Anti-MerTK antibodies and uses thereof

Abstract

The present disclosure provides anti-MerTK antibodies, variants and humanized forms thereof. The newly disclosed antibodies exhibit high affinity for the MerTK protein and are useful in the treatment of cancer, particularly solid tumors.

Description

Background Art

[0001] Currently, most cancer immuno-oncology therapies focus on modulating the activity of adaptive immune cells by blocking inhibitory pathways (including checkpoint molecules) or redirecting adaptive immune cells (especially T cells) to fight tumors through the binding of tumor-associated antigens (TAAs). However, data obtained from numerous studies in cancer mouse models and cancer patients provide convincing evidence that specific innate immune cell types, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), mainly function as exogenous tumor suppressor mechanisms, resulting in limited clinical benefits of adaptive immune cell-based therapies. Targeting the innate immune system may complement adaptive immuno-oncology therapies to achieve durable anti-tumor responses.

[0002] Macrophages in the innate immune system are a collection of multiple cell types that play a variety of functional roles under homeostatic and pathological conditions. M1 macrophages are a classically activated macrophage that plays a pro-inflammatory role to clear intracellular pathogens. In contrast, M2 macrophages are alternatively activated macrophages that contribute to tissue repair and efferocytosis. Macrophages are professional phagocytes that are highly specialized to clear dying or dead cells and cell debris that are produced in large quantities under normal physiological conditions. In addition, an extremely abundant number of macrophages has been detected in many types of solid tumors. Macrophages in the tumor microenvironment are called tumor-associated macrophages (TAMs), which usually promote the initiation and proliferation of cancer cells, accelerate angiogenesis, and inhibit anti-tumor immunity, thereby promoting tumor progression and metastasis. There is increasing evidence that TAMs may be one of the reasons for the relatively low response rate of T cell-based therapies. In solid tumors, uncontrolled tumor growth is often accompanied by increased cell death due to hypoxia and metabolic stress. In order to escape immune surveillance, tumors take advantage of the non-immunogenic nature of apoptosis. TAMs actively remove dying tumor cells while reducing the production of inflammatory cytokines that would alert the immune system.

[0003] MerTK (Mer proto-oncogene tyrosine protein kinase) has been shown to play a role in the clearance of apoptotic cells. MerTK is a member of the TYRO3 / AXL / MER (TAM) receptor kinase family and encodes a transmembrane protein that contains two fibronectin type III domains, two Ig-like C2-type (immunoglobulin-like) domains, and a tyrosine kinase domain. MerTK expression has been observed to be higher on M2 macrophages than on M1 macrophages. MerTK helps to clear dying or damaged cells that display an “eat me” signal (i.e., phosphatidylserine, PtdSer) on the cell surface, a process that requires the help of the bridge molecule growth arrest-specific protein 6 (Gas6) or protein S. Macrophages expressing MerTK engulf apoptotic cells by efferocytosis. In tumors, uncontrolled proliferation leads to increased apoptosis of cancer cells, but TAMs rely on MerTK to clear dying cells, which may inhibit immune activation.

[0004] Blocking MerTK leads to accumulation of apoptotic cells within tumors and triggers a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibodies stimulates T cell activation and improves the efficacy of anti-PD-1 and anti-PD-L1 therapies. Therefore, blocking MerTK enhances tumor immunogenicity and enhances anti-tumor immunity, and also provides a therapeutic approach to enhance tumor immunogenicity and improve cancer immunotherapy. Summary of the invention

[0005] In various embodiments, the present disclosure provides antibodies and antigen-binding fragments specific for human MerTK protein. One embodiment provides an antibody or an antigen-binding fragment thereof, which is specific to human Mer proto-oncogene tyrosine protein kinase (MerTK) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the following amino acid sequences: SEQ ID NO: 25-30; SEQ ID NO: 59-64; SEQ ID NO: 65-70; SEQ ID NO: 31-33, 28-29, 34; SEQ ID NO: 35-40; SEQ ID NO: 41-46; SEQ ID NO: 47-52; SEQ ID NO: 53-58; SEQ ID NO: 71, 66, 72, 73, 69, 74; SEQ ID NO: 65, 75, 67, 76, 69, 77; SEQ ID NO: 71, 78-79, 73, 69, 74; or SEQ ID NO: 65-66, 80-81, 69-70.

[0006] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:25; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:26; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:29; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:30.

[0007] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 82-87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 89-91.

[0008] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO: 83, and the VL comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 83, and the VL comprises the amino acid sequence of SEQ ID NO: 91.

[0009] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:59; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:60; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:61; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:62; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:63; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:64.

[0010] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 102, and 104, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 106. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 102, and the VL comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 104, and the VL comprises the amino acid sequence of SEQ ID NO: 106.

[0011] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:65; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:66; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:67; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:68; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:69; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:70.

[0012] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 93-96, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 98-100. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 95, and the VL comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 96, and the VL comprises the amino acid sequence of SEQ ID NO: 99.

[0013] In some embodiments, the antibody or fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

[0014] In one embodiment, a multispecific antibody is also provided, which comprises the antigen-binding fragment of the present disclosure and one or more antibodies or antigen-binding fragments having binding specificity to a target antigen other than MerTK.

[0015] Still further provided is a chimeric antigen receptor (CAR), which comprises the antigen binding fragment of the present disclosure, a transmembrane domain, a co-stimulatory domain and a CD3ξ intracellular domain.

[0016] Also provided are one or more polynucleotides encoding antibodies or antigen-binding fragments thereof or CARs disclosed herein. In some embodiments, the polynucleotides are one or more mRNAs. In some embodiments, the mRNA is chemically modified.

[0017] In one embodiment, a method for treating cancer or inflammatory conditions in a patient in need is also provided, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof or CAR disclosed herein. In some embodiments, the cancer is a solid tumor, such as bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer and thyroid cancer.

[0018] In some embodiments, the inflammatory disorder is selected from the group consisting of: Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It was shown that all the anti-MerTK antibodies tested could effectively bind to the human MerTK protein.

[0020] Figure 2 It was shown that all the tested anti-MerTK antibodies could effectively bind to the cynomolgus monkey MerTK protein.

[0021] Figure 3 It was shown that most of the tested MerTK chimeric antibodies had higher maximal binding capacity and binding efficacy to human MerTK expressed on CHO-K1 cells compared to the benchmark antibodies Ab2000-A7, M6 and h13B4.v16.

[0022] Figure 4It was shown that some of the tested MerTK chimeric antibodies had higher binding potency to human MerTK expressed on SK-MEL-5 cells compared to the benchmark antibodies Ab2000-A7, M6 and h13B4.v16.

[0023] Figure 5 It was shown that all of these antibodies were able to effectively inhibit human MerTK binding to human Gas6 expressed on cells.

[0024] Figure 6 It was shown that some of the tested MerTK antibodies had higher blocking efficiency in efferocytosis than the benchmark antibody h13B4.v16.

[0025] Figure 7 It was shown that all humanized antibodies tested had binding potency to human MerTK protein comparable to that of the chimeric antibodies.

[0026] Figure 8 It was shown that some of the humanized antibodies tested had comparable binding activity to their parental chimeric counterparts to human MerTK expressed on CHOK1 cells. DETAILED DESCRIPTION definition

[0027] It should be noted that the term "a" or "an" entity refers to one or more of said entities; for example, "an antibody" should be understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0028] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody as well as any antigen-binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0029] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment can bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0030] The term antibody encompasses a wide variety of polypeptide classes that can be distinguished by biochemical methods. Those skilled in the art will recognize that the heavy chain is classified as γ, μ, α, δ, or ε, with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, IgG, IgM, IgA, IgG, or IgE, respectively.

[0031] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. In view of the present disclosure, those skilled in the art can easily identify modified versions of these classes and isotypes, and accordingly, these modified versions are encompassed within the scope of the present disclosure. All immunoglobulin classes are obviously encompassed within the scope of the present disclosure, and the following discussion will generally be directed to immunoglobulin molecules of the IgG class. Regarding IgG, a standard immunoglobulin molecule includes two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. These four chains are typically connected in a "Y" configuration by disulfide bonds, wherein the light chain wraps around the heavy chain from the opening of the "Y" and extends to the variable region.

[0032] The antibodies, antigen-binding polypeptides, variants or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies for LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecule disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0033] As used herein, the term "chimeric antibody" should be understood as any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is of human origin.

[0034] The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, these antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In some embodiments, the variable region may be of condricthoid origin (e.g., from sharks).

[0035] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be produced by combining polynucleotides that do not normally occur together.

[0036] Hybridoma technology can be performed under different "stringency" conditions. Generally speaking, low stringency hybridization reactions are performed at about 40°C in a solution of about 10x SSC or equivalent ionic strength / temperature. Medium stringency hybridizations are usually performed at about 50°C in about 6x SSC, and high stringency hybridization reactions are usually performed at about 60°C in about 1x SSC. It is well known to those skilled in the art that hybridization reactions can also be performed under "physiological conditions". Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg2+ concentrations typically found in cells. Anti-MerTK antibody

[0037] As demonstrated in the attached experimental examples, the inventors were able to generate anti-MerTK antibodies 10F7D9, 85H8D5, 216G3D6, 247E5A8, 252C12B10, 252H9D5, 254B4D9, 265F11B5, 276C2D1, 280C6A3, 293C2B7 and 300A5A3 (Table 1). Equally important, many of these antibodies exhibited higher binding affinity to human MerTK proteins expressed on cells than benchmark antibodies (including M6, Ab2000-A7 and h13B4.v16 (as disclosed in WO 2019084307A1, WO 2016106221A1 and WO 2020214995A1)). Moreover, some of these antibodies exhibited higher ligand binding blocking activity and efferocytosis inhibition efficiency than these benchmark antibodies.

[0038] According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or its antigen-binding fragment has binding specificity to human MerTK protein. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VLCDR3.

[0039] In some embodiments, antibodies or antigen-binding fragments derived from antibody 10F7D9 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 25; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 27; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 29; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30.

[0040] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 1 and 82-87. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 2 and 89-91.

[0041] In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 1 and 82-87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 1 and 82-87, while retaining the corresponding VH CDRs. In some embodiments, the VL comprises an amino acid sequence of any one of SEQ ID NOs: 2 and 89-91, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 2 and 89-91, while retaining the corresponding VL CDRs.

[0042] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 83, and VL comprises the amino acid sequence of any one of SEQ ID NO: 2 and 89-91. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 83, and VL comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 83, and VL comprises the amino acid sequence of SEQ ID NO: 91.

[0043] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 1 and 82-87, and VL comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 82-87, and VL comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 1 and 82-87, and VL comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 82-87, and VL comprises the amino acid sequence of SEQ ID NO: 91.

[0044] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 10F7D9. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 10F7D9 for binding to MerTK.

[0045] In some embodiments, antibodies or antigen-binding fragments derived from antibody 85H8D5 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 31; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 32; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 33; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 28; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 29; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 34.

[0046] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 3. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 4, while retaining the corresponding VL CDRs.

[0047] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK that 85H8D5 binds to. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 85H8D5 for binding to MerTK.

[0048] In some embodiments, antibodies or antigen-binding fragments derived from antibody 216G3D6 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 35; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 36; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 37; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 38; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 39; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40.

[0049] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 5. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6, while retaining the corresponding VL CDRs.

[0050] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK that 216G3D6 binds to. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 216G3D6 for binding to MerTK.

[0051] In some embodiments, antibodies or antigen-binding fragments derived from antibody 247E5A8 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 41; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 42; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 45; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.

[0052] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 7. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8, while retaining the corresponding VL CDRs.

[0053] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 247E5A8. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 247E5A8 for binding to MerTK.

[0054] In some embodiments, antibodies or antigen-binding fragments derived from antibody 252C12B10 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 47; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 48; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 49; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 51; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52.

[0055] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 9. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10, while retaining the corresponding VL CDRs.

[0056] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 252C12B10. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 252C12B10 for binding to MerTK.

[0057] In some embodiments, antibodies or antigen-binding fragments derived from antibody 252H9D5 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 53; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 55; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 56; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 57; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 58.

[0058] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 11. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 11, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12, while retaining the corresponding VL CDRs.

[0059] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK that 252H9D5 binds to. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 252H9D5 for binding to MerTK.

[0060] In some embodiments, antibodies or antigen-binding fragments derived from antibody 254B4D9 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 59; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 60; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 61; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 62; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 63; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 64.

[0061] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 13, 102, and 104. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 14 and 106.

[0062] In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 13, 102, and 104, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 13, 102, and 104, while retaining the corresponding VH CDRs. In some embodiments, the VL comprises an amino acid sequence of any one of SEQ ID NOs: 14 and 106, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 14 and 106, while retaining the corresponding VL CDRs.

[0063] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 102, and VL comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 104, and VL comprises the amino acid sequence of SEQ ID NO: 106.

[0064] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 254B4D9. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 254B4D9 for binding to MerTK.

[0065] In some embodiments, antibodies or antigen-binding fragments derived from antibody 265F11B5 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 65; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 66; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70.

[0066] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 15 and 93-96. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NOs: 16 and 98-100.

[0067] In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 15 and 93-96, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 15 and 93-96, while retaining the corresponding VH CDRs. In some embodiments, the VL comprises an amino acid sequence of any one of SEQ ID NOs: 16 and 98-100, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs: 16 and 98-100, while retaining the corresponding VL CDRs.

[0068] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 95, and VL comprises the amino acid sequence of any one of SEQ ID NO: 16 and 98-100. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 95, and VL comprises the amino acid sequence of SEQ ID NO: 99.

[0069] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 96, and VL comprises the amino acid sequence of any one of SEQ ID NO: 16 and 98-100. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 96, and VL comprises the amino acid sequence of SEQ ID NO: 99.

[0070] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 15 and 93-96, and VL comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NOs: 93-96, and VL comprises the amino acid sequence of SEQ ID NO: 99.

[0071] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 265F11B5. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 265F11B5 for binding to MerTK.

[0072] In some embodiments, antibodies or antigen-binding fragments derived from antibody 276C2D1 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 66; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 72; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74.

[0073] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 17. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 17, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18, while retaining the corresponding VL CDRs.

[0074] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK that 276C2D1 binds to. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 276C2D1 for binding to MerTK.

[0075] In some embodiments, antibodies or antigen-binding fragments derived from antibody 280C6A3 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 65; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 75; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 76; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 77.

[0076] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 19. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 19, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 20, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 20, while retaining the corresponding VL CDRs.

[0077] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 280C6A3. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 280C6A3 for binding to MerTK.

[0078] In some embodiments, antibodies or antigen-binding fragments derived from antibody 293C2B7 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 78; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 79; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 73; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74.

[0079] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 21. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 22. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 21, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 21, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 22, while retaining the corresponding VL CDRs.

[0080] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 293C2B7. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 293C2B7 for binding to MerTK.

[0081] In some embodiments, antibodies or antigen-binding fragments derived from antibody 300A5A3 are provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 65; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 66; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 80; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 81; VLCDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70.

[0082] Exemplary VH sequences include the amino acid sequence of SEQ ID NO: 23. Exemplary VL sequences include the amino acid sequence of SEQ ID NO: 24. In some embodiments, the VH includes the amino acid sequence of SEQ ID NO: 23, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 23, while retaining the corresponding VH CDRs. In some embodiments, the VL includes the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 24, while retaining the corresponding VL CDRs.

[0083] Thus, in some embodiments, antibodies and antigen binding fragments are also provided that bind to the same epitope on MerTK as 300A5A3. Thus, in some embodiments, antibodies and antigen binding fragments are also provided that compete with 300A5A3 for binding to MerTK.

[0084] In some embodiments, antibodies and antigen-binding fragments are also provided that include CDR sequences derived from the CDR sequences of the present disclosure, having one, two or three amino acid substitutions, deletions and / or additions.

[0085] In some embodiments, the antibody or fragment thereof is capable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody or fragment thereof is not capable of inducing antibody-dependent cellular cytotoxicity (ADCC). Multifunctional molecules

[0086] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for MerTK, such as those disclosed herein, and one or more antibodies or antigen-binding fragments specific for a second antigen.

[0087] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0088] In some embodiments, the second antigen is CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR or VEGF. In one embodiment, the second antigen is PD1. In one embodiment, the second antigen is PD-L1.

[0089] Different forms of bispecific antibodies are also provided. In some embodiments, the anti-MerTK fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0090] Also provided are bifunctional molecules including not only antibodies or antigen binding fragments. As molecules targeting tumor antigens, antibodies or antigen binding fragments (such as those described herein) with specificity for MerTK can be optionally combined with immune cytokines or ligands through peptide linkers. The immune cytokines or ligands connected include but are not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bifunctional molecules can combine immune checkpoint blocking effects with local immunomodulation at the tumor site. Chimeric Antigen Receptor

[0091] In one embodiment, a chimeric antigen receptor (CAR) is also provided, which includes an antibody or a fragment thereof disclosed herein as a targeting unit. In certain embodiments, CAR includes an antibody or a fragment thereof disclosed herein, a transmembrane domain, a costimulatory domain, and a CD3 ξ intracellular domain.

[0092] The membrane-spanning domain can be designed to be fused with the extracellular domain including an antibody or a fragment, optionally through a hinge domain. It can also be fused with an intracellular domain, such as a costimulatory domain. In certain embodiments, the membrane-spanning domain may include a natural membrane-spanning region of a costimulatory domain (e.g., a TM region of CD28T or 4-1BB used as a costimulatory domain) or a natural membrane-spanning domain of a hinge region (e.g., a TM region of CD8α or CD28T used as a hinge domain).

[0093] In some embodiments, a transmembrane domain may include a sequence that spans the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane may include a transmembrane sequence, which itself may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include one or more amino acids that extend out of the inner or outer surface of the membrane itself; such sequences may still be considered "transmembrane domains".

[0094] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker preferably between 2 and 10 amino acids in length can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplet (GSG) or alanine-alanine-alanine triplet (AAA) provide suitable linkers.

[0095] In some embodiments, CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Examples of co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2) 、CD49a(ITGA1)、CD49d(ITGA4)、CD49f(ITGA6)、CD66a(CEACAM1)、CD66b(CEACAM8)、CD66c(CEACAM6)、CD66d(CEACAM3)、CD66e(CEACAM5)、CD69(CLEC2)、CD79A(B cell antigen receptor complex-associated α chain)、CD79B(B cell antigen receptor complex-associated β chain)、CD84(SLAMF5)、CD96(Tactile)、CD 100(SEMA4D)、CD 103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1 ), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(S LAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (KG2D), CD319 (SLAMF7), CD335 (K-p46), CD336 (K-p44), CD337 (K-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF 18), inducible T cell co-stimulator (ICOS), LFA-1 (CD 1la / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0096] In some embodiments, the cytoplasmic portion of CAR also includes a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 ξ domain, or its amino acid sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a CD3 ξ domain. Polynucleotides, mRNA, and methods for expressing or preparing antibodies

[0097] The disclosure also provides polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof or CARs disclosed herein. The polynucleotides disclosed herein can encode the entire heavy chain and light chain variable region of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides disclosed herein can encode the heavy chain and light chain variable region of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0098] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell to express the antibody or fragment thereof.

[0099] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0100] In some embodiments, to prepare mRNA encoding an antibody, a DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter (e.g., T3, T7, or SP6 promoter) for in vitro transcription, followed by a desired nucleotide sequence and termination signal for mRNA encoding a desired antibody (e.g., encoding a heavy chain or a light chain).

[0101] The mRNA sequence of the desired antibody (for example, encoding heavy chain or light chain) can be determined using standard methods and incorporated into the DNA template. For example, starting from the desired amino acid sequence (for example, the desired heavy chain or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. Then, the optimization algorithm can be used to select suitable codons. Typically, the G / C content can be optimized on the one hand to achieve the highest possible G / C content, and the frequency of tRNA can be considered as much as possible according to the usage of codons on the other hand. The optimized RNA sequence can, for example, be set up and displayed by means of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stability and instability characteristics of RNA, or to calculate its region respectively.

[0102] mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. Therefore, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, uracil, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, whitinosine as well as amidophosphates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanine, 5-methylcytosine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entireties.

[0103] In certain embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are modifications to the phosphates of the backbone of the nucleotides contained in RNA that are chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications from groups composed of methylphosphonates, methylphosphoramidates, phosphoramidates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphates)), borophosphates, positively charged guanidine groups, etc., which means replacing phosphodiester bonds with other anions, cations, or neutral groups.

[0104] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides contained therein, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), ), 2'-O-alkyl oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligonucleotides and their isomers (2'-arabinocytidine 5'-triphosphate, 2'-arabinouridine 5'-triphosphate) or azidotriphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0105] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine ribonucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthine nucleoside 5'-triphosphate.

[0106] Typically, mRNA synthesis involves the addition of a "cap" at the N-terminal (5') end and a "tail" at the C-terminal (3') end. The presence of the cap is important to provide resistance to nucleases found in most eukaryotic cells. The presence of the "tail" is to protect the mRNA from exonuclease degradation.

[0107] Thus, in some embodiments, an mRNA (e.g., an mRNA encoding a heavy chain and a light chain) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylate transferase to produce a 5'5'5 triphosphate bond; then the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5)A and G(5)ppp(5')G.

[0108] Therefore, in some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) includes a 3' poly (A) tail structure. The poly A tail at the 3' end of the mRNA generally includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding heavy and light chains) includes a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of the mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail can be added to the poly-A tail or can replace the poly-A tail.

[0109] Therefore, in some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) includes 5' and / or 3' untranslated regions. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron response elements. In some embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides (e.g., about 50 to 400 nucleotides in length, about 50 to 300 nucleotides in length, about 50 to 200 nucleotides in length, or about 50 to 100 nucleotides in length).

[0110] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In a specific embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is directly or indirectly linked to the sequence encoding the heavy or light chain at the N-terminus.

[0111] The technology of the present invention can be used to deliver any antibody known in the art and antibodies that can be generated against a desired antigen using standard methods. The present invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or compositions, human or humanized antibodies, chimeric antibodies or bispecific antibodies.

[0112] Methods for preparing antibodies are well known in the art and described herein. In certain embodiments, the variable regions and constant regions of the antigen-binding polypeptides disclosed herein are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies to a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, ​​the contents of which are incorporated herein by reference in their entirety. Treatment and uses

[0113] As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0114] The present disclosure further relates to antibody-based therapies, which involve administering the antibodies or fragments of the present disclosure to patients (e.g., animals, mammals, and humans) to treat one or more disorders or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0115] The antibodies disclosed herein can also be used to treat or inhibit cancer. As mentioned above, MerTK is rarely expressed in normal adult tissues, but is highly expressed in the placenta and most common tumors, and is usually highly expressed in more than 80% of renal cancer, breast cancer, colon cancer, prostate cancer and ovarian cancer.

[0116] Thus, in some embodiments, a method of treating cancer in a patient in need thereof is provided. In one embodiment, the method requires administering to the patient an effective amount of an antibody or fragment of the disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient overexpresses MerTK.

[0117] Cell therapy, such as chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used, the cells are transduced with a vector encoding CAR or contacted with CAR (or alternatively engineered to express the anti-MerTK antibody disclosed herein), and the CAR includes the anti-MerTK antibody disclosed herein. After such contact or engineering, the cells can be introduced into cancer patients in need of treatment. Cancer patients may suffer from any type of cancer as disclosed herein. Cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0118] In some embodiments, the cells are isolated from the cancer patient's own body. In some embodiments, the cells are provided by a donor or come from a cell bank. When the cells are isolated from the cancer patient's body, adverse immune responses can be minimized.

[0119] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer.

[0120] Additional diseases or conditions associated with increased cell survival (which may be treated, prevented, diagnosed and / or predicted using the antibodies of the present disclosure or variants or derivatives thereof) include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma , osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0121] In some embodiments, the antibodies, polynucleotides or compositions disclosed herein can be used to treat inflammatory diseases or conditions. In some embodiments, the inflammatory diseases or conditions to be treated using the disclosed antibodies, fragments and compositions include one or more of the following: Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis and ulcerative colitis.

[0122] In some embodiments, the autoimmune diseases or disorders to be treated using the disclosed antibodies, fragments, and compositions include one or more of alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's granulomatosis).

[0123] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. RA typically causes warmth, swelling, and pain in the joints. The pain and stiffness usually worsen after rest. Most commonly, it affects the wrists and hands, and often the same joints on both sides of the body. The disease may also affect other parts of the body. Although the cause of rheumatoid arthritis is unknown, it is thought to involve a combination of genetic and environmental factors. The underlying mechanism involves the body's immune system attacking the joints. This causes inflammation and thickening of the joint capsule. The goals of treatment are to reduce pain, reduce inflammation, and improve a person's overall function. Pain relievers, steroids, and NSAIDs are often used to relieve symptoms. A group of drugs called disease-modifying antirheumatic drugs (DMARDs), such as hydroxychloroquine and methotrexate, can be used to try to slow the progression of the disease.

[0124] Osteoarthritis (OA) is a group of joint diseases caused by the breakdown and degeneration of joint cartilage and underlying bone. The most common symptoms are joint pain and stiffness. At first, symptoms may only occur after exercise, but over time, they may become constant. Other symptoms may include swollen joints, reduced range of motion, and, when the spine is affected, weakness or numbness in the arms and legs. Causes include previous joint injuries, abnormal joint or limb development, and genetic factors. People who are overweight, have different leg lengths, and work in jobs that put stress on their joints are at a higher risk. Osteoarthritis is thought to be caused by mechanical stress on the joints and a low-grade inflammatory process. Treatment includes exercise, minimizing stress on the joints, support groups, and pain medications.

[0125] Multiple sclerosis (MS) is a demyelinating disease in which the insulating outer layer of nerve cells in the brain and spinal cord becomes damaged. This damage interferes with the ability of the parts of the nervous system to communicate, resulting in a range of signs and symptoms including physical, mental, and sometimes psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness, sensory impairment, or difficulty with coordination. Although the cause is unknown, the underlying mechanisms are thought to be damage by the immune system or malfunction of the cells that produce myelin. There is currently no known cure for multiple sclerosis. Treatment aims to improve physical function after an episode and to prevent new episodes.

[0126] Asthma is a common, long-term inflammatory disease of the airways of both lungs. Asthma is characterized by variable and recurrent symptoms, reversible airflow obstruction and bronchospasm. Symptoms include wheezing attacks, cough, chest tightness and shortness of breath. Asthma is believed to be caused by a combination of genetic and environmental factors. Environmental factors include exposure to air pollution and allergens. Asthma is classified according to the frequency of symptoms, forced expiratory volume in one second (FEV1) and peak expiratory flow rate. Asthma can also be classified as atopic or non-atopic, where atopy refers to the tendency to have type 1 hypersensitivity reactions. There is currently no cure for asthma. Symptoms can be prevented by avoiding triggers such as allergens and irritants and by using inhaled corticosteroids. If asthma symptoms are still not controlled, long-acting beta-agonists (LABA) or anti-leukotriene drugs can also be used in addition to inhaled corticosteroids. For rapidly exacerbated symptoms, treatment is usually with inhaled short-acting beta-2 agonists such as salbutamol and oral corticosteroids. In very severe cases, intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.

[0127] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long-term lack of airflow. COPD can include two main conditions, emphysema and chronic bronchitis. In emphysema, the walls between many of the alveoli are damaged. As a result, the alveoli lose their shape and become loose. This damage can also damage the alveolar walls, resulting in fewer and larger alveoli instead of many tiny alveoli. If this happens, the amount of gas exchange in the lungs decreases. In chronic bronchitis, the lining of the airways is constantly irritated and inflamed, which causes the lining to swell. A lot of thick mucus forms in the airways, making it difficult to breathe. There is currently no known cure for COPD, but its symptoms can be treated and its progression can be slowed.

[0128] Pain is a distressing sensation that is usually triggered by an intense or noxious stimulus, such as a stubbed toe, burned finger, alcohol on a cut, or a "muscle hit." Pain is a complex, subjective phenomenon, and defining it has always been a challenge. Pain is also known as an uncomfortable sensory and emotional experience associated with actual or potential tissue damage. Pain is sometimes considered a symptom of an underlying disease, such as inflammation.

[0129] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health, sex, diet and administration time, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of the medical care staff on such factors is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of preparation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0130] The method of applying antibody or fragment includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route. Antigen-binding polypeptide or composition can be applied by any convenient approach, such as by infusion or push injection, absorbed by epithelial or mucocutaneous lining (for example, oral mucosa, rectum and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Therefore, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be applied orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneal, locally (such as by powder, ointment, drops or transdermal patch), to the cheek or as a mouth or nasal spray.

[0131] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

[0132] Administration may be systemic or local. In addition, it is desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection may be facilitated, for example, by a ventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration may also be employed, for example, by using an inhaler or nebulizer, and formulations with an atomizing agent.

[0133] It is desirable to administer the antigen-binding polypeptide or composition of the present disclosure locally to the area in need of treatment; this can be achieved, for example but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous or gelatinous material, including a membrane, such as a sialastic membrane or fiber. Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that the proteins do not absorb.

[0134] The amount of the antibody or fragment of the present disclosure that effectively treats, inhibits and prevents inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro assays can also be optionally used to help determine the ideal dosage range. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease, disorder or condition, and should be determined according to the physician's judgment and the circumstances of each patient. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0135] As a general suggestion, the dosage of the antibody or fragment of the present disclosure administered to the patient is usually 0.001 mg / kg to 100 mg / kg patient weight, 0.01 mg / kg to 20 mg / kg patient weight or 0.5 mg / kg to 10 mg / kg patient weight. In general, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than the half-life of antibodies from other species. Therefore, the dosage of human antibodies can usually be reduced and the frequency of administration can be reduced. In addition, the dosage and frequency of administration of the antibody of the present disclosure can be reduced by modifying (e.g., lipidation) to enhance the uptake and tissue penetration (e.g., into the brain) of the antibody.

[0136] In another embodiment, the composition of the present disclosure is administered in combination with a cytokine. The cytokines that can be administered with the composition of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0137] In further embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens (eg, radiation therapy). Composition

[0138] The present disclosure also provides a pharmaceutical composition. Such a composition comprises an effective amount of an antibody or fragment and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0139] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In addition, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0140] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting tension, such as sodium chloride or dextrose, are also contemplated. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated into suppositories with conventional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in purified form, and an appropriate amount of carrier to provide a suitable administration form for the patient. The formulation should be suitable for the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0141] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally speaking, the composition is provided in a unit dosage form alone or mixed together, for example, provided in a sealed container (such as an ampoule or a pouch) with a dry lyophilized powder or anhydrous concentrate, and the amount of the active agent is indicated. When the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline may be used for distribution. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the composition is mixed before administration.

[0142] The compounds disclosed herein can be formulated in neutral form or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples Example 1: Generation of mouse monoclonal antibodies (mAbs) against human MerTK

[0143] This example describes the generation of anti-human MerTK mouse monoclonal antibodies using hybridoma technology.

[0144] Antigens: human MerTK-Fc protein and human MerTK-his protein, which comprise the extracellular domain (ECD) of human MerTK fused to human IgG1 Fc or a his tag at the C-terminus.

[0145] Immunization: To generate mouse monoclonal antibodies against human MerTK, Balb / c mice, SJL mice, C57BL / 6 mice, and SD mice were immunized intraperitoneally and subcutaneously with human MerTK-Fc or MerTK-his protein at biweekly intervals. Serum titers of immunized mice were monitored using ELISA against human MerTK-his and cynomolgus MerTK-his proteins and FACS against human MerTK overexpressed on CHO-K1 cell line (CHO-K1-hMerTK), while the CHO-K1 parental cell line served as a negative control. After 2-4 rounds of immunization, mice with sufficient titers were boosted with 25 μg of human MerTK-his protein and then selected for fusion.

[0146] Cell fusion and hybridoma screening: The spleen cells of the selected mice were fused with the mouse myeloma cell line Sp2 / 0 by electrofusion. First, the supernatants of these hybridoma cells were screened using ELISA for human MerTK-his protein. Then, the supernatants of positive clones were screened using ELISA for cynomolgus monkey MerTK-his, and their function of blocking MerTK binding to its ligand growth arrest-specific protein 6 (Gas6) was detected in an ELISA-based receptor blocking assay. The positive primary clones produced by each fusion were subcloned, and the subclones were further confirmed by ELISA-based receptor blocking assays and affinity grading. Those hybridoma clones with efficient binding and blocking capabilities were sequenced and selected for further analysis. The amino acid sequences of the variable regions of these clones are provided in Table 1 below. Table 1. Sequences of variable regions of selected clones (underlined / bold indicates CDRs) Table 1A. CDR sequences of selected clones Example 2: Antigen Binding Properties of Anti-MerTK Chimeric Monoclonal Antibodies

[0147] The variable region of the mouse MerTK antibody was fused to the human IgG1 constant region containing the L234A / L235A / P329G (LALAPG) mutations to generate a chimeric monoclonal antibody without Fc binding ability. Benchmark antibodies (including M6, Ab2000-A7 and h13B4.v16) were prepared separately using the sequences described in patent applications WO 2019084307A1 / WO 2016106221A1 / WO2020214995A1 according to the above method. This example tests the binding properties of anti-MerTK chimeric monoclonal antibodies. ELISA binding activity of anti-MerTK chimeric antibody to human and cynomolgus monkey MerTK proteins

[0148] To evaluate binding activity, these chimeric mAbs were subjected to ELISA testing. Briefly, 96-well plates were coated with 2 μg / mL of human or cynomolgus monkey MerTK-his protein in PBS at 100 μL / well overnight at 4°C and then blocked with 150 μL / well of 1% BSA. Five-fold dilutions of MerTK antibody (starting at 20 nM) were added to each well and incubated at room temperature for 1 hour. The plates were washed with PBS / Tween-20 and then incubated with goat anti-human IgG Fc antibody conjugated to horseradish peroxidase (HRP) at room temperature for 30 minutes (min). After washing, the plates were developed using TMB substrate, followed by the addition of stop solution to complete the reaction. The signal from each well was read at OD 450nm using a spectrophotometer. Figure 1 , Figure 2 As shown in Table 2, most of the tested MerTK antibodies showed binding activity to human or cynomolgus monkey MerTK-his protein comparable to that of the benchmark Ab2000-7, M6 and h13B4.v16 antibodies. Table 2. Cross-species activity of anti-MerTK chimeric monoclonal antibodies Using Biacore TM Affinity ranking of anti-MerTK chimeric antibodies

[0149] Using the capture method, using Biacroe TM The binding affinity of the MerTK chimeric antibody to the human MerTK-his protein was tested by T200. Briefly, the antibody was captured using a Pro-A chip. Two doses (12.5 nM and 50 nM, or 25 nM and 50 nM) of human MerTK-his protein were injected onto the captured antibody at a flow rate of 30 μL / min for 180 s. The antigen was allowed to dissociate for 420-800 s. The Biacore TM T200 evaluation software was used for data analysis. The results are shown in Table 3 below. Table 3. Binding affinity of antibodies to hMerTK protein Binding to CHO-K1 cells overexpressing human MerTK

[0150] In order to evaluate the binding properties to antigens expressed on these cells, FACS was used to analyze the binding of chimeric monoclonal antibodies to human MerTK overexpressed on CHO-K1 cells. Briefly, CHO-K1 (CHO-K1-MerTK) cells overexpressing human MerTK were first incubated with 5-fold serial dilutions (starting at 20 nM) of mAb at 4°C for 1 hour. After washing with FACS buffer, PE-conjugated anti-human IgG-Fc secondary antibody was added to each well and incubated at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE was evaluated using QuantAnalyzer16. Figure 3 As shown, most of the tested MerTK chimeric antibodies showed higher maximal binding ability (Top) and potency (EC50) to human MerTK expressed on CHOK1 cells than the benchmark Ab2000-A7, M6 and h13B4.v16 antibodies. Binding to tumor cells overexpressing human MerTK

[0151] To evaluate the binding properties to MerTK expressed on cells, FACS analysis was used to analyze the binding of chimeric monoclonal antibodies to the human melanoma cell line SK-MEL-5 expressing MerTK. Briefly, SK-MEL-5 cells were first incubated with 5-fold serial dilutions (starting from 20 nM) of chimeric mAb at 4°C for 30 min. After washing with FACS buffer, PE-conjugated anti-human IgG secondary antibodies were incubated with the cell-antibody complexes in these wells at 4°C for 30 min to detect antibodies bound to these cells. The MFI of PE was evaluated using QuantAnalyzer 16. Figure 4 As shown, most of the tested MerTK chimeric antibodies showed higher maximal binding capacity to human MerTK expressed on SK-MEL-5 cells than the benchmark Ab2000-A7, M6 and h13B4.v16 antibodies. Notably, some of them showed significantly improved binding potency (EC50) when compared to the benchmark antibodies. Example 3. Blocking activity of anti-MerTK chimeric monoclonal antibodies Blocking MerTK binding to its ligand Gas6

[0152] The TAM (Tyro3, ​​Axl, and MerTK) family shares a common ligand named Gas6. This ligand consists of an N-terminal GLA domain, four EGF-like repeats, and two C-terminal laminin G domains. The N-terminal GLA domain is able to bind to PtdSer exposed on the plasma membrane under different conditions, including apoptosis, immune activation, and coagulation. At the C-terminus, one of the laminin G domains interacts with the Ig-like domain of MerTK to form a heterotetrameric complex and lead to downstream signaling activation.

[0153] To evaluate the blocking effect of anti-hMerTK mAbs on hMerTK binding to its ligand hGas6, a receptor blocking assay was set up. Briefly, Jurkat cells engineered to overexpress human Gas6 were cultured at 5 × 10 4 The density of cells / well was added to a 96-well microplate. Human MerTK-mouse Fc fusion protein (50 μL / well, 1 μg / mL) and 3-fold serial dilutions of MerTK chimeric antibody starting from 120 nM in a volume of 50 μL were added to the 96-well plate and incubated at 4°C for 30 min. After washing with FACS buffer, the plates were incubated with diluted Alex Fluor 488-conjugated goat anti-mouse IgG antibody for 30 min at 4°C. After washing, the plates were analyzed using QuantAnalyzer 16. Figure 5 As shown, all antibodies could effectively inhibit the binding of human MerTK to human Gas6 expressed on cells. Example 4: Functional properties of anti-MerTK antibodies

[0154] In this example, an efferocytosis assay was performed to evaluate the inhibitory activity of anti-MerTK antibodies on macrophage-mediated phagocytosis of apoptotic cells in vitro.

[0155] Briefly, Jurkat cells were induced to undergo apoptosis by treatment with 1 μM staurosporine for 4 h. The cells were then washed twice with DPBS and incubated at 1.0 × 10 6 The cells were resuspended in DPBS at a density of cells / mL. Apoptotic cells were then labeled with 0.5 μM CFSE for 5 min at 37°C in the dark. After labeling, apoptotic cells were washed three times with culture medium and resuspended in culture medium. CD14+ monocytes were isolated from the buffy coat of healthy donors using human CD14 microbeads (Miltenyi Biotec). CD14+ monocytes were cultured in the presence of 100 ng / ml M-CSF for 7 days to differentiate into M0 macrophages showing upregulated MerTK expression. The macrophages were then incubated at 4.0×10 4The cells were seeded into 96-well microplates at a density of 10 cells / well. Serial dilutions of the antibodies were incubated with macrophages in the 96-well plate for 20 min. Then, freshly prepared CFSE-labeled apoptotic Jurkat cells were incubated at 2.0 × 10 5 The cells were added to the microwells at a density of 10 cells / well and co-cultured at 37°C for 90 min to allow macrophages to phagocytose apoptotic cells. After incubation, APC anti-human CD14 antibody was used to label macrophages in the co-culture system. Phagocytosis events were quantified using QuantAnalyzer 16. CD14 + CFSE + The cells represent macrophages that have engulfed apoptotic Jurkat cells. Epoptosis assay demonstrated that anti-MerTK antibodies can inhibit the engulfment of apoptotic cells by human macrophages. Figure 6 The results in Figure 5 show that some of the tested MerTK antibodies displayed more effective blocking efficacy in efferocytosis when compared to h13B4.v16. Example 5: Humanization of chimeric MerTK antibody

[0156] The variable regions of the chimeric antibodies 10F7D9, 265F11B5 and 254B4D9 were selected for humanization. In brief, the amino acid sequences of VH and VL were compared with the existing human Ig gene sequence database to determine the best overall matching human germline Ig gene sequence. For each clone, the CDRs of the light and heavy chains were transplanted to the candidate germline. A 3D model was then generated to determine if there were any key mouse amino acids in the framework region, which could affect binding and / or CDR conformation by replacing them with human amino acids. In order to maintain the structure and function of the humanized antibodies, key amino acids were selected for back mutation. The humanized variable regions of the antibodies were then fused to the constant regions of human IgG1 LALAPG for antibody production.

[0157] For the light chain of 10F7D9, the candidate germline is the IGKV6-21*01 gene. For the heavy chain, the candidate germline is the IGHV7-4-1*02 gene. In the case of the light chain, L45P, L46W, K48Y and Y86F in the framework region are involved in back mutations. In the case of the heavy chain, V2I, V20I, R38K, E46K, S84N, S85N, Y95F and R98T in the framework region are involved in back mutations.

[0158] For the light chain of 265F11B5, the candidate germline is the IGKV6-21*01 gene. For the heavy chain, the candidate germline is the IGHV1-18*01 gene. In the case of the light chain, K50Y and F72Y in the framework region are involved in back mutations. In the case of the heavy chain, M48I, V68A, M70L, T71A, T72V, Y95F and A97T in the framework region are involved in back mutations.

[0159] For the light chain of 254B4D9, the candidate germline was the IGKV1-33*01 gene, and for the heavy chain, the candidate germline was the IGKV7-4-1*02 or IGKV7-81*01 gene. In the case of the light chain, Y49H, T69R, and Y87F in the framework region were involved in back mutations. In the case of the heavy chain, R38K, P38K, E46Q, M72L, Y80F, and Y95F in the framework region were included as back mutations. (Tables 4 and 5). Table 4. Humanized antibody sequences (underlined / bold indicates CDR; bold / italic indicates back mutation) Table 5. Pairing of humanized antibody VH and VL 254B4D9 HU-VL1 V L HU-VH1 Hu-1 HU-VH2 Hu-2 VH Chimerism Example 6: Antigen Binding Properties of Humanized Antibodies Binding to recombinant human MerTK

[0160] To evaluate antigen binding activity, humanized antibodies were tested by ELISA as described above. Figure 7 As shown, the humanized antibody showed comparable binding potency to human MerTK as its parent chimeric antibody. Using Biacore TM Affinity grading of humanized antibodies

[0161] To explore whether the humanized antibody can maintain its binding kinetics, Biacore TM Affinity fractionation was performed. Protein A chips were used to capture antibodies. Human MerTK-his protein at 50 nM was injected over the captured antibodies at a flow rate of 30 μL / min for 180 s. The antigen was allowed to dissociate for 600 s. TM Experiments were performed on 8K. Using Biacore TMData analysis was performed using 8K evaluation software. The results are shown in Table 6. 265F11B5Hu-4, 265F11B5Hu-7, 265F11B5Hu-8, 254B4D9Hu-1, and 254B4D9Hu-2 showed comparable affinities to their chimeric antibodies. Table 6. Affinity ranking results of humanized antibodies Binding to CHO-K1 cells overexpressing human MerTK

[0162] To evaluate the binding properties to antigens expressed on cells, humanized antibodies were analyzed using FACS as described previously. Figure 8 As shown, humanized antibodies 265F11B5 and 254B4D9 showed comparable cell binding potency to their chimeric antibodies. Meanwhile, some humanized antibodies of 10F7D9 showed comparable cell binding potency to their chimeric antibodies.

[0163] The scope of the present disclosure is not limited by the particular embodiments described, which are intended to be a single illustration of the various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they are within the scope of the appended claims and their equivalents.

[0164] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or an antigen-binding fragment thereof, which is specific to human Mer proto-oncogene tyrosine protein kinase (MerTK) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the following amino acid sequences: SEQ ID NO:25-30; SEQ ID NO:59-64; SEQ ID NO:65-70; SEQ ID NO:31-33, 28-29, 34; SEQ ID NO:35-40; SEQ ID NO:41-46; SEQ ID NO:47-52; SEQ ID NO:53-58; SEQ ID NO:71, 66, 72, 73, 69, 74; SEQ ID NO: 65, 75, 67, 76, 69, 77; SEQ ID NO:71, 78-79, 73, 69, 74; or SEQ ID NO:65-66, 80-81, 69-70.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: the VH CDR1 comprises the amino acid sequence of SEQ ID NO:25; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:26; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 27; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 29; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

30.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 82-87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 89-91.

4. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises the amino acid sequence of SEQ ID NO: 83, and the VL comprises the amino acid sequence of SEQ ID NO:

90.

5. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises the amino acid sequence of SEQ ID NO: 83, and the VL comprises the amino acid sequence of SEQ ID NO:

91.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein: the VH CDR1 comprises the amino acid sequence of SEQ ID NO:59; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 60; The VH CDR3 comprises the amino acid sequence of SEQ ID NO:61; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 62; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 63; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

64.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 102 and 104, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 106.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 102, and the VL comprises the amino acid sequence of SEQ ID NO:

106.

9. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO: 104, and the VL comprises the amino acid sequence of SEQ ID NO:

106.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein: the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 65; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:66; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 69; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

70.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 93-96, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 98-100.

12. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises the amino acid sequence of SEQ ID NO: 95, and the VL comprises the amino acid sequence of SEQ ID NO:

99.

13. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises the amino acid sequence of SEQ ID NO: 96, and the VL comprises the amino acid sequence of SEQ ID NO:

99.

14. The antibody or fragment thereof according to any one of claims 1 to 13, wherein the antibody or fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

15. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1 to 14 and one or more antibodies or antigen-binding fragments having binding specificity to a non-MerTK target antigen.

16. A chimeric antigen receptor (CAR), comprising an antigen binding fragment according to any one of claims 1-14, a transmembrane domain, a co-stimulatory domain and a CD3ξ intracellular domain.

17. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the CAR according to claim 16.

18. The one or more polynucleotides of claim 17, which are one or more mRNAs.

19. The one or more polynucleotides of claim 18, wherein the mRNA is chemically modified.

20. A cell comprising one or more polynucleotides according to any one of claims 17-19.

21. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the CAR according to claim 16 and a pharmaceutically acceptable carrier.

22. A method of treating cancer or an inflammatory disorder in a patient in need thereof, the method comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the CAR according to claim 16.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-15 or the CAR according to claim 16 for the preparation of a medicament for treating cancer or inflammatory disorders.

24. The method of claim 22 or the use of claim 23, wherein the cancer is a solid tumor.

25. The method or use of claim 24, wherein the solid tumor is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, gastric cancer, cervical cancer, uterine cancer and thyroid cancer.

26. The method of claim 22 or the use of claim 23, wherein the inflammatory disorder is selected from the group consisting of Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

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