well. After centrifugation (2000 rpm, 5 minutes), the supernatant was discarded, and the cells were resuspended in the serially diluted serum and incubated at 4°C for 1 hour. After incubation, the cell plate was centrifuged as above and washed three times with PBS. 100 μl / well of AF647-labeled goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch, 111-605-144) diluted 1:1000 was added and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the cells were resuspended in 80 μl PBS and read by flow cytometry using iQue Screener PLUS. The results were analyzed using GradPad Prism. Figure 2 As shown, Figure 2 A and 2B are the binding of two rabbit pre-immune sera (PB), three-immune sera (TB1), and four-immune sera (TB2), respectively, to recombinant CHO human MerTK overexpressing cells (clone number: 1-T-53). The post-immune sera all showed specific binding. Figure 2 Figures C and D show the binding of two rabbit sera to CHO null cells, respectively, before immunization, after three immunizations, and after four immunizations. The post-immunization sera essentially did not bind to the null cells. After four immunizations with human MerTK ECD mFc protein, both rabbit sera showed specific binding to human overexpressed cells, with the rabbit sera from rabbit number E18979 showing the strongest binding.

[0095] Example 3 Screening of single B cells

[0096] (1) B cell sorting

[0097] Human MerTK ECD his protein (SEQ ID NO: 2) was diluted to 2 μg / mL in PBS, and 2 mL / well was coated on a 6-well plate. The plate was incubated overnight at 4°C. The next day, the cells were centrifuged at 400g for 3 minutes, and the supernatant was removed. The cells were resuspended in B cell culture medium (University). The resuspended cells were incubated on a 6-well plate pre-coated with human MerTK ECD his protein at 37°C, 5% CO2 for 60 minutes. The supernatant was then removed, and the plate surface was gently washed once with 1xPBS. The cells were then digested with trypsin (Gibco, Cat: 25300062) and harvested. The cells were centrifuged at 400g for 3 minutes, and the supernatant was removed. The cells were resuspended in 1 mL of FACS buffer (1xPBS + 0.5% BSA), and 1 μL of fluorescent secondary antibody (University) was added. The cells were incubated at 4°C for 20 minutes. After centrifugation as above, cells were washed twice with 1 mL of FACS buffer (1xPBS + 0.5% BSA) and resuspended in 1 mL of buffer (1xPBS + 2% FBS). Next, cells were filtered through a flow cytometer with a strainer, sorted using a flow cytometer, and harvested (96-well plate, 1 cell / well). After culturing the sorted B cells for 12 days, they were centrifuged at 400 g for 2 minutes at room temperature. 80 μl of the supernatant was collected and stored separately at -20°C for ELISA and FACS analysis. The cells were then resuspended in 100 μl of DNA / RNA extraction buffer (Urisys) and stored in PCR tubes at -80°C for subsequent RNA extraction.

[0098] (II) B cell supernatant ELISA test (conventional ELISA)

[0099] ELISA binding assay of B cell supernatants was performed using human MerTK ECD his protein. Specifically, 1 μg / ml human MerTK ECD his protein was coated in a 384-well plate, 25 μl / well, and incubated at 4°C overnight. The next day, the coated 384-well plate was washed five times with 1xPBST, then blocked with 1% BSA (prepared in 1xPBST) and incubated at 37°C for 1 hour; after incubation, it was washed five times with 1xPBST, and diluted B cell supernatant (10 μl of B cell supernatant + 15 μl of reaction buffer) was added at 25 μl / well and incubated at room temperature for 1 hour; after incubation and washing, 1:5000 diluted goat anti-rabbit IgG (H+L) HRP antibody (Jackson ImmunoResearch, 111-035-045) was added at 25 μl / well and incubated at room temperature for 1 hour; after washing with PBST, 25 μl of TMB substrate (TMB, Thermo Fisher, CAT: 34029) was added to each well to detect antibody binding at 25 μl / well, and color was developed at room temperature in the dark for 3 minutes, and then 10 μl / well of stop solution (2M The reaction was terminated with H2SO4 and the plate was read at OD450nm / 630nm. The result was the background at 450nm minus 630nm.

[0100] Simultaneously, ELISA binding assays of B cell supernatants were performed in 96-well plates using mouse MerTK ECD-his protein. 1 μg / ml mouse MerTK ECD-his protein was coated into 96-well plates and incubated overnight. The next day, the plates were washed and then 100 μl / well of 1:10 diluted cell supernatant was added for 1 hour at 37°C. After washing, 100 μl / well of 1:5000 diluted goat anti-rabbit IgG (H+L) HRP antibody (Jackson ImmunoResearch, 111-035-144) was added for 1 hour at 37°C. After washing with PBST, antibody binding was detected by adding 100 μl of TMB substrate (TMB, Thermo Fisher, CAT: 34029) to each well. Color was developed at room temperature in the dark, and 100 μl / well of 1N HCl stop solution was added. The plate was read at OD 450 nm.

[0101] (III) B cell supernatant ELISA (Capture ELISA)

[0102] 384-well plates (Corning, CAT: 3700) were coated with secondary antibody (Goat Anti-Rabbit IgG Fragment, Jackson ImmunoResearch, 111-005-046) diluted in PBS at 1 μg / mL, 25 μl / well, and incubated overnight at 4°C. The next day, the coated 384-well plates were washed five times and nonspecific binding sites were blocked with 50 μl / well of blocking buffer. The plates were incubated for 1 hour at room temperature. After washing five times as above, diluted B cell supernatant (10 μl B cell supernatant + 15 μl reaction buffer) was added at 25 μl / well and incubated for 1 hour at room temperature. Following incubation and washing, Biotin human MerTKECD his protein was added at 1 μg / mL, 25 μl / well, and incubated for 1 hour at room temperature. Similarly, the plates were washed five times, and secondary antibody (Neutravidin-HRP, Thermo Fisher, 31001) diluted 1:10,000 was added at 25 μl / well, and incubated at room temperature in the dark for 1 hour. After washing, antibody binding was detected by adding 25 μl / well of TMB substrate (TMB, Thermo Fisher, CAT: 34029). The plate was allowed to develop for 3 minutes at room temperature in the dark, and then 10 μl / well of stop solution (2 M H2SO4) was added to terminate the reaction. The plate was read at OD 450 nm / 630 nm, and the OD at 450 nm was subtracted from the OD at 630 nm.

[0103] (IV) FACS analysis of B cell supernatant

[0104] Cell-based binding assays were performed using recombinant CHO human MerTK-overexpressing cells. Specifically, exponentially growing recombinant CHO human MerTK-overexpressing cells (clone 1-T-53) and CHO null cells were harvested, adjusted to a cell density of 2E6 cells / ml, and plated into 96-well plates at 100 μl / well. After centrifugation (2000 rpm, 5 min), the supernatant was discarded and the cells were resuspended in 100 μl of 1:10 diluted B cell supernatant. The plates were incubated at 4°C for 1 hour. Following incubation, the plates were centrifuged as above and washed three times with PBS. Then, 100 μl / well of AF647-conjugated goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch, 111-605-144) at a dilution of 1:1000 was added and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the cells were resuspended in 80 μl of PBS and flow cytometry was performed using iQue Screener PLUS. The results were analyzed using GradPad Prism.

[0105] (V) Evaluation of B cell supernatant blocking activity

[0106] The blocking activity of B cell supernatants on Gas6-MerTK binding was detected by competitive ELISA. First, 96-well plates were coated with 2 μg / ml goat anti-mouse IgG Fcγ fragment antibody (Jackson ImmunoResearch, 115-005-008) at 100 μl / well and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1xPBST, then blocked with 1% BSA (prepared in 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1xPBST, and 100 μl of 2 μg / ml recombinant human MerTK mFc protein was added to each well and incubated at 37°C for 1 hour. After incubation and washing, 50 μl of 2.5-fold diluted B cell supernatant and 50 μl of 1.7 μg / ml human Gas6 his protein (Novoprotein, cat# C01W) were added to each well and incubated at 37°C for 1 hour. After incubation and washing, 1:10,000 diluted rabbit anti-6xhis HRP antibody (abcam, cat# ab1187) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μl of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were analyzed by Spectra The M5e instrument measures OD450nm. A mixture of 50μl of 1% BSA and 50μl of 3.4μg / ml recombinant human GAS6his protein is used as the maximum signal (Max). Wells containing only 1% BSA have the lowest absorbance (Mini). Wells with a lower absorbance than the Max value correspond to serum dilutions containing antibodies that inhibit the binding of human GAS6 and human MerTK. Inhibition rate (%) = (OD450 Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100.

[0107] Based on the results of the above ELISA binding, cell binding and blocking assays, 41 B cell clones were selected for the next step of gene fishing and antibody preparation and activity identification.

[0108] Example 4 Preparation of rabbit anti-human MerTK antibody

[0109] The positive B cell clones screened were used for amplification of antibody fragments, construction of expression vectors and production of rabbit anti-human MerTK antibodies.

[0110] (I) Extraction of positive clone genes and amplification of VH / VL cDNA

[0111] RNA from single B cells was extracted using an RNA extraction kit and reverse transcribed using a reverse transcription kit (ABScript II RT, ABclonal, RK21400). First, mix 1 was prepared as follows. After centrifugation, the mixture was placed in a PCR instrument and programmed to run program 01 (65°C, 5 min). Next, mix 2 was prepared by adding 7 μl of mix 2 to 13 μl of mix 1. The mixture was then centrifuged and placed in a PCR instrument. Programmes 03 (42°C, 1 h; 85°C, 5 min) were started.

[0112]

[0113] After obtaining the synthesized cDNA from the above steps, use 2x Gloria Nov (a Gloria Nova HS 2×HF, ABclonal, RK20715) to amplify the VH and VL genes:

[0114]

[0115] After the program is finished, electrophoresis is performed on a 1% agarose gel. The target band size is approximately 500 bp. The VH / VL fragments are then recovered using a gel recovery kit.

[0116] (II) Construction of pYURK expression vector

[0117] First, pYURK_OSLIC_H / L was digested with NheI and XbaI and recovered using a gel extraction kit. Then, 4 μl of VH / VL and 1 μl of the digested vector were mixed and added to 50 μl of thawed Trelief 5α competent cells and incubated on ice for 30 minutes. The mixture was then heat-shocked in a 42°C water bath for 1 minute. After the heat shock, it was incubated on ice for 2 minutes. 400 μl of LB medium preheated at 37°C was added to the centrifuge tube and incubated at 37°C with shaking at 220 rpm for 1 hour. The bacterial suspension was spread onto an Amp-resistant LB plate preheated at 37°C and incubated overnight at 37°C. Two to three single colonies were picked from the plate and inoculated into LB medium at 37°C at 220 rpm overnight. The plasmid was extracted using a plasmid extraction kit and then sequenced.

[0118] (III) Construction of pTT5 expression vector

[0119] To facilitate subsequent expression, the IgG-H and IgG-L genes were constructed in-house into the pTT5 expression vector. First, IgG_H and IgG_L genes were amplified using 2x Gloria Nova (Gloria Nova HS 2×HF, ABclonal, RK20715) and specific primers (primer sequences are shown in Table 2). The fragments were then electrophoresed on a 1% agarose gel. The heavy chain was approximately 1500 bp, and the light chain was approximately 750 bp. IgG_H and IgG_L fragments were recovered using a gel extraction kit. Simultaneously, pTT5 was double-digested with EcoRI and NotI and recovered using a gel extraction kit. Mix 4 μl of IgG-H / IgG-L and 1 μl of double-digested vector and add to 50 μl of melted Trelief 5α competent medium. Incubate on ice for 30 minutes. Heat shock the mixture in a 42°C water bath for 1 minute. After the heat shock, incubate on ice for 2 minutes. Add 400 μl of LB medium preheated at 37°C to the centrifuge tube and incubate at 37°C with shaking at 220 rpm for 1 hour. Spread the bacterial suspension onto an Amp-resistant LB plate preheated at 37°C and incubate at 37°C overnight. Pick two to three single colonies from the plate and inoculate them into LB medium. Incubate at 37°C at 220 rpm overnight. Extract the plasmid using a plasmid extraction kit before sequencing.

[0120] Table 2 Specific primers used for IgG_H and IgG_L gene amplification

[0121] Primers sequence 5' OSLIC pTT5_IgG_H_2 (SEQ ID NO: 44) TAAACGGATCTCTAGCGAATTCACCATGGAGACTGGGCTGCGCTG 3' OSLIC pTT5_IgG_H (SEQ ID NO: 45) GAGGTCGAGGTCGGTGCGGCCGCCTATTTACCCGGAGAGCGGG 5' OSLIC pTT5_IgG_L_2 (SEQ ID NO: 46) TAAACGGATCTCTAGCGAATTCACCATGGACACGAGGGCCCCCAC 3' OSLIC pTT5_IgG_L (SEQ ID NO: 47) GAGGTCGAGGTCGGTGCGGCCGCCTAACAGTCACCCCTATTGA

[0122] (IV) Expression and purification of anti-human MerTK antibodies

[0123] After sequence analysis, the correctly sequenced plasmid was amplified and extracted. The heavy and light chain plasmids were transfected into HEK293 cells at a density of 2E6 / ml using PEI reagent (Polysciences, Cat#24885) at a 2:3 ratio (1 μg plasmid: 4 μg PEI). The transfected cells were cultured in an orbital incubator at 37°C with 5% CO2 for 5–7 days. The culture supernatant was centrifuged and filtered through a 0.22 μm filter. The supernatant was then purified using a Protein G agarose column (GE Healthcare Biosciences, 17-0618-05). The column was equilibrated with 1xPBS (pH 7.4), then loaded with the filtered culture supernatant. The column was then washed with 1xPBS (pH 7.4) and eluted with elution buffer (50 nM sodium citrate, pH 2.5). The eluted sample was then neutralized with 1M Tris-HCl, pH 9.0. The neutralized sample was replaced with 1xPBS (pH 7.4), sterilized by filtration using a 0.22 μm filter, and the concentration of the purified antibody was measured using Nanodrop (Thermo Fisher Scientific Inc) and set aside.

[0124] Example 5 Binding Identification of Rabbit Anti-Human MerTK Antibody

[0125] (I) ELISA of purified rabbit anti-human MerTK antibody binding to recombinant human, monkey, and mouse MerTK proteins

[0126] Antibody binding assays were performed using recombinant human, monkey, and mouse MerTK proteins. Specifically, 1 μg / ml recombinant human MerTK ECD his protein, monkey MerTK mFc protein (R&D, cat#10576-MR), and mouse MerTK ECD his protein were coated in 96-well plates at 100 μl / well and incubated overnight. The next day, the coated 96-well plate was washed three times with 1xPBST, then blocked with 1% BSA (prepared in 1xPBST) and incubated at 37 degrees for 1 hour; after incubation, it was washed three times with 1xPBST, and a gradient dilution of purified antibody was added and incubated at 37 degrees for 1 hour; after incubation and washing, a 1:5000 dilution of goat anti-rabbit IgG (H+L) specific HRP antibody (Jackson ImmunoResearch, Cat#111-035-144) was added and incubated at 37 degrees for 1 hour; after washing with PBST, 100 μl of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured at OD450nm using a Spectra M5e instrument. The control antibodies PC1 and PC2 were the antibody h10F7.v16 in Genentech patent application (WO2020214995A1) (VH sequence is shown in SEQ ID NO: 92 in the patent application, and VL sequence is shown in SEQ ID NO: The antibodies tested specifically bound to human and cyno MerTK, while some showed cross-binding to mouse MerTK. Table 3 shows that all antibodies tested specifically bound to human and cyno MerTK, and some showed cross-binding to mouse MerTK.

[0127] Table 3 ELISA binding of purified antibodies to human, monkey, and mouse MerTK proteins

[0128]

[0129] (II) Binding of purified rabbit anti-human MerTK antibody to recombinant human MerTK-overexpressing cells

[0130] Cell-based binding assays for rabbit anti-human MerTK antibodies were performed using recombinant CHO-human MerTK-overexpressing cells (clone 1-T-53) and CHO-null cells. Specifically, the antibody was diluted three-fold starting at 10 μg / ml and eleven times. Recombinant CHO-human MerTK-overexpressing cells and CHO-null cells were harvested at the exponential growth phase, adjusted to a cell density of 2E6 cells / ml, and plated into 96-well plates at 100 μl / well. The plates were centrifuged (2000 rpm, 5 min), the supernatant discarded, and the cells resuspended in serially diluted antibody. The plates were incubated at 4°C for 1 hour. Following incubation, the plates were centrifuged as above and washed three times with PBS. A 1:1000 dilution of goat anti-rabbit Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 115-136-164) was added at 100 μl / well and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the cells were resuspended in 80 μl of PBS and analyzed by flow cytometry using the iQue Screener PLUS. The results were analyzed using GraphPad Prism. The control antibody PC1 was h10F7.v16, a Genentech patent (WO2020214995A1).

[0131] Table 4 EC50 and top MFI values ​​of purified rabbit anti-human MerTK antibodies binding to overexpressing cells and empty cells

[0132]

[0133]

[0134] The results are as follows Figure 3 As shown in Table 4, most antibodies showed specific binding to human MerTK cells. Based on the results, three antibodies with high nonspecific binding to CHO empty cells, 1H5, 22G11, and 18D8, and two antibodies with low MFI Top values, 32E9 and 4A3, were removed; the remaining 20 antibodies were evaluated for ligand-receptor binding blocking.

[0135] Example 6 Blocking Identification of Rabbit Anti-Human MerTK Antibodies

[0136] The blocking effect of rabbit anti-human MerTK antibodies on the binding of human GAS6 and human MerTK proteins was assessed using an ELISA. Specifically, 2 μg / ml anti-mouse Fcγ fragment antibody (Jackson ImmunoResearch, 115-005-008) was coated into a 96-well plate at 100 μl / well and incubated overnight at 4°C. The next day, the coated 96-well plate was washed three times with 1xPBST, then blocked with 1% BSA (in 1xPBST) and incubated at 37°C for 1 hour. Following incubation, the plate was washed three times with 1xPBST, and 100 μl of 2 μg / ml human MerTK ECD mFc protein was added to each well and incubated at 37°C for 1 hour. After washing as above, 50 μl of a mixture of serially diluted antibody and 50 μl of recombinant human GAS6 his protein (Novoprotein, Cat#C01W) at a final concentration of 1.7 μg / ml was added to each well and incubated at 37°C for 1 hour. Genentech's patented (WO2020214995A1) antibody h10F7.v16 at a final concentration of 7.5 μg / ml served as a positive control, and hIgG1 at a final concentration of 7.5 μg / ml served as a negative control. A mixture of 50 μl of 1% BSA and 50 μl of recombinant human GAS6 his protein at a final concentration of 1.7 μg / ml was used as the maximum signal (Max). Wells containing only 1% BSA had the lowest absorbance (Mini). After incubation and washing, goat anti-6xhis HRP antibody (abcam, Cat#ab1187) diluted 1:5000 was added and incubated at 37 degrees for 1 hour. After washing with PBST, 100 μl of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured using a Spectra M5e instrument to measure the absorbance at OD450 nm. Inhibition rate (%) = (OD450 Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100.

[0137] The results are as follows Figure 4 As shown, purified rabbit anti-human MerTK antibodies demonstrated specific blocking activity against GAS6-MerTK binding in this system, with antibodies 8F7 and 26A10 showing the greatest inhibitory activity. Table 5 summarizes the antibody inhibition rates and IC50 values. Based on comprehensive consideration of the antibody binding results on cells and the RBA blocking results, 3E6, 8A7, 8F7, and 26A10 were selected for subsequent testing and evaluation.

[0138] Table 5 Summary of IC50 and inhibition rate of rabbit anti-human MerTK antibody blocking GAS6-MerTK binding

[0139] serial number name IC50(nM) Inhibition rate (%) 1 3E6 NA 54 2 6A6 0.24 78 3 6D2 0.15 47 4 6F2 0.37 75 5 7G12 0.35 69 6 8A7 0.36 83 7 8F7 0.51 117 8 9C2 0.34 68 9 9H9 0.06 67 10 9H10 1.02 38 11 20E8 0.38 92 12 26A1 0.50 88 13 26A10 0.68 118 14 27A9 0.58 68 15 27B2 0.91 84 16 27H6 0.49 64 17 28H2 0.56 71 18 30A4 1.08 81 19 31A11 0.89 86 20 31C3 0.63 109 PC1 h10F7.v16 1.14 113

[0140] Example 7 Epitope Analysis of Rabbit Anti-Human MerTK Antibody

[0141] Epitope analysis between antibodies was performed using a tandem assay on an Octet Red 96 (Forte Bio, Serial No. FB-50482). The antigen was immobilized and then competitively bound to different test antibodies. Specifically, each cycle began with capturing 100 nM human MerTK ECD his protein using an NTA sensor (Forte Bio, REF Num: 18-5101) for 180 s (binding height 0.3 nm). This was followed by binding of the first antibody at 10 μg / ml for 180 s, followed by binding of the second antibody at 10 μg / ml for 180 s. Finally, the sensor was regenerated in pH 1.5 Glycine-HCl for 30 s. Before the next cycle, the NTA sensor was activated in a NiSO4 solution, followed by a new round of antigen binding and antibody competition binding. The results were analyzed and summarized with reference to the epitope analysis in the OctetRed 96 operation guide. The results are shown in Table 6. The antibodies showed good self-inhibition (inhibition rate >80%), and the four antibodies did not inhibit each other, that is, 8F7, 26A10, 8A7, and 3E6 belong to different epitopes.

[0142] Table 6 Epitope analysis results of rabbit anti-human MerTK antibody

[0143] Antibody 8F7 26A10 8A7 3E6 8F7 92.5 6.0 25.2 -61.9 26A10 10.9 87.6 7.7 -47.2 8A7 32.3 31.2 82.3 -29.0 3E6 50.4 49.8 46.4 95.4

[0144] Example 8 Humanized Engineering of Rabbit Anti-Human MerTK Antibody

[0145] Antibody humanization is achieved through CDR region transplantation. Specifically, an antibody structural model is constructed based on the VH / VL sequences (MOE software). The humanized antibody with the highest homology is selected to provide the framework. The CDR regions of the rabbit anti-VH / VL, designated according to the CCG nomenclature, are then transplanted into the humanized antibody framework, forming a humanized variable region sequence consisting of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Simultaneously, based on the established variable region structural model, potential backmutation residues are selected to ensure the activity of the humanized antibody. Backmutations are primarily based on the following three principles: first, amino acids within the framework region located at the VH-VL interface, near or directly interacting with the CDRs; second, amino acids embedded within the protein are selected for backmutation to reduce immunogenicity; and third, mutations that lower the molecular energy level are selected to ensure stability and expression levels. Post-translational modification sites (such as NS, DS, and NG) within the sequence are also mutated to minimize the impact of humanization on the antibody's physicochemical properties and maintain stability. Humanized antibodies containing different mutations were tested for binding to cells expressing MerTK, affinity assessment for human and monkey MerTK, and ligand-receptor binding blocking activity. Humanized antibodies with comparable or better affinity, characterization, and functional activity compared to the pre-transformed antibodies were selected. The heavy chain amino acid sequence of the original rabbit anti-MerTK antibody, rb8F7, is shown in SEQ ID NO: 10, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 11, and HCDR1-HCDR3 are shown in SEQ ID NOs: 14-16, respectively. The light chain amino acid sequence of rb8F7 is shown in SEQ ID NO: 12, the light chain variable region amino acid sequence is shown in SEQ ID NO: 13, and LCDR1-LCDR3 are shown in SEQ ID NOs: 17-19, respectively. The CDRs of rb8F7 were transplanted onto a humanized framework to generate hz8F1 H1L1, with the heavy chain amino acid sequence shown in SEQ ID NO: 20, the heavy chain variable region amino acid sequence shown in SEQ ID NO: 21, the light chain amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region amino acid sequence shown in SEQ ID NO: 23. Modification mutations at the PTM sites in hz8F1 H1L1 were performed to generate hz8F1 H3L1, which retains activity comparable to that of rb8F7 and hz8F1 H1L1. The heavy chain amino acid sequence is shown in SEQ ID NO: 24, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 25, the light chain amino acid sequence is shown in SEQ ID NO: 22, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 23.The CDR region sequences of the heavy and light chains of antibodies hz8F1 H1L1 hz8F1 H3L1 are consistent with those of the rb8F7 antibody, with HCDR1-HCDR3 being SEQ ID NOs: 14-16, and LCDR1-LCDR3 being SEQ ID NOs: 17-19, respectively.

[0146] Example 9 Cell Binding Assay of Humanized Antibodies

[0147] The humanized antibody was tested for binding at the cellular level using recombinant CHO-human MerTK overexpressing cells (clone 1-T-53), recombinant CHO-human MerTK underexpressing cells (clone 1-T-3), and Colo699 tumor cells (ATCC, Cat#TIB-67), which naturally express MerTK. Specifically, the antibody was diluted three-fold starting at 10 μg / ml and then diluted through 11 steps. Recombinant CHO-human MerTK overexpressing cells and Colo699 cells were harvested at the exponential growth phase, adjusted to a cell density of 2E6 cells / ml, and plated into 96-well plates at 100 μl per well. The plates were centrifuged (2000 rpm, 5 minutes), the supernatant discarded, and the cells resuspended in serially diluted antibody. The plates were incubated at 4°C for 1 hour. After incubation, the cell plates were centrifuged as above and washed three times with PBS. 100 μl / well of a 1:1000 dilution of goat anti-rabbit IgG (H+L)-specific Alexa Fluor 647 antibody (Jackson ImmunoResearch, 111-605-144) or goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added and incubated at 4°C in the dark for 30 min. After washing three times with PBS, the cells were resuspended in 80 μl of PBS and analyzed by flow cytometry using the iQue Screener PLUS. The results were analyzed using GraphPad Prism.

[0148] The results are as follows Figure 6 As shown, different humanized antibodies bind differently to the same cell. For 8F7, H1L1 is the antibody before the PTM site is removed. Different PTM site mutations affect the binding activity of the antibody to the cell. Finally, H3L1 was selected for the binding evaluation on MerTK high and low expression cells and tumor cells ( Figure 6 A, C, E). For 3E6, H2L2 is the antibody before the PTM site was removed. Based on H2L2, the PTM site was further mutated, and H8L2 was finally selected for the binding evaluation on MerTK high- and low-expressing cells and tumor cells ( Figure 6Results (B, D, F) show that the humanized antibodies exhibited specific binding activity comparable to that of the pre-PTM-deleted antibodies in recombinant MerTK-overexpressing and low-expressing cells, as well as tumor cells. Based on these results, 8F7 H3L1 and 3E6 H8L2 were selected for further antibody functional evaluation.

[0149] Example 10 Detection of blocking activity of humanized antibodies

[0150] Gas6, Protein S, and TULP1 are all specific ligands for MerTK, playing important roles in MerTK-mediated efferocytosis and downstream signaling. The following will evaluate the blocking activity of humanized antibodies against the binding of these three ligands to the receptor.

[0151] (I) Blocking assay of humanized antibodies against the binding of human GAS6 protein to human MerTK protein, and human Protein S protein to human MerTK protein

[0152] The blocking effect of humanized antibodies on the binding of human GAS6 and human MerTK proteins was assessed by ELISA. Specifically, 2 μg / ml anti-mouse Fcγ fragment antibody (Jackson ImmunoResearch, 115-005-008) was coated in 96-well plates at 100 μl / well and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1xPBST, blocked with 1% BSA in 1xPBST, and incubated at 37°C for 1 hour. Following incubation, the plates were washed three times with 1xPBST, and 100 μl of 2 μg / ml human MerTK ECD mFc protein was added to each well and incubated at 37°C for 1 hour. After washing as above, 50 μl of a mixture of serially diluted antibody and 50 μl of recombinant human GAS6 his protein (Novoprotein, Cat#C01W) at a final concentration of 1.7 μg / ml was added to each well and incubated at 37°C for 1 hour. Alternatively, add 50 μl of serially diluted antibody and 50 μl of recombinant human Protein S his protein (R&D, Cat# 9489-PS-100) at a final concentration of 4 μg / ml to each well and incubate at 37°C for 1 hour. Genentech's patented (WO2020214995A1) antibody h10F7.v16 at a final concentration of 10 μg / ml serves as a positive control, and hIgG1 LALA at a final concentration of 10 μg / ml serves as a negative control. A mixture of 50 μl of 1% BSA and 50 μl of recombinant human GAS6 his protein at a final concentration of 1.7 μg / ml represents the maximum signal (Max). Wells containing only 1% BSA exhibit the lowest absorbance (Mini). After incubation and washing, goat anti-6xhis HRP antibody (abcam, Cat#ab1187) diluted 1:5000 was added and incubated at 37 degrees for 1 hour. After washing with PBST, 100 μl of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured using a Spectra M5e instrument to measure the absorbance at OD450 nm. Inhibition rate (%) = (OD450 Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100. The result is as follows Figure 7 The results showed that the humanized MerTK antibody showed specific blocking activity on the binding of GAS6-MerTK and Protein S-MerTK in this system, which was comparable to that of the control molecule.

[0153] Table 7 Summary of the blocking activity of humanized antibodies on ligand-receptor binding

[0154]

[0155] (II) Blocking assay of humanized antibodies against binding of human TULP1 and MerTK proteins

[0156] The blocking effect of humanized antibodies on the binding of human TULP1 and human MerTK proteins was assessed using an ELISA method. Specifically, 4 μg / ml TULP1 his protein (Kactus, TUP-HE101) was coated in a 96-well plate at 100 μl / well and incubated overnight at 4°C. The next day, the coated 96-well plate was washed three times with 1xPBST, blocked with 1% BSA (in 1xPBST), and incubated at 37°C for 1 hour. Following incubation, the plate was washed three times with 1xPBST, and 50 μl of a mixture of serially diluted antibodies and recombinant human MerTK mFc protein (final concentration: 20 μg / ml) was added to each well and incubated at 37°C for 1 hour. Antibody 20A77 (VH sequence as shown in SEQ ID NO: 21 in Chiome Bioscience patent application WO202363026A1, VL sequence as shown in SEQ ID NO: 22) at a final concentration of 60 μg / ml served as a positive control, indicating no inhibition of TULP1-MerTK binding. hIgG1 LALA at a final concentration of 60 μg / ml served as a negative control. A mixture of 50 μl of 1% BSA and 50 μl of recombinant human MerTK mFc protein (final concentration of 20 μg / ml) was used as the maximum signal (Max). Wells containing only 1% BSA had the lowest absorbance (Mini). After incubation and washing, goat anti-mouse Fcr fragment HRP antibody (Jackson ImmunoResearch, 115-035-164) diluted 1:5000 was added and incubated at 37 degrees for 1 hour. After washing with PBST, 100 μl of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured using a Spectra M5e instrument to measure the absorbance at OD450 nm. The relative binding rate of ligand receptor (%) = 100-(OD450 Max -OD450 Sample ) / (OD450 Max -OD450 Mini )*100. The result is as follows Figure 7 E shows that under the experimental conditions, the control antibody 20A77 did not inhibit the binding of TULP1-MerTK, and the humanized MerTK antibody 8F7 H3L1 LALA of the present invention also did not inhibit the binding of TULP1-MerTK.

[0157] Example 11 Affinity Analysis of Humanized Antibodies

[0158] Antibody affinity was assessed by measuring kinetic binding using Octet Red 96 (Forte Bio, Serial No. FB-50482). Humanized antibodies were captured using an AHC sensor (Forte Bio, REF Num: 18-5060) and then loaded with recombinant human MerTK his protein (2-fold dilution, 100 nM to 1.56 nM) or recombinant monkey MerTK his protein (Acrobiosystems, cat# MEK-H52H6, 2-fold dilution, 50 nM to 0.78 nM). Association constants (Kon) and dissociation constants (Koff) were calculated using Octet Red 96 analysis software using a 1:1 binding model. The equilibrium dissociation constant (KD) (Koff / Kon ratio) was then calculated. The results are shown in Table 8. Although the KD of the final humanized version of 8F7, H3L1, is greater than that of the pre-PTM site removal version, H1L1, considering the limitations of the measurement machine and the combined binding curve results (not shown), it is speculated that the theoretical affinity of the two is not significantly different. The two humanized antibodies of 3E6 have comparable affinities.

[0159] Table 8 Affinity results of humanized antibodies to human-monkey MerTK

[0160]

[0161] Example 12 Evaluation of the Effect of Humanized Antibodies on Phosphorylation of AKT, a Downstream Signal of MerTK

[0162] The effects of humanized antibodies on Gas6-MerTK-activated AKT phosphorylation were evaluated using Colo699 tumor cells (ATCC, Cat#TIB-67), which naturally express MerTK. Colo699 cells were harvested by trypsinization, centrifuged, and resuspended in DMEM medium supplemented with 10% FBS. After counting, the cells were adjusted to a density of 5E5 / ml and plated at 100 μl per well in a 96-well flat-bottom plate (Costar, Cat.# 3959). The plates were incubated overnight at 37°C in a 5% CO2 incubator. The next day, the plates were washed twice with serum-free RPMI1640 medium, and 200 μl of serum-free RPMI1640 medium was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. After serum-free incubation, humanized MerTK antibody was added at final concentrations of 40 μg / ml, 8 μg / ml, and 1.6 μg / ml (50 μl / well), with triplicates for each concentration point. The cells were incubated for 1 hour at 37°C in a 5% CO2 incubator. Subsequently, human Gas6 His protein (R&D, Cat#885-GSB) diluted in serum-free RPMI1640 medium was added at a final concentration of 40 μg / ml (50 μl / well), and the cells were incubated for 15 minutes at 37°C in a 5% CO2 incubator. Genentech's proprietary antibody h10F7.v16 was used as a positive control, and hIgG1 LALA was used as a negative control. After incubation, changes in AKT phosphorylation were detected using the TR-FRET method in the pAKT (Ser473) kit (Bioauxilium, KIT-AKTS473P-100) according to the manufacturer's instructions. First, gently remove the culture supernatant from the wells of the culture plate and immediately add 50 μl of lysis buffer to each well. Incubate at room temperature with shaking for 30 minutes. Pipet to homogenize the solution, and transfer 15 μl / well of the lysate to a white detection plate (Cisbio, Ref: 66PL96025). Next, add 5 μl of a 4x concentration (vol / vol) antibody mixture to each well and incubate at room temperature in the dark for 4 hours. Fluorescence emission at wavelengths of 665 nm and 615 nm is read on a Spectra M5e instrument. The HTRF ratio is calculated based on the kit's principle: Signal 665 nm / Signal 615 nm x 10. 4Wells stimulated with only Gas6 his protein (Gas6 only) showed the highest AKT phosphorylation levels, i.e., the highest HTRF ratio. AKT phosphorylation was inhibited after incubation with the antibody. Phosphorylation levels in cells incubated with serum-free medium (no Gas6) represent the baseline for AKT phosphorylation. pATK activity was then calculated based on the HTRF ratio: pAKT activity (%) = HTRF ratio (sample) / HTRF ratio (Gas6 only).

[0163] The results are as follows Figure 8 Humanized antibodies 8F7 H3L1 and 3E6 H8L7 both specifically inhibited AKT phosphorylation. Humanized antibody 8F7 H3L1 inhibited pAKT activity by approximately 60% at all three concentrations, comparable to the activity of the proprietary antibody h10F7.v16. Humanized antibody 3E6 H8L7 exhibited dose-dependent inhibition of pAKT activity, with 50%, 40%, and 30% inhibition at 40 μg / ml, 8 μg / ml, and 1.6 μg / ml, respectively. Compared to treatment with medium alone, pAKT activity after treatment with the control antibody h10F7.v16 and humanized antibody 8F7 H3L1 remained at baseline levels, indicating that the antibodies completely inhibited Gas6-his-induced AKT phosphorylation in colo699 cells.

[0164] Example 13 Humanized Antibody Epitope Binding and Specific Binding Analysis

[0165] MerTK has four distinct extracellular domains. It has been reported that different antibody binding sites can lead to varying functions. Rabbit anti-epitope analysis results in Example 6 showed that rb8F7 and rb3E6 belong to different epitopes. ELISA was used to further identify the specific binding sites of the two antibodies. Non-specific binding of the MerTK antibody to Axl and Tyro3, ​​members of the TAM receptor tyrosine kinase family, was also evaluated. Specifically, 1 μg / ml recombinant human MerTK IG1-2 his protein (Q12866 Pro81-Ser273, SEQ ID NO: 5), human MerTK IG1 hFc protein (Q12866 Gly76-Gly195, SEQ ID NO: 6), human MerTK IG2 hFc protein (SEQ ID NO: 7), human MerTK FNIII-1 hFc protein (SEQ ID NO: 8), and human MerTK FNIII-2 hFc protein (SEQ ID NO: 9), as well as human Axl his protein (AcroBiosystems, AXL-H5226), and human TYRO3 his protein (Biorbyt, orb55941) were coated in 96-well plates at 100 μl / well and incubated overnight. The next day, the coated 96-well plate was washed three times with 1xPBST, then blocked with 1% BSA (prepared in 1xPBST) and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with 1xPBST, and 10 μg / ml of purified antibody was added and incubated at 37°C for 1 hour. After incubation and washing, a 1:5000 dilution of goat anti-human IgG Fcγ-specific HRP antibody (Jackson ImmunoResearch, Cat# 109-035-098) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μl of TMB substrate (Thermo Fisher, REF Num: 34029) was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were measured at OD450nm using a Spectra M5e instrument.Antibody z10 specifically binds to the IG1 domain (sequence from RGENIX patent# WO2016106221 A1, VH sequence as shown in SEQ ID NO:105, VL sequence as shown in SEQ ID NO:106). Antibody 2D9 (sequence from BMS patent# WO2020076799 A1, VH sequence as shown in SEQ ID NO: 253, VL sequence as shown in SEQ ID NO: 254) specifically binds to the IG2 domain as positive controls. Human Axl antibodies (R&D, AF154) and human Tyro3 antibodies (R&D, MAB859) specifically bind to Axl and Tyro3, ​​respectively, as positive controls. hIgG1 LALA, mIgG1 (abcam, cat# ab170190), and goat IgG (R&D, AB-108-C) served as negative controls. The results are shown in the table below. Figure 9 The results showed that 8F7 H3L1 bound to both the MerTK IG1-2 his protein and the human MerTK IG1hFc protein, indicating that its epitope is located in the IG1 domain. However, 3E6 H8L7 did not specifically bind to any of the tested proteins, suggesting that it may bind to a conformational epitope or to an epitope located between different domains.

[0166] Example 14 Evaluation of the efficacy of MerTK humanized antibody in mice

[0167] The mouse MC38 colon cancer cell model was used to evaluate the inhibitory effect of anti-MerTK antibodies on tumor growth. MC38 cells in the logarithmic growth phase were taken, resuspended in PBS, and the cells were counted and the cell concentration was adjusted to 1.0×10 7 / mL; the cell suspension was inoculated subcutaneously in the right flank of C57-HuMerTK mice using a 1 mL syringe, 100 μL per mouse, and approximately 1.0×10 6 When the average tumor volume reached 86 mm 3 At the same time, mice with moderate tumor volumes were selected and randomly assigned to experimental groups according to tumor volume, with 5 mice in each group. Drug administration began on the day of grouping. The specific dosing schedule is shown in the table below:

[0168] Group Test substance quantity Dosage (mg / kg) Route of administration Dosing frequency Number of doses 1 hIgG1 5 20 ip BIW 6 4 8F7 H3L1 5 20 ip BIW 6

[0169] The mice were weighed on the day of administration and before each administration, and the tumor size was measured twice a week. The experiment was terminated or the tumor size exceeded 2000 mm. 3 The animals were euthanized and the tumors were removed and weighed. Figure 10Results showed that treatment with the antibody 8F7 H3L1 inhibited tumor growth in mice compared to the control, with a tumor growth inhibition rate (TGI) of approximately 56%, indicating that inhibiting MerTK activity can have an anti-tumor effect. The effects of combined treatment with a MerTK antibody and a PD-1 antibody on tumor growth were also investigated in this model. However, due to the high dose of the PD-1 antibody used, the effect of the combined treatment was too significant to demonstrate the efficacy of the combined treatment, and the results are not presented here. Studies have shown that MerTK signaling can induce upregulation of PD-L1 expression in tumor cells and PD-1 expression in T cells (Kasikara et al., 2017; Lee-Sherick et al., 2018). Therefore, it is speculated that the combination of a MerTK antibody and a low-dose PD-1 antibody would significantly inhibit tumor growth compared to either agent alone. This finding requires further verification.

[0170] Figure 10 To evaluate the tumor growth inhibitory activity of MerTK humanized antibody, Figure 10 A is the effect of antibody treatment on mouse tumor growth. Compared with the results of control antibody treatment, 8F7 H3L1 antibody treatment has an approximately 56% tumor growth inhibition rate, p=0.13. Figure 10 B is the result of weighing the tumor of each group of mice. Compared with the hIgG1 treatment, the mice treated with 8F7 H3L1 antibody had a tumor weight inhibition rate of about 50% (IR=(W C -W T ) / W C ×100%, where WC represents the tumor weight of the control group; WT represents the tumor weight of the treatment group), p=0.1. Figure 10 C / D shows the tumor inhibition rate of each mouse in the G4 / G1 group. Compared with G1, the tumor growth of one of the five mice in the G4 group was almost completely inhibited. Figure 10 E is the weight change of the two groups of mice. There was no obvious weight change of the mice after drug treatment.

[0171] In summary, the MerTK antibody 8F7 H3L1 has certain tumor inhibitory activity against MC38 tumors in C57-HuMerTK mice. More sufficient data are needed to further verify its tumor inhibitory effect in more mice or other models.

[0172] name SEQ ID NO sequence human mertk ECD mFc 1 AITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNAASVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK human mertk ECD his 2 AITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNAASGSHHHHHH mouse mertk ECD his 3 MVLAPLLLGLLLLPALWSGGTAEKWEETELDQLFSGPLPGRLPVNHRPFSAPHSSRDQLPPPQTGRSHPAHTAAPQVTSTASKLLPPVAFNHTIGHIVLSEHKNVKFNCSINIPNTYQETAGISWWKDGKELLGAHHSITQFYPDEEGVSIIALFSIASVQRSDNGSYFCKMKVNNREIVSDPIYVEVQGLPYFIKQPESVNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEKPERSPSVLTVPGLTETAVFSCEAHNDKGLTVSKGVHINIKVIPSPPTEVHILNSTAHSILVSWVPGFDGYSPLQNCSIQVKEADRLSNGSVMVFNTSASPHLYEIQQLQALANYSIAVSCRNEIGWSAVSPWILASTTEGAPSVAPLNITVFLNESNNILDIRWTKPPIKRQDGELVGYRISHVWESAGTYKELSEEVSQNGSWAQIPVQIHNATCTVRIAAITKGGIGPFSEPVNIIIPEHSKVDYAPSSTPAPGNTDSMFASGSHHHHHH human mertk Fulllength 4 MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFADDSSEGSEVLM human mertk IG1-IG2his 5 PQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSASGSHHHHHH human mertk IG1 hFc 6 GNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAIQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGEPCSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK human mertk IG2 hFc 7 LPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK human mertk FNIII-1hFc 8 PPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK human mertk FNIII-2hFc 9 LNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK rb8F7 HC 10 QSVEESGGRLVTPGTPLTLTCTASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTISKTSTTVDLRITSPTTEDTATYFCARDRVIGLYRLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK rb8F7 VH 11 QSVEESGGRLVTPGTPLTLTCTASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTISKTSTTVDLRITSPTTEDTATYFCARDRVIGLYRLDLWGQGTLVTVSS rb8F7 LC 12 AAVLTQTPSPVSAAVGGTVSISCQSSESVYNNNYLSWYQQKPGQPPKLLIYGAANLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYKSDSDNGFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC rb8F7 VL 13 AAVLTQTPSPVSAAVGGTVSISCQSSESVYNNNYLSWYQQKPGQPPKLLIYGAANLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGGYKSDSDNGFGGGTEVVVK rb8F7 HCDR1 14 TYGMT rb8F7 HCDR2 15 MINPLDSPYYATWAKG rb8F7 HCDR3 16 DRVIGLYRLDL rb8F7 LCDR1 17 QSSESVYNNNYLS rb8F7 LCDR2 18 GAANLAS rb8F7 LCDR3 19 AGGYKSDSDNG hz8F7 H1L1 HC 20 QQLLESGGGLVQPGGSLRLSCAASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTFSIDNSKNTVYLQMTSLRAEDTAVYFCARDRVIGLYRLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hz8F7 H1L1 VH 21 QQLLESGGGLVQPGGSLRLSCAASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTFSIDNSKNTVYLQMTSLRAEDTAVYFCARDRVIGLYRLDLWGQGTLVTVSS hz8F7 H1L1 LC 22 AQLTQSPSTLSASVGDRVTITCQSSESVYNNNYLSWYQQKPGKAPKLLIYGAANLASGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCAGGYKSDSDNGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hz8F7 H1L1 VL 23 AQLTQSPSTLSASVGDRVTITCQSSESVYNNNYLSWYQQKPGKAPKLLIYGAANLASGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCAGGYKSDSDNGFGGGTKVEIK hz8F7 H3L1 HC 24 QQLLESGGGLVQPGGSLRLSCAASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTFSIDQSKNTVYLQMTSLRAEDTAVYFCARDRVIGLYRLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hz8F7 H3L1 VH 25 QQLLESGGGLVQPGGSLRLSCAASEFSLSTYGMTWVRQAPGKGLEWIGMINPLDSPYYATWAKGRFTFSIDQSKNTVYLQMTSLRAEDTAVYFCARDRVIGLYRLDLWGQGTLVTVSS rb3E6 HC 26 QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKTSSTSVDLKMTSLTTEDTATYFCARYDSSGYHALWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK rb3E6 VH 27 QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKTSSTSVDLKMTSLTTEDTATYFCARYDSSGYHALWGQGTLVTVSS rb3E6 LC 28 AQVLTQTPASVSAAVGGTVTINCQASQSVYNNKNSAWYQQKPGQPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCYSADCIAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC rb3E6 VL 29 AQVLTQTPASVSAAVGGTVTINCQASQSVYNNKNSAWYQQKPGQPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCYSADCIAFGGGTEVVVK rb3E6 HCDR1 30 NYAMS rb3E6 HCDR2 31 VTGGGDTVYCASWAKG rb3E6 HCDR3 32 YDSSGYHAL rb3E6 LCDR1 33 QASQSVYNNKNSA rb3E6 LCDR2 34 SASTLAS rb3E6 LCDR3 35 QGEFSCYSADCIA hz3E6 H2L2 HC 36 EVQLVESGGGLVQPGPSLRLSCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKDDSKSIVYLQMNSLKTEDTATYFCARYDSSGYHALWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hz3E6 H2L2 VH 37 EVQLVESGGGLVQPGPSLRLSCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKDDSKSIVYLQMNSLKTEDTATYFCARYDSSGYHALWGQGTLVTVSS hz3E6 H2L2 LC 38 AQVLTQSPSSLSASVGDRVTITCQASQSVYNNKNSAWYQQKPGKPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISSLQPDDAATYYCQGEFSCYSADCIAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hz3E6 H2L2 VL 39 AQVLTQSPSSLSASVGDRVTITCQASQSVYNNKNSAWYQQKPGKPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISSLQPDDAATYYCQGEFSCYSADCIAFGGGTKVEIK hz3E6 H8L7 HC 40 EVQLVESGGGLVQPGPSLRLSCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKDDTKSIVYLQMNTLKTEDTATYFCARYDTSGYHALWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hz3E6 H8L7 VH 41 EVQLVESGGGLVQPGPSLRLSCTVSGFSLSNYAMSWVRQAPGKGLEWIGVTGGGDTVYCASWAKGRFTISKDDTKSIVYLQMNTLKTEDTATYFCARYDTSGYHALWGQGTLVTVSS hz3E6 H8L7 LC 42 AQVLTQSPSSLSASVGDRVTITCQASQSVYNNKNTAWYQQKPGKPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISSLQPDDAATYYCQGEFSCYSADCIAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC hz3E6 H8L7 VL 43 AQVLTQSPSSLSASVGDRVTITCQASQSVYNNKNTAWYQQKPGKPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISSLQPDDAATYYCQGEFSCYSADCIAFGGGTKVEIK Ab 8N42 VH 48 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSIYYWSWIRQPPGKGLELIGEINDEGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGTGDIHAFDIWGQGTMVTVSS Ab 8N42 VL 49 DIQMTQSPSSLSASVGDRVTITCRASQGISKWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPWTFGQGTKVEIK h10F7.v16 VH 50 EVQLVESGGGLVQPGGSLRLSCAVSGFSLSGYAMSWVRQAPGKGLEYIGVISSSGSSYYPSWAKGRFTISKDNSKNTLYLQMGSLRAEDMAVYYCARVQFYVGYAVYGYGIIDRLDLWGQGTLVTVSS h10F7.v16 VL 51 DIVLTQSPDSLAVSLGERATINCQSSKSVYNNNWLSWYQQKPGQPPKLLIYRASTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCAGGYSSSSSANAFGGGTKVEIK h13B4.v16 VH 52 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS h13B4.v16 VL 53 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK 20A77 VH 54 QVQLVQSGAEVKKPGSSVKVSCKASGNTFTSYWMHWVRQAPGQGLEWIGNINPSSGGTNYNEKFKSRVTITVDKSTSTAYMELSSLRSEDTAVYYCARLNYYGFDGFAFWGQGTLVTVSS 20A77 VL 55 DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQYDEFPFTFGGGTKVEIK z10 VH 56 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTFTTDTSTSTAYMELRSLRSDDMAVYYCARKSTVVSRYFDVWGQGTTVTVSS z10 VL 57 DIQMTQSPSFLSASVGDRVTITCKASQDVGDAVTWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRSYPLTFGQGTKLEIK 2D9 VH 58 QVQLKESGPGLVAPSQSLSITCTVSGISLSSFAISWVRQPPGKGLEWLGVIWTGGGTDYNSALKSRLTISKDTSKNQVFLKMNSLQTDDTARYYCASHWYLDVWGTGTTVTVSS 2D9 VL 59 QAVVTQESALTTSPGETVTLTCRSSTGAVSTSNYANWVQEKPDHLFTGLIGGANSRAPGIPARFSGSLIGDKAALTITGAQTEDEAIYFCALWFSNHWVFGGGTKLTVL hz3E6 H8L7 HCDR3 60 YDTSGYHAL hz3E6 H8L7 LCDR1 61 QASQSVYNNKNTA

[0173] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-human MerTK antibody, characterized in that The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 14, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 15, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 16; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 17, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 18, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 19; Or, the heavy chain variable region includes HCDR1 with an amino acid sequence as shown in SEQ ID NO: 30, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 60; the light chain variable region includes LCDR1 with an amino acid sequence as shown in SEQ ID NO: 33 or SEQ ID NO: 61, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

35.

2. The anti-human MerTK antibody according to claim 1, wherein The antibody is selected from the following: (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39; Or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43; (2) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 13; Or, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 21; the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 23; Or, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 25; the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 23; Or, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 27; the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 29; Or, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 37; the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 39; Alternatively, the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 41; and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

43.

3. The anti-human MerTK antibody according to claim 2, wherein The antibody is selected from one or more of the following: (1) Full-length antibody, Fab, F(ab)2, Fv; (2) Monoclonal antibodies or polyclonal antibodies; (3) Bispecific antibodies or multispecific antibodies.

4. The anti-human MerTK antibody according to claim 3, wherein The antibody is a full-length antibody, which also includes at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE constant regions and their related variants.

5. The anti-human MerTK antibody according to claim 4, wherein The heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 10; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 12; Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 20; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22; Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 24; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22; Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 26; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 28; Or, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 36; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 38; Alternatively, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 40; the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

42.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human MerTK antibody according to any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 6; Preferably, the recombinant expression vector is any one or a combination of at least two of a viral vector, a plasmid, a DNA fragment, or an RNA; More preferably, the recombinant expression vector is a plasmid.

8. A transformant, characterized in that Containing the recombinant expression vector according to claim 7 in a host cell; Preferably, the host cell is a mammalian cell.

9. A method for preparing an anti-human MerTK antibody, characterized in that: The method comprises culturing the transformant according to claim 8, and obtaining an anti-human MerTK antibody from the culture.

10. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the anti-human MerTK antibody according to any one of claims 1 to 5.

11. A genetically modified cell, characterized in that It comprises the chimeric antigen receptor as claimed in claim 10; preferably, the host of the genetically modified cell is a T cell or a NK cell.

12. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent or a label, and the anti-human MerTK antibody according to any one of claims 1 to 5.

13. A pharmaceutical composition or a kit containing the same, characterized in that: The pharmaceutical composition or a kit containing the same comprises the anti-human MerTK antibody according to any one of claims 1 to 5, the genetically modified cell according to claim 11, or the antibody-drug conjugate according to claim 12, and at least one of a pharmaceutically acceptable carrier, excipient, or diluent; Preferably, the pharmaceutical composition or the kit containing the same further contains one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

14. Use of the anti-human MerTK antibody according to any one of claims 1 to 5, or the genetically modified cell according to claim 11, or the antibody-drug conjugate according to claim 12, or the pharmaceutical composition according to claim 13, or a kit containing the same, in the preparation of a medicament for preventing and / or treating tumors, wherein the tumor is a MerTK-overexpressing tumor; Preferably, the tumor is one of the following: B or T lymphocytic leukemia, gastric cancer, NSCLC, colon cancer, prostate cancer, melanoma, glioblastoma.

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