Anti-MerTK Antibodies and Their Uses

Abstract

The present disclosure provides anti-MerTK antibodies, variants thereof, and humanized forms. The newly disclosed antibodies exhibit high affinity for the MerTK protein and can be used for treating cancer, particularly solid tumors.

Description

Background Art

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

[0002] Macrophages in the innate immune system are a collection of various cell types that play multiple functional roles under both homeostatic and pathological conditions. M1 macrophages are classically activated macrophages that play a pro-inflammatory role in clearing intracellular pathogens. In contrast, M2 macrophages are alternatively activated macrophages that contribute to tissue repair and efferocytosis. Macrophages are professional phagocytes, highly specialized in clearing dying or dead cells and cell debris that are produced in large amounts under normal physiological conditions. In addition, a large number of macrophages have been detected in various types of solid tumors. Macrophages in the tumor microenvironment are called tumor-associated macrophages (TAMs), which usually promote cancer cell initiation and proliferation, accelerate angiogenesis, and inhibit anti-tumor immunity, thus driving tumor progression and metastasis. Increasing evidence suggests 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 usually accompanied by increased cell death due to hypoxia and metabolic stress. To evade immune surveillance, tumors take advantage of the non-immunogenic nature of apoptosis. TAMs actively clear dying tumor cells while reducing the production of inflammatory cytokines to avoid alerting 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. It has been observed that MerTK is more highly expressed on M2 macrophages than on M1 macrophages. MerTK helps to clear dying or damaged cells that display "eat me" signals (i.e., phosphatidylserine, PtdSer) on their cell surface, a process that requires the bridging molecules growth arrest-specific protein 6 (Gas6) or protein S. Macrophages expressing MerTK phagocytose apoptotic cells through efferocytosis. In tumors, uncontrolled proliferation leads to increased apoptosis of cancer cells, but TAM-dependent MerTK to clear dying cells may inhibit immune activation.

[0004] Blocking MerTK leads to the accumulation of apoptotic cells within tumors and triggers a type I interferon response. Treatment of tumor-bearing mice with an anti-MerTK antibody stimulates T cell activation and enhances the efficacy of anti-PD-1 and anti-PD-L1 therapies. Thus, blocking MerTK can enhance tumor immunogenicity and enhance 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 that are specific for the human MerTK protein. One embodiment provides an antibody or an antigen-binding fragment thereof that is specific for the human proto-oncogene tyrosine-protein kinase (MerTK) protein and comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and the light-chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the following amino acid sequences, respectively: 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 NO:1 and 82-87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 89-91.

[0008] 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: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 NO:13, 102, and 104, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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 NO:15 and 93 - 96, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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 its fragment 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 an antigen-binding fragment disclosed herein and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than MerTK.

[0015] A chimeric antigen receptor (CAR) is further provided, which comprises an antigen-binding fragment disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

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

[0017] In one embodiment, a method of treating cancer or an inflammatory disorder in a patient in need thereof is also provided, the method comprising administering to the patient an effective amount of an antibody or its antigen-binding fragment 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 is shown that all tested anti-MerTK antibodies can effectively bind to human MerTK protein.

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

[0021] Figure 3 It is shown that compared with the reference antibodies Ab2000-A7, M6, and h13B4.v16, most tested MerTK chimeric antibodies have higher maximum binding capacity and binding efficacy for human MerTK expressed on CHO-K1 cells.

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

[0023] Figure 5 It is shown that all of these antibodies can effectively inhibit the binding of human Gas6 expressed on human MerTK-binding cells.

[0024] Figure 6 It is shown that some of the tested MerTK antibodies have higher blocking efficiency in efferocytosis compared to the reference antibody h13B4.v16.

[0025] Figure 7 It is shown that all of the tested humanized antibodies have binding efficacy to human MerTK protein comparable to that of the chimeric antibodies.

[0026] Figure 8 It is shown that some of the tested humanized antibodies have binding activity to human MerTK expressed on CHOK1 cells comparable to that of their parental chimeric counterparts. Detailed Description

[0027] Definitions

[0028] 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" may be used interchangeably herein.

[0029] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a full antibody as well as any antigen-binding fragment or its single chain. Thus, the term "antibody" includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule having biological activity for binding an antigen. Such examples include, but are not limited to, complementarity-determining regions (CDRs) of the heavy or light chain or their ligand-binding portions, variable regions of the heavy or light chain, constant regions of the heavy or light chain, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

[0030] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a part of an antibody, such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc. Regardless of 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.

[0031] The term antibody encompasses a wide variety of polypeptide classes that can be biochemically distinguished. Those skilled in the art will recognize that heavy chains are 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 to be IgG, IgM, IgA, IgG, or IgE, respectively.

[0032] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Given this disclosure, those skilled in the art can readily identify modified versions of these classes and isotypes, and accordingly, these modified versions are encompassed within the scope of this disclosure. All immunoglobulin classes are clearly encompassed within the scope of this disclosure, and the following discussion will generally be directed to immunoglobulin molecules of the IgG class. With respect to IgG, a standard immunoglobulin molecule comprises 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 linked by disulfide bonds in a "Y" configuration, where the light chains wrap around the heavy chains starting from the opening of the "Y" and extend into the variable regions.

[0033] The antibodies, antigen-binding polypeptides, variants, or derivatives of this disclosure 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 generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0034] As used herein, the term "chimeric antibody" shall be understood to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which, according to the present disclosure, may be full-length, partial, or modified) 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 will be of human origin.

[0035] 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 chondricthoid origin (e.g., from a shark).

[0036] As used herein, the term "recombinant," when referring to a polypeptide or polynucleotide, means a form of polypeptide or polynucleotide that is not naturally occurring, non-limiting examples of which can be produced by combining polynucleotides that do not normally occur together.

[0037] Hybridoma technology can be carried out under different "stringency" conditions. Generally, low-stringency hybridization reactions are carried out at about 40 °C in a solution of about 10x SSC or equivalent ionic strength / temperature. Medium-stringency hybridizations are typically carried out at about 50 °C in about 6x SSC, and high-stringency hybridization reactions are typically carried out at about 60 °C in about 1x SSC. As is well known to those skilled in the art, hybridization reactions can also be carried out under "physiological conditions." Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg2+ concentration that are typically found in cells.

[0038] Anti-MerTK antibody

[0039] As demonstrated in the appended 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 importantly, many of these antibodies exhibited higher binding affinities for human MerTK protein 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 inhibitory efficiency than these benchmark antibodies.

[0040] According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or an antigen-binding fragment thereof has binding specificity for the human MerTK protein. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3.

[0041] In some embodiments, an antibody or an antigen-binding fragment derived from antibody 10F7D9 is 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; VL CDR2 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.

[0042] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NO:1 and 82 - 87. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NO:2 and 89 - 91.

[0043] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:1 and 82 - 87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:1 and 82 - 87, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:2 and 89 - 91, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:2 and 89 - 91, while retaining the corresponding VL CDR.

[0044] 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.

[0045] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:1 and any one of 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 82-87, 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:1 and any one of 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 82-87, and VL comprises the amino acid sequence of SEQ ID NO:91.

[0046] Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided, which bind to the same epitope on MerTK as that bound by 10F7D9. Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided, which compete with 10F7D9 for binding to MerTK.

[0047] 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; VL CDR2 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.

[0048] Exemplary VH sequences comprise the amino acid sequence of SEQ ID NO:3. Exemplary VL sequences comprise the amino acid sequence of SEQ ID NO:4. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:3, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:4, while retaining the corresponding VL CDR.

[0049] 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.

[0050] 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; VL CDR2 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.

[0051] Exemplary VH sequences comprise the amino acid sequence of SEQ ID NO:5. Exemplary VL sequences comprise the amino acid sequence of SEQ ID NO:6. In some embodiments, VH comprises 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, VL comprises 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.

[0052] 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.

[0053] 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; VL CDR2 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.

[0054] 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, VH comprises 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 CDR. In some embodiments, VL comprises 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 CDR.

[0055] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind the same epitope on MerTK to which 247E5A8 binds. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 247E5A8 for binding to MerTK.

[0056] 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; VL CDR2 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.

[0057] 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, 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, 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.

[0058] Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on MerTK as that bound by 252C12B10. Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 252C12B10 for binding to MerTK.

[0059] In some embodiments, antibodies or antigen-binding fragments derived from antibody 252H9D5 are provided. In some embodiments, VH CDR1 includes the amino acid sequence of SEQ ID NO:53; VH CDR2 includes the amino acid sequence of SEQ ID NO:54; VH CDR3 includes the amino acid sequence of SEQ ID NO:55; VL CDR1 includes the amino acid sequence of SEQ ID NO:56; VL CDR2 includes the amino acid sequence of SEQ ID NO:57; and VL CDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO:58.

[0060] 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, 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, 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.

[0061] Accordingly, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on MerTK that 252H9D5 binds to. Accordingly, in some embodiments, antibodies and antigen-binding fragments are provided that compete with 252H9D5 for binding to MerTK.

[0062] 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; VL CDR2 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.

[0063] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NO:13, 102, and 104. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NO:14 and 106.

[0064] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO: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 NO:13, 102, and 104, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:14 and 106, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NO:14 and 106, while retaining the corresponding VL CDR.

[0065] 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.

[0066] Accordingly, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on MerTK that 254B4D9 binds to. Accordingly, in some embodiments, antibodies and antigen-binding fragments are provided that compete with 254B4D9 for binding to MerTK.

[0067] 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; VL CDR2 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.

[0068] Exemplary VH sequences include amino acid sequences selected from the group consisting of SEQ ID NO:15 and 93 - 96. Exemplary VL sequences include amino acid sequences selected from the group consisting of SEQ ID NO:16 and 98 - 100.

[0069] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO: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 NO:15 and 93 - 96, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO: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 NO:16 and 98 - 100, while retaining the corresponding VL CDR.

[0070] 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.

[0071] 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.

[0072] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO: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 NO:93 - 96, and VL comprises the amino acid sequence of SEQ ID NO:99.

[0073] Thus, in some embodiments, antibodies and antigen - binding fragments are also provided, which antibodies and antigen - binding fragments bind to the same epitope on MerTK that 265F11B5 binds to. Thus, in some embodiments, antibodies and antigen - binding fragments are also provided, which antibodies and antigen - binding fragments compete with 265F11B5 for binding to MerTK.

[0074] 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; VL CDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:74.

[0075] 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, VH comprises the amino acid sequence of SEQ ID NO:17, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:17, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:18, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:18, while retaining the corresponding VL CDR.

[0076] Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds 276C2D1. Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 276C2D1 for binding to MerTK.

[0077] 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; VL CDR2 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.

[0078] Exemplary VH sequences comprise the amino acid sequence of SEQ ID NO:19. Exemplary VL sequences comprise the amino acid sequence of SEQ ID NO:20. In some embodiments, VH comprises 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 CDR. In some embodiments, VL comprises 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 CDR.

[0079] Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds 280C6A3. Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 280C6A3 for binding to MerTK.

[0080] 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; VL CDR2 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.

[0081] 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, VH comprises the amino acid sequence of SEQ ID NO:21, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:21, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:22, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:22, while retaining the corresponding VL CDR.

[0082] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind the same epitope on MerTK to which 293C2B7 binds. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 293C2B7 for binding to MerTK.

[0083] 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; VL CDR2 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.

[0084] 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, 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, 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.

[0085] Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that bind the same epitope on MerTK that 300A5A3 binds to. Accordingly, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 300A5A3 for binding to MerTK.

[0086] In some embodiments, antibodies and antigen-binding fragments are also provided that include CDR sequences derived from the CDR sequences disclosed herein and having one, two or three amino acid substitutions, deletions and / or additions.

[0087] In some embodiments, the antibody or fragment thereof is capable of inducing antibody-dependent cell cytotoxicity (ADCC). In some embodiments, the antibody or fragment thereof is not capable of inducing antibody-dependent cell cytotoxicity (ADCC).

[0088] Multifunctional group molecule

[0089] A multifunctional group molecule that includes an antibody or antigen-binding fragment specific for MerTK, such as those disclosed herein, and one or more antibody or antigen-binding fragments specific for a second antigen.

[0090] In some embodiments, the second antigen is a protein expressed on an immune cell, such as a T cell, B cell, monocyte, macrophage, neutrophil, dendritic cell, phagocyte, natural killer cell, eosinophil, basophil and mast cell.

[0091] 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.

[0092] Also provided are different forms of bispecific antibodies. 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.

[0093] Also provided are bifunctional molecules that include not only antibodies or antigen-binding fragments. As molecules targeting tumor antigens, an antibody or antigen-binding fragment specific for MerTK (such as those described herein) can optionally be combined with an immunocytokine or ligand via a peptide linker. The linked immunocytokine or ligand includes but is 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 the immune checkpoint blockade effect with local immunomodulation at the tumor site.

[0094] Chimeric antigen receptor

[0095] In one embodiment, a chimeric antigen receptor (CAR) is also provided, which comprises an antibody or a fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR comprises an antibody or a fragment thereof disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

[0096] The transmembrane domain can be designed to fuse with the extracellular domain comprising the antibody or fragment, optionally via a hinge domain. It can also fuse with the intracellular domain, such as the co-stimulatory domain. In some embodiments, the transmembrane domain can include the native transmembrane region of the co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB used as the co-stimulatory domain) or the native transmembrane domain of the hinge region (e.g., the TM region of CD8α or CD28T used as the hinge domain).

[0097] In some embodiments, the transmembrane domain may include sequences that span the cell membrane but extend into the cytoplasm and / or extracellular space of the cell. For example, the transmembrane may include transmembrane sequences, which in turn 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, the transmembrane domain includes a transmembrane region and may further contain 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".

[0098] 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, may form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0099] In some embodiments, the CAR further comprises 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 alpha chain), CD79B (B cell antigen receptor complex associated beta 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 (SLAMF3), 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-stimulatory molecule (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 I molecule, MHC class II molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0100] In some embodiments, the cytoplasmic portion of the CAR further comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is the CD3ξ domain, or its amino acid sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the CD3ξ domain.

[0101] Polynucleotides, mRNA, and methods of expressing or preparing antibodies

[0102] The present disclosure also provides polynucleotides or nucleic acid molecules encoding the antibodies, variants or derivatives thereof, or CARs of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0103] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into target cells to express an antibody or a fragment thereof.

[0104] 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 generally uses a linear or circular DNA template containing a promoter, a pool of ribonucleoside triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNase inhibitor. The exact conditions will vary depending on the specific application.

[0105] In some embodiments, for preparing mRNA encoding an antibody, the DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription (e.g., T3, T7 or SP6 promoter), followed by the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding the heavy or light chain) and a termination signal.

[0106] Standard methods can be used to determine the mRNA sequence encoding the desired antibody (e.g., encoding the heavy or light chain) and incorporate it into a DNA template. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. Then, optimization algorithms can be used to select appropriate codons. Typically, on the one hand, the G / C content can be optimized to achieve as high a G / C content as possible, and on the other hand, the frequency of tRNAs can be considered as much as possible according to codon usage. The optimized RNA sequence can be established and displayed, for example, with the aid of appropriate display means, and compared with the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stability and instability characteristics of the RNA, or its regions, respectively.

[0107] 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. Thus, 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), which include, but are not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-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, 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-oxyacetic acid 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, wybutosine, and phosphoramidates, thiophosphates, 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. Patent 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.

[0108] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain RNA backbone modifications. Typically, backbone modifications are modifications that chemically modify the phosphate of the backbone of the nucleotides contained in the RNA. Exemplary backbone modifications generally include, but are not limited to, modifications from the group consisting of methylphosphonate, methylaminophosphate, aminophosphate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidine groups, etc., which means replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0109] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain sugar modifications. Typical sugar modifications are chemical modifications to the sugar of the nucleotides it contains, 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'-deaminated-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'-arabinosylcytidine 5'-triphosphate, 2'-arabinosyluridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0110] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain modifications of nucleobases (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-aminoallyl cytidine 5'-triphosphate, 5-aminoallyl uridine 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 ribonucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaaadenosine 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 xanthosine 5'-triphosphate.

[0111] Typically, mRNA synthesis includes adding a "cap" at the N-terminus (5') end and a "tail" at the C-terminus (3') end. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" is to protect the mRNA from exonuclease degradation.

[0112] Thus, in some embodiments, the mRNA (e.g., 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 a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, generating 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.

[0113] Thus, in some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) includes a 3' poly(A) tail structure. The polyA tail on the 3' end of the mRNA typically 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, the mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) includes a 3' poly(C) tail structure. A suitable polyC tail on the 3' end of the mRNA typically includes 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 polyC tail can be added to the polyA tail or can replace the polyA tail.

[0114] Thus, in some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron response element. In some embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides (e.g., a length of about 50 to 400 nucleotides, a length of about 50 to 300 nucleotides, a length of about 50 to 200 nucleotides, or a length of about 50 to 100 nucleotides).

[0115] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding a heavy chain and a light chain) includes a sequence encoding a signal peptide, such as those described herein. In certain embodiments, 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 at the N-terminus to the sequence encoding the heavy chain or the light chain.

[0116] The techniques 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.

[0117] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against 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 antigen challenge, but whose endogenous loci have been inactivated. Exemplary techniques useful for preparing 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.

[0118] Therapeutics and Uses

[0119] As described herein, the antibodies, variants or derivatives of the present disclosure can be used in certain therapeutic and diagnostic methods.

[0120] The present disclosure further relates to antibody-based therapies that involve administering the antibodies or fragments of the present disclosure to a patient (e.g., an animal, mammal, and human) to treat one or more disorders or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives as described herein).

[0121] The antibodies of the present disclosure can also be used to treat or inhibit cancer. As described above, MerTK is rarely expressed in normal adult tissues, but is highly expressed in the placenta and most common tumors, typically being highly expressed in more than 80% of renal cell carcinomas, breast cancers, colon cancers, prostate cancers, and ovarian cancers.

[0122] Accordingly, in some embodiments, methods for treating cancer in a patient in need thereof are provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody or fragment of the present disclosure. In some embodiments, at least one type of cancer cell (e.g., stromal cells) in the patient overexpresses MerTK.

[0123] Also provided in the present disclosure are cell therapies, such as chimeric antigen receptor (CAR) T cell therapies. Suitable cells can be used, which are transduced with a vector encoding a CAR or contacted with a CAR (or alternatively engineered to express the anti-MerTK antibody of the present disclosure), where the CAR comprises the anti-MerTK antibody of the present disclosure. After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient can have any type of cancer as disclosed herein. The 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.

[0124] 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 are from a cell bank. When isolating cells from a cancer patient, adverse immune reactions can be minimized.

[0125] Non-limiting examples of cancers 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.

[0126] Additional diseases or conditions associated with increased cell survival (which can be treated, prevented, diagnosed, and / or predicted using the antibodies or variants or derivatives thereof disclosed herein) include, but are not limited to, the progression and / or metastasis of malignancies and related disorders, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblasts, promyelocytes, myelomonocytes, monocytes, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, 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, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, 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.

[0127] In some embodiments, the antibodies, polynucleotides or compositions of the present disclosure can be used to treat inflammatory diseases or disorders. In some embodiments, the inflammatory diseases or disorders 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.

[0128] In some embodiments, the autoimmune diseases or disorders to be treated using the disclosed antibodies, fragments and compositions include one or more of the following: alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (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).

[0129] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. RA typically causes joint heat, swelling, and pain. Pain and stiffness usually worsen after rest. Most commonly, the wrists and hands are affected, and usually the same joints on both sides of the body. The disease can also affect other parts of the body. Although the cause of rheumatoid arthritis is unknown, it is thought to be related to the combined effects of genetic and environmental factors. Its underlying mechanism involves the body's immune system attacking these joints. This causes inflammation and thickening of the joint capsule. The goals of treatment are to reduce pain, relieve inflammation, and improve a person's overall function. Painkillers, 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.

[0130] Osteoarthritis (OA) is a type of joint disease caused by the breakdown and degeneration of joint cartilage and the underlying bone. The most common symptoms are joint pain and stiffness. Initially, symptoms may only occur after exercise, but over time, they may become persistent. Other symptoms may include joint swelling, 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 those who work in jobs that put high pressure on joints are at higher risk. Osteoarthritis is thought to be caused by mechanical stress on the joints and low-grade inflammatory processes. Treatments include exercise, minimizing joint stress, support groups, and painkillers.

[0131] Multiple sclerosis (MS) is a demyelinating disease in which the insulating outer layer of nerve cells in the brain and spinal cord is damaged. This damage interferes with the ability of parts of the nervous system to communicate with each other, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms can include double vision, loss of vision in one eye, muscle weakness, sensory disturbances, or problems with coordination. Although the cause is unknown, the underlying mechanism is thought to be the damaging effects of the immune system or malfunction of the cells that produce myelin. There is currently no known cure for multiple sclerosis. Treatments aim to improve physical function after an attack and prevent new attacks.

[0132] Asthma is a common, long-term inflammatory disease of the airways in both lungs. Asthma is characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include wheezing attacks, coughing, chest tightness, and shortness of breath. Asthma is thought 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 develop 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 remain uncontrolled, in addition to inhaled corticosteroids, long-acting beta-agonists (LABA) or leukotriene modifiers may be used. For rapidly worsening symptoms, treatment is usually with inhaled short-acting beta-2 agonists such as salbutamol and oral corticosteroids. In extremely severe cases, intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.

[0133] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disorder characterized by long-term airflow limitation. COPD can include two main conditions, emphysema and chronic bronchitis. In emphysema, the walls between many of the alveoli are damaged. As a result, these alveoli lose their shape and become floppy. This damage also breaks down the alveolar walls, causing a reduction in the number of alveoli and an increase in their size, such that they are no longer many tiny alveoli. If this occurs, the amount of gas exchange in both lungs is reduced. In chronic bronchitis, the inner walls of the airways are constantly irritated and inflamed, which causes the inner walls to swell. A large amount of thick mucus forms in the airways, resulting in difficulty breathing. There is currently no known cure for COPD, but its symptoms can be treated and its progression can be slowed down.

[0134] Pain is a distressing feeling that is usually triggered by intense or noxious stimuli, such as stubbing a toe, burning a finger, getting alcohol in a wound, or hitting the "funny bone". Pain is a complex subjective phenomenon, and defining pain has always been a challenge. Pain is also referred to as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Pain is sometimes considered a symptom of an underlying disease (such as inflammation).

[0135] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variants or derivatives being used, the patient's age, weight, general health, sex, diet and time of administration, rate of excretion, drug combinations, and the severity of the particular disease being treated. The judgment of medical caregivers regarding such factors is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound being 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.

[0136] Methods of administering an antibody or fragment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or skin mucosal linings (such as oral mucosa, rectal, and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Thus, a pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be administered orally, rectally, parenterally, intracisternally, vaginally, intraperitoneally, topically (such as by powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray.

[0137] As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0138] Administration can be systemic or local. Additionally, it is desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intracerebroventricular injection and intrathecal injection; intracerebroventricular injection can be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and formulations with nebulizing agents.

[0139] It is desirable to locally administer the antigen-binding polypeptides or compositions of the present disclosure 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 combination with a wound dressing after surgery), by injection, via a catheter, via a suppository, or via an implant, which is a porous, non-porous, or gelatinous material, including membranes, such as a sialastic membrane or fibers. Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that do not absorb the protein.

[0140] The amount of the antibodies or fragments of the present disclosure effective for treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be employed to assist in determining the optimal dose range. The precise 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 judgment of the physician and the circumstances of each patient. The effective dose can be inferred from the dose-response curve derived from in vitro or animal model test systems.

[0141] As a general recommendation, the dose of the antibodies or fragments of the present disclosure administered to a patient is typically from 0.001 mg / kg to 100 mg / kg of patient body weight, from 0.01 mg / kg to 20 mg / kg of patient body weight, or from 0.5 mg / kg to 10 mg / kg of patient body weight. Generally, due to the immune response to foreign polypeptides, the half-life of human antibodies in the human body is longer than that of antibodies from other species. Therefore, the dose of human antibodies can usually be reduced and the frequency of administration can be decreased. Additionally, the dose and frequency of administration of the antibodies 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.

[0142] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered together with the compositions 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.

[0143] In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens (e.g., radiotherapy).

[0144] Composition

[0145] The disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody or fragment and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0146] In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals and more particularly in humans. Additionally, a "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation adjuvant.

[0147] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, 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 aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetate, citrate or phosphate. Bacteriostatic agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for the adjustment of the osmotic pressure, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E.W. Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) and a suitable amount of carrier in order to provide the proper form for administration to the patient. The formulation should be suitable for the mode of administration. Parenteral formulations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0148] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans 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 solubilizing agents and local anesthetics, such as lidocaine, to reduce the pain at the injection site. Generally, the ingredients are provided singly or in combination in unit dosage forms, for example, as dry lyophilized powders or anhydrous concentrates in sealed containers (such as ampoules or sachets), 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 can be used for dispensing. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix the ingredients before administration.

[0149] The compounds of the present disclosure can be formulated in neutral form or in salt form. Pharmaceutically acceptable salts include salts formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0150] Examples

[0151] Example 1: Generation of murine monoclonal antibodies (mAbs) against human MerTK

[0152] This example describes the preparation of anti-human MerTK murine monoclonal antibodies using hybridoma technology.

[0153] Antigens: Human MerTK-Fc protein and human MerTK-his protein, which contain the extracellular domain (ECD) of human MerTK fused to human IgG1 Fc or a his tag at the C-terminus.

[0154] Immunization: To generate murine monoclonal antibodies against human MerTK, Balb / c mice, SJL mice, C57BL / 6 mice, and SD mice were immunized intraperitoneally and subcutaneously at two-week intervals with human MerTK-Fc or MerTK-his protein. ELISA against human MerTK-his and cynomolgus monkey MerTK-his proteins and FACS against human MerTK overexpressed in the CHO-K1 cell line (CHO-K1-hMerTK) were used to monitor the serum titers of the immunized mice, while the CHO-K1 parental cell line served as a negative control. After 2 - 4 rounds of immunization, mice with sufficient titers were boost-immunized with 25 μg of human MerTK-his protein and then selected for fusion.

[0155] Cell fusion and hybridoma screening: The splenocytes 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 against human MerTK-his protein. Then, the supernatants of the positive clones were screened using ELISA against cynomolgus monkey MerTK-his, and their function of blocking MerTK from binding its ligand growth arrest-specific protein 6 (Gas6) was detected in an ELISA-based receptor blocking assay. The positive primary clones generated from each fusion were subcloned and further confirmed by ELISA-based receptor blocking assay and affinity ranking. The hybridoma clones with high binding and blocking ability were sequenced and selected for further analysis. The amino acid sequences of the variable regions of these clones are provided in Table 1 below.

[0156] Table 1. Sequences of the variable regions of the selected clones (CDRs are underlined / bold)

[0157]

[0158]

[0159]

[0160] Table 1A. CDR sequences of the selected clones

[0161]

[0162]

[0163]

[0164] Example 2: Antigen-binding properties of anti-MerTK chimeric monoclonal antibodies

[0165] The variable regions of the mouse MerTK antibody were fused to the human IgG1 constant region containing the L234A / L235A / P329G (LALAPG) mutation to generate chimeric monoclonal antibodies without Fc-binding ability. The reference antibodies (including M6, Ab2000-A7, and h13B4.v16) were individually prepared according to the above method using the sequences described in patent applications WO 2019084307A1 / WO 2016106221A1 / WO2020214995A1. The binding properties of the anti-MerTK chimeric monoclonal antibodies were tested in this example.

[0166] ELISA binding activity of anti-MerTK chimeric antibodies to human and cynomolgus monkey MerTK proteins

[0167] To evaluate the 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 the MerTK antibodies (starting from 20 nM) were added to each well and incubated for 1 hour at room temperature. The plates were washed with PBS / Tween-20 and then incubated with goat anti-human IgG Fc antibody conjugated to horseradish peroxidase (HRP) for 30 minutes (min) at room temperature. After washing, the plates were developed using TMB substrate, and then the stop solution was added to complete the reaction. The signal from each well was read at OD 450 nm using a spectrophotometer. As Figure 1 , Figure 2 and Table 2 show, most of the tested MerTK antibodies showed binding activity to human or cynomolgus monkey MerTK-his protein comparable to that of the reference Abs 2000-7, M6, and h13B4.v16 antibodies.

[0168] Table 2. Cross-species activity of anti-MerTK chimeric monoclonal antibodies

[0169]

[0170] Affinity ranking of anti-MerTK chimeric antibodies using Biacore TM

[0171] Using the capture method, the binding affinity of MerTK chimeric antibodies to human MerTK-his protein was tested with Biacroe TM T200. Briefly, a Pro-A chip was used to capture the antibodies. Two doses (12.5 nM and 50 nM, or 25 nM and 50 nM) of human MerTK-his protein were injected onto the captured antibodies at a flow rate of 30 μL / min for 180 s. The antigen was allowed to dissociate for 420 - 800 s. Data analysis was performed using Biacore TM T200 evaluation software. The results are shown in Table 3 below.

[0172] Table 3. Binding affinity of antibodies to hMerTK protein

[0173]

[0174] Binding to CHO-K1 cells overexpressing human MerTK

[0175] To evaluate the binding characteristics to the antigens expressed on these cells, FACS was used to analyze the binding of the chimeric monoclonal antibody 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 from 20 nM) of the mAb at 4 °C for 1 hour. After washing with FACS buffer, a PE-conjugated anti-human IgG-Fc secondary antibody was added to each well and incubated at 4 °C for 30 min. The mean fluorescence intensity (MFI) of PE was evaluated using QuantAnalyzer 16. As Figure 3 shown, most of the tested MerTK chimeric antibodies showed higher maximum binding capacity (Top) and potency (EC50) to human MerTK expressed on CHO K1 cells than the benchmark Ab2000-A7, M6, and h13B4.v16 antibodies.

[0176] Binding to tumor cells overexpressing human MerTK

[0177] To evaluate the binding characteristics to MerTK expressed on cells, FACS analysis was used to analyze the binding of the chimeric monoclonal antibody 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 the chimeric mAb at 4 °C for 30 min. After washing with FACS buffer, a PE-conjugated anti-human IgG secondary antibody was incubated with the cell-antibody complexes in these wells at 4 °C for 30 min to detect the antibodies binding to these cells. The MFI of PE was evaluated using QuantAnalyzer 16. As Figure 4 shown, most of the tested MerTK chimeric antibodies showed higher maximum binding capacity to human MerTK expressed on SK-MEL-5 cells than the benchmark Ab2000-A7, M6, and h13B4.v16 antibodies. Notably, some of these antibodies showed significantly improved binding potency (EC50) when compared to the benchmark antibodies.

[0178] Example 3. Blocking activity of anti-MerTK chimeric monoclonal antibodies

[0179] Blocking MerTK from binding to its ligand Gas6

[0180] The TAM (Tyro3, Axl, and MerTK) family shares a common ligand called 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 can bind to PtdSer exposed on the plasma membrane under different conditions, including apoptosis, immune activation, and blood coagulation. At the C-terminal, one of the laminin G domains interacts with the Ig-like domain of MerTK, forming a heterotetrameric complex and leading to downstream signal activation.

[0181] To evaluate the blocking effect of anti-hMerTK mAb on the binding of hMerTK to its ligand hGas6, a receptor blocking assay was set up. Briefly, Jurkat cells engineered to overexpress human Gas6 were added to 96-well microplates at a density of 5×10 4 cells / well. Human MerTK-mouse Fc fusion protein (50 μL / well, 1 μg / mL) and 50 μL of MerTK chimeric antibody serially diluted 3-fold starting from 120 nM were added to the 96-well plates and incubated at 4 °C for 30 min. After washing with FACS buffer, these plates were incubated with diluted Alex Fluor 488-conjugated goat anti-mouse IgG antibody at 4 °C for 30 min. After washing, QuantAnalyzer 16 was used to analyze these plates. As Figure 5 shown, all antibodies could effectively inhibit the binding of human MerTK to human Gas6 expressed on the cells.

[0182] Example 4: Functional properties of anti-MerTK antibodies

[0183] 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.

[0184] Briefly, Jurkat cells were induced to undergo apoptosis by treatment with 1 μM staurosporine for 4 hours. Then the cells were washed twice with DPBS and resuspended in DPBS at a density of 1.0×10 6 cells / mL. Then, the apoptotic cells were labeled with 0.5 μM CFSE at 37 °C for 5 min in the dark. After labeling, the apoptotic cells were washed 3 times with medium and resuspended in medium. Using human CD14 microbeads (Miltenyi Biotec), CD14+ monocytes were isolated from the buffy coat of healthy donors. The CD14+ monocytes were cultured for 7 days in the presence of 100 ng / ml M-CSF to differentiate into M0 macrophages showing upregulated MerTK expression. Then the macrophages were at 4.0×104 Cells were seeded into 96-well microplates at a density of 4 cells / well. Serial dilutions of the antibody were incubated with macrophages in 96-well plates for 20 min. Then, freshly prepared CFSE-labeled apoptotic Jurkat cells were added to the wells at a density of 2.0×10 5 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. QuantAnalyzer 16 was used to quantify phagocytosis events. CD14 + CFSE + cells represent macrophages that have phagocytosed apoptotic Jurkat cells. The efferocytosis assay demonstrated that the anti-MerTK antibody was able to inhibit the phagocytosis of apoptotic cells by human macrophages. Figure 6 The results in Figure 6 showed that some of the tested MerTK antibodies exhibited more potent blocking efficacy in efferocytosis when compared to h13B4.v16.

[0185] Example 5: Humanization of Chimeric MerTK Antibodies

[0186] The variable regions of the chimeric antibodies 10F7D9, 265F11B5, and 254B4D9 were selected for humanization. Briefly, the amino acid sequences of VH and VL were aligned with existing human Ig gene sequence databases to identify the most globally matching human germline Ig gene sequences. For each clone, the CDRs of the light and heavy chains were grafted onto the candidate human germlines. Then, 3D models were generated to determine if any critical murine amino acids were present in the framework regions, whose replacement with human amino acids might affect binding and / or CDR conformation. To maintain the structure and function of the humanized antibodies, key amino acids were selected for back-mutation. Then, the humanized variable regions of the antibodies were fused to the constant region of human IgG1 LALAPG for antibody production.

[0187] For the light chain of 10F7D9, the candidate germline was the IGKV6-21*01 gene. For the heavy chain, the candidate germline was the IGHV7-4-1*02 gene. In the case of the light chain, L45P, L46W, K48Y, and Y86F in the framework region were involved in back-mutation. In the case of the heavy chain, V2I, V20I, R38K, E46K, S84N, S85N, Y95F, and R98T in the framework region were involved in back-mutation.

[0188] 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.

[0189] For the light chain of 254B4D9, the candidate germline is the IGKV1-33*01 gene, and for the heavy chain, the candidate germlines are the IGKV7-4-1*02 or IGKV7-81*01 genes. In the case of the light chain, Y49H, T69R, and Y87F in the framework region are involved in back mutations. In the case of the heavy chain, R38K, P38K, E46Q, M72L, Y80F, and Y95F are included as back mutations. (Tables 4 and 5).

[0190] Table 4. Humanized antibody sequences (underlined / bold indicate CDRs; bold / italic indicate back mutations)

[0191]

[0192]

[0193]

[0194] Table 5. Pairing of VH and VL of humanized antibodies

[0195]

[0196]

[0197] 254B4D9 HU-VL1 VL HU-VH1 Hu-1 HU-VH2 Hu-2 VH Chimeric

[0198] Example 6: Antigen-binding properties of humanized antibodies

[0199] Binding to recombinant human MerTK

[0200] To evaluate the antigen-binding activity, as described previously, ELISA tests were performed on the humanized antibodies. As Figure 7 shown, the humanized antibodies showed comparable binding efficacy to human MerTK as their parental chimeric antibodies.

[0201] Using Biacore TM To grade the affinity of the humanized antibodies

[0202] To explore whether the humanized antibodies could maintain their binding kinetics, Biacore was used TMAffinity grading was performed. A protein A chip was used to capture the antibodies. The human MerTK-his protein at 50 nM was injected onto the captured antibodies at a flow rate of 30 μL / min for 180 s. The antigen was dissociated for 600 s. Experiments were carried out on a Biacore TM 8K. Data analysis was performed using the Biacore TM 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.

[0203] Table 6. Affinity ranking results of humanized antibodies

[0204]

[0205] Binding to CHO-K1 cells overexpressing human MerTK

[0206] To evaluate the binding characteristics to the antigen expressed on the cells, as described above, FACS was used to analyze the humanized antibodies. As Figure 8 shown, the humanized antibodies 265F11B5 and 254B4D9 showed comparable cell binding efficacies to their chimeric antibodies. At the same time, some humanized antibodies of 10F7D9 showed comparable cell binding efficacies to their chimeric antibodies.

[0207] ***

[0208] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be illustrative 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 can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, 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.

[0209] All publications and patent applications mentioned in this specification are incorporated herein 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 antigen-binding fragment thereof that is specific for human proto-oncogene tyrosine-protein kinase (MerTK) protein and comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and the light-chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein: the VH CDR1 is the amino acid sequence of SEQ ID NO:25; the VH CDR2 is the amino acid sequence of SEQ ID NO:26; the VH CDR3 is the amino acid sequence of SEQ ID NO:27; the VL CDR1 is the amino acid sequence of SEQ ID NO:28; the VL CDR2 is the amino acid sequence of SEQ ID NO:29; and the VL CDR3 is the amino acid sequence of SEQ ID NO:

30.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 82 - 87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 89 - 91.

3. The antibody or antigen-binding fragment thereof according to claim 1, 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.

4. The antibody or antigen-binding fragment thereof according to claim 1, 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.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 - 4, wherein the antibody or antigen-binding fragment 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.

6. One or more polynucleotides that encode the antibody or antigen-binding fragment thereof according to any one of claims 1 - 5.

7. The one or more polynucleotides according to claim 6, wherein the one or more polynucleotides are one or more mRNAs.

8. The one or more polynucleotides according to claim 7, wherein the mRNA is chemically modified.

9. A cell that comprises the one or more polynucleotides according to any one of claims 6 - 8.

10. A composition that comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 - 5, and a pharmaceutically acceptable carrier.

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