Anti-mer-tk antibodies and uses thereof

Abstract

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

Description

[0001] This patent application is a divisional application of the patent application filed on July 28, 2023, with application number 202380056183.6 and invention title "Anti-MerTK Antibody and Its Use". Technical Field

[0002] This invention relates to an anti-MerTK antibody and its uses. Background Technology

[0003] Currently, most cancer immuno-oncology therapies focus on modulating the activity of adaptive immune cells (especially T cells) by blocking inhibitory pathways (including checkpoint molecules) or by redirecting the binding of tumor-associated antigens (TAAs) to fight tumors. However, data from numerous studies in mouse models and cancer patients provide compelling evidence that specific innate immune cell types, including tumor-associated macrophages (TAMs) and bone marrow-derived suppressor cells (MDSCs), primarily function as exogenous tumor suppressor mechanisms, limiting the clinical benefit of adaptive immune cell-based therapies. Targeting the innate immune system may complement adaptive immuno-oncology therapies, thereby achieving a durable anti-tumor response.

[0004] Macrophages in the innate immune system are a collection of various cell types that play diverse functional roles under both homeostatic and pathological conditions. M1 macrophages are classically activated macrophages that exert pro-inflammatory effects to clear intracellular pathogens. In contrast, M2 macrophages are alternatively activated macrophages that contribute to tissue repair and necrolysis. Macrophages are professional phagocytic cells, highly specialized to clear the large quantities of dying or dead cells and cellular debris generated under normal physiological conditions. Furthermore, macrophages are extremely abundant in many types of solid tumors. Macrophages in the tumor microenvironment are known as tumor-associated macrophages (TAMs), which typically promote cancer cell initiation and proliferation, accelerate angiogenesis, and suppress anti-tumor immunity, thereby driving tumor progression and metastasis. Increasing evidence suggests that TAMs may be one of the reasons for the relatively low response rates to T-cell-based therapies. In solid tumors, uncontrolled tumor growth is often accompanied by increased cell death due to hypoxia and metabolic stress. To evade immune surveillance, tumors utilize the non-immunogenic nature of apoptosis. TAM actively eliminates dying tumor cells while reducing the production of inflammatory cytokines to avoid alarming the immune system.

[0005] MerTK (Mer proto-oncogene tyrosine protein kinase) has been shown to play a role in clearing apoptotic cells. MerTK is a member of the TYRO3 / AXL / MER (TAM) receptor kinase family and encodes a transmembrane protein containing two fibronectin type III domains, two Ig-like C2 (immunoglobulin-like) domains, and one tyrosine kinase domain. MerTK expression has been observed to be higher in M2 macrophages than in M1 macrophages. MerTK helps clear dying or damaged cells that display an "eat me" signal (i.e., phosphatidylserine, PtdSer) on their cell surface, a process that requires the assistance of bridging molecule growth arrest-specific protein 6 (Gas6) or protein S. Macrophages expressing MerTK engulf apoptotic cells through endocytosis. In tumors, uncontrolled proliferation leads to increased cancer cell apoptosis, but TAM-dependent MerTK clearance of dying cells may suppress immune activation.

[0006] Blocking MerTK leads to the accumulation of apoptotic cells within tumors and triggers a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibodies stimulates T cell activation and enhances the efficacy of anti-PD-1 and anti-PD-L1 therapies. Therefore, blocking MerTK can enhance tumor immunogenicity and boost anti-tumor immunity, providing a therapeutic pathway to enhance tumor immunogenicity and improve cancer immunotherapy. Summary of the Invention

[0007] In various embodiments, this disclosure provides antibody- and antigen-binding fragments specific to human MerTK protein. One embodiment provides an antibody or antigen-binding fragment thereof that is specific for human Mer 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 comprises VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3. The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO:25-30; SEQ ID NO:59-64; SEQ ID NO:65-70; SEQ ID NO:31-33, 28-29, 34; SEQ ID NO:35-40; SEQ ID NO:41-46; SEQ ID NO:47-52; SEQ ID NO:53-58 ... 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.

[0008] In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO:25; VH CDR2 contains the amino acid sequence of SEQ ID NO:26; VH CDR3 contains the amino acid sequence of SEQ ID NO:27; VL CDR1 contains the amino acid sequence of SEQ ID NO:28; VL CDR2 contains the amino acid sequence of SEQ ID NO:29; and VL CDR3 contains the amino acid sequence of SEQ ID NO:30.

[0009] 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.

[0010] 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.

[0011] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO:59; the VH CDR2 contains the amino acid sequence of SEQ ID NO:60; the VH CDR3 contains the amino acid sequence of SEQ ID NO:61; the VL CDR1 contains the amino acid sequence of SEQ ID NO:62; the VL CDR2 contains the amino acid sequence of SEQ ID NO:63; and the VL CDR3 contains the amino acid sequence of SEQ ID NO:64.

[0012] 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.

[0013] In some embodiments, the VH CDR1 contains the amino acid sequence of SEQ ID NO:65; the VH CDR2 contains the amino acid sequence of SEQ ID NO:66; the VH CDR3 contains the amino acid sequence of SEQ ID NO:67; the VL CDR1 contains the amino acid sequence of SEQ ID NO:68; the VL CDR2 contains the amino acid sequence of SEQ ID NO:69; and the VL CDR3 contains the amino acid sequence of SEQ ID NO:70.

[0014] 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.

[0015] In some embodiments, the antibody or a fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

[0016] In one embodiment, a multispecific antibody is also provided, comprising the antigen-binding fragment disclosed herein and one or more antibodies or antigen-binding fragments having binding specificity to non-MerTK target antigens.

[0017] Furthermore, a chimeric antigen receptor (CAR) is provided, which includes the antigen-binding fragment, transmembrane domain, co-stimulatory domain and CD3ξ intracellular domain disclosed herein.

[0018] Also provided are one or more polynucleotides encoding the antibody or its antigen-binding fragment or CAR disclosed herein. In some embodiments, the polynucleotide is one or more mRNAs. In some embodiments, the mRNA is chemically modified.

[0019] In one embodiment, a method for treating cancer or an inflammatory condition in a patient in need is also provided, the method comprising administering to the patient an effective amount of the disclosed antibody or its antigen-binding fragment or CAR. 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, stomach cancer, cervical cancer, uterine cancer, and thyroid cancer.

[0020] In some embodiments, the inflammatory condition 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. Attached Figure Description

[0021] Figure 1 This demonstrates that all tested anti-MerTK antibodies can effectively bind to the human MerTK protein.

[0022] Figure 2 The results showed that all the tested anti-MerTK antibodies could effectively bind to the cynomolgus monkey MerTK protein.

[0023] Figure 3 The results showed that most of the tested MerTK chimeric antibodies had higher maximum binding capacity and binding potency for human MerTK expressed on CHO-K1 cells compared to the benchmark antibodies Ab2000-A7, M6 and h13B4.v16.

[0024] Figure 4The results showed that some of the tested MerTK chimeric antibodies exhibited higher binding efficacy to human MerTK expressed on SK-MEL-5 cells compared to the benchmark antibodies Ab2000-A7, M6, and h13B4.v16.

[0025] Figure 5 It was shown that all of these antibodies could effectively inhibit human MerTK from binding to human Gas6 expressed on cells.

[0026] Figure 6 Some of the tested MerTK antibodies were shown to have higher blocking efficiency than the baseline antibody h13B4.v16 in terms of cell closure.

[0027] Figure 7 This demonstrates that all the humanized antibodies tested possessed binding efficacy to the human MerTK protein comparable to that of the chimeric antibodies.

[0028] Figure 8 Some of the tested humanized antibodies were shown to have binding activity against human MerTK expressed on CHOK1 cells comparable to their parental chimeric counterparts. Detailed Implementation

[0029] definition

[0030] It should be noted that the terms "a" or "an" refer to one or more of the entities described; for example, "an antibody" should be understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0031] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody as well as any antigen-binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule having the biological activity of binding an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region, or any portion thereof, or at least a portion of the binding protein.

[0032] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that exerts its antibody effect by binding to a specific antigen to form a complex.

[0033] The term antibody encompasses a wide range of polypeptide classes that can be distinguished by biochemical methods. Those skilled in the art will recognize that heavy chains are classified as γ, μ, α, δ, or ε, with several subclasses (e.g., γ1–γ4). It is the properties of this chain that determine the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE.

[0034] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specialization. In view of this disclosure, those skilled in the art can readily identify modified versions of these classes and isotypes, which are accordingly covered within the scope of this disclosure. All immunoglobulin classes are obviously covered within the scope of this disclosure, and the following discussion will generally refer to immunoglobulin molecules of the IgG class. Regarding 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 molecular weights of 53,000–70,000 Daltons. These four chains are typically linked by disulfide bonds in a “Y” configuration, wherein the light chain begins at the opening of the “Y”, wraps around the heavy chain, and extends into the variable region.

[0035] The antibodies, antigen-binding peptides, variants, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate, 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 containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0036] As used herein, the term "chimeric antibody" should be understood to mean any antibody in which the immune-reactive region or site is derived from or obtained from a first species, and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be of human origin.

[0037] The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, these antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region may be of condricthoid origin (e.g., from sharks).

[0038] As used herein, the term “recombinant” in relation to polypeptides or polynucleotides refers to a form of polypeptide or polynucleotide that is not naturally occurring, and non-limiting examples of which can be produced by combining polynucleotides that do not normally occur together.

[0039] Hybridoma technology can be performed under varying “strictness” conditions. Generally, low-strictness hybridization reactions are carried out at approximately 40°C in solutions with approximately 10x SSC or equivalent ionic strength / temperature. Medium-strictness hybridization is typically carried out at approximately 50°C in solutions with approximately 6x SSC, and high-strictness hybridization reactions are typically carried out at approximately 60°C in solutions with approximately 1x SSC. Those skilled in the art will recognize that hybridization reactions can also be performed under “physiological conditions.” Non-limiting examples of physiological conditions are temperature, ionic strength, pH, and Mg2+ concentration commonly found in cells.

[0040] anti-MerTK antibody

[0041] As demonstrated in the accompanying experimental examples, the inventors were able to generate anti-MerTK antibodies 10F7D9, 85H8D5, 216G3D6, 247E5A8, 252C12B10, 252H9D5, 254B4D9, 265F11B5, 276C2D1, 280C6A3, 293C2B7, and 300A5A3 (Table 1). Equally important, many of these antibodies exhibited higher binding affinity to human MerTK protein expressed on cells than benchmark antibodies (including M6, Ab2000-A7, and h13B4.v16 (as disclosed in WO 2019084307A1, WO 2016106221A1, and WO 2020214995A1)). Furthermore, some of these antibodies exhibited higher ligand-binding blocking activity and cell burial inhibition efficiency than these benchmark antibodies.

[0042] According to one embodiment of this disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or the antigen-binding fragment thereof has binding specificity to human MerTK protein. In some embodiments, the antibody or the antigen-binding fragment thereof includes 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 VLCDR3.

[0043] In some embodiments, an antibody or 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; VLCDR2 comprises the amino acid sequence of SEQ ID NO:29; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:30.

[0044] An exemplary VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 82-87. An exemplary VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 89-91.

[0045] 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.

[0046] 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.

[0047] In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:1 and 82-87, and VL comprises the amino acid sequence of SEQ ID NO:90. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:82-87, and VL comprises the amino acid sequence of SEQ ID NO:90. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:1 and 82-87, and VL comprises the amino acid sequence of SEQ ID NO:91. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:82-87, and VL comprises the amino acid sequence of SEQ ID NO:91.

[0048] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 10F7D9. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 10F7D9 for binding to MerTK.

[0049] In some embodiments, an antibody or antigen-binding fragment derived from antibody 85H8D5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:31; VH CDR2 comprises the amino acid sequence of SEQ ID NO:32; VH CDR3 comprises the amino acid sequence of SEQ ID NO:33; VL CDR1 comprises the amino acid sequence of SEQ ID NO:28; VLCDR2 comprises the amino acid sequence of SEQ ID NO:29; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34.

[0050] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:3. An exemplary VL sequence comprises 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.

[0051] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 85H8D5. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 85H8D5 for binding to MerTK.

[0052] In some embodiments, an antibody or antigen-binding fragment derived from antibody 216G3D6 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:35; VH CDR2 comprises the amino acid sequence of SEQ ID NO:36; VH CDR3 comprises the amino acid sequence of SEQ ID NO:37; VL CDR1 comprises the amino acid sequence of SEQ ID NO:38; VLCDR2 comprises the amino acid sequence of SEQ ID NO:39; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40.

[0053] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:5. An exemplary VL sequence comprises 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 with SEQ ID NO:5, while retaining the corresponding VH CDR. 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 with SEQ ID NO:6, while retaining the corresponding VL CDR.

[0054] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 216G3D6. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 216G3D6 for binding to MerTK.

[0055] In some embodiments, an antibody or antigen-binding fragment derived from antibody 247E5A8 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of SEQ ID NO:42; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VLCDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:46.

[0056] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:7. An exemplary VL sequence comprises 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 with 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 with SEQ ID NO:8, while retaining the corresponding VL CDR.

[0057] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 247E5A8. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 247E5A8 for binding to MerTK.

[0058] In some embodiments, an antibody or antigen-binding fragment derived from antibody 252C12B10 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:47; VH CDR2 comprises the amino acid sequence of SEQ ID NO:48; VH CDR3 comprises the amino acid sequence of SEQ ID NO:49; VL CDR1 comprises the amino acid sequence of SEQ ID NO:50; VLCDR2 comprises the amino acid sequence of SEQ ID NO:51; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:52.

[0059] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:9. An exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10, while retaining the corresponding VL CDR.

[0060] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 252C12B10. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 252C12B10 for binding to MerTK.

[0061] In some embodiments, an antibody or antigen-binding fragment derived from antibody 252H9D5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:53; VH CDR2 comprises the amino acid sequence of SEQ ID NO:54; VH CDR3 comprises the amino acid sequence of SEQ ID NO:55; VL CDR1 comprises the amino acid sequence of SEQ ID NO:56; VLCDR2 comprises the amino acid sequence of SEQ ID NO:57; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:58.

[0062] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:11. An exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:11, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:11, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:12, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12, while retaining the corresponding VL CDR.

[0063] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 252H9D5. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 252H9D5 for binding to MerTK.

[0064] In some embodiments, an antibody or antigen-binding fragment derived from antibody 254B4D9 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:59; VH CDR2 comprises the amino acid sequence of SEQ ID NO:60; VH CDR3 comprises the amino acid sequence of SEQ ID NO:61; VL CDR1 comprises the amino acid sequence of SEQ ID NO:62; VLCDR2 comprises the amino acid sequence of SEQ ID NO:63; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:64.

[0065] An exemplary VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 102, and 104. An exemplary VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and 106.

[0066] 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 with 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 with any one of SEQ ID NO:14 and 106, while retaining the corresponding VL CDR.

[0067] 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.

[0068] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 254B4D9. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 254B4D9 for binding to MerTK.

[0069] In some embodiments, an antibody or antigen-binding fragment derived from antibody 265F11B5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:65; VH CDR2 comprises the amino acid sequence of SEQ ID NO:66; VH CDR3 comprises the amino acid sequence of SEQ ID NO:67; VL CDR1 comprises the amino acid sequence of SEQ ID NO:68; VLCDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:70.

[0070] An exemplary VH sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO:15 and 93-96. An exemplary VL sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO:16 and 98-100.

[0071] 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 with 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 with any one of SEQ ID NO:16 and 98-100, while retaining the corresponding VL CDR.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Therefore, in some embodiments, antibody-antigen binding fragments are also provided that bind to the same epitope on MerTK that binds to 265F11B5. Therefore, in some embodiments, antibody-antigen binding fragments are also provided that compete with 265F11B5 for binding to MerTK.

[0076] In some embodiments, an antibody or antigen-binding fragment derived from antibody 276C2D1 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:71; VH CDR2 comprises the amino acid sequence of SEQ ID NO:66; VH CDR3 comprises the amino acid sequence of SEQ ID NO:72; VL CDR1 comprises the amino acid sequence of SEQ ID NO:73; VLCDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74.

[0077] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:17. An exemplary VL sequence comprises 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.

[0078] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 276C2D1. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 276C2D1 for binding to MerTK.

[0079] In some embodiments, an antibody or antigen-binding fragment derived from antibody 280C6A3 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:65; VH CDR2 comprises the amino acid sequence of SEQ ID NO:75; VH CDR3 comprises the amino acid sequence of SEQ ID NO:67; VL CDR1 comprises the amino acid sequence of SEQ ID NO:76; VLCDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:77.

[0080] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:19. An exemplary VL sequence comprises 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 with 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 with SEQ ID NO:20, while retaining the corresponding VL CDR.

[0081] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 280C6A3. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 280C6A3 for binding to MerTK.

[0082] In some embodiments, an antibody or antigen-binding fragment derived from antibody 293C2B7 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:71; VH CDR2 comprises the amino acid sequence of SEQ ID NO:78; VH CDR3 comprises the amino acid sequence of SEQ ID NO:79; VL CDR1 comprises the amino acid sequence of SEQ ID NO:73; VLCDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74.

[0083] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:21. An exemplary VL sequence comprises 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.

[0084] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 293C2B7. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 293C2B7 for binding to MerTK.

[0085] In some embodiments, an antibody or antigen-binding fragment derived from antibody 300A5A3 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:65; VH CDR2 comprises the amino acid sequence of SEQ ID NO:66; VH CDR3 comprises the amino acid sequence of SEQ ID NO:80; VL CDR1 comprises the amino acid sequence of SEQ ID NO:81; VLCDR2 comprises the amino acid sequence of SEQ ID NO:69; and VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:70.

[0086] An exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:23. An exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:23, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:23, while retaining the corresponding VH CDR. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:24, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:24, while retaining the corresponding VL CDR.

[0087] Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that bind to the same epitope on MerTK that binds to 300A5A3. Therefore, in some embodiments, antibody and antigen-binding fragments are also provided that compete with 300A5A3 for binding to MerTK.

[0088] In some embodiments, an antibody-antigen binding fragment is also provided, comprising a CDR sequence derived from the CDR sequence disclosed herein, having one, two, or three amino acid substitutions, deletions, and / or additions.

[0089] In some embodiments, the antibody or a fragment thereof can induce antibody-dependent cytotoxicity (ADCC). In some embodiments, the antibody or a fragment thereof cannot induce antibody-dependent cytotoxicity (ADCC).

[0090] Multifunctional group molecules

[0091] Multifunctional molecules, including antibodies or antigen-binding fragments specific to MerTK, such as those disclosed herein, and one or more antibodies or antigen-binding fragments specific to a second antigen.

[0092] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0093] 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 another embodiment, the second antigen is PD-L1.

[0094] Different forms of bispecific antibodies are also provided. In some embodiments, the anti-MerTK fragment and the second fragment are each independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody further includes an Fc fragment.

[0095] Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As molecules targeting tumor antigens, antibodies or antigen-binding fragments specific to MerTK (such as those described herein) can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blockade effects with local immunomodulation at the tumor site.

[0096] Chimeric antigen receptor

[0097] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR comprises an antibody or fragment thereof disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

[0098] The transmembrane domain can be designed to fuse with an extracellular domain, including an antibody or fragment, optionally via a hinge domain. It can also fuse with an intracellular domain, such as a co-stimulatory domain. In some embodiments, the transmembrane domain may include a native transmembrane region of a co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB used as a co-stimulatory domain) or a native transmembrane domain of a hinge region (e.g., the TM region of CD8α or CD28T used as a hinge domain).

[0099] In some embodiments, a transmembrane domain may include a sequence that crosses the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane domain may include a transmembrane sequence that may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids extending into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include one or more amino acids extending into the inner or outer surface of the membrane itself; such sequences may still be considered "transmembrane domains".

[0100] In some embodiments, the transmembrane domain fuses with the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine duplexes (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0101] In some embodiments, the CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Examples of co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), and CD48 (SLAMF2). CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD 103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1 ), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(S LAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (KG2D), CD319 (SLAMF7), CD335 (K-p46), CD336 (K-p44), CD337 (K-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF 18), Inducible T cell co-stimulatory molecules (ICOS), LFA-1 (CD11a / CD12)18) KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and its fragments or combinations.

[0102] In some embodiments, the cytoplasmic portion of the CAR further includes a signal transduction / activation domain. In one embodiment, the signal transduction / activation domain is a CD3ξ domain, or its amino acid sequence has at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the CD3ξ domain.

[0103] Polynucleotides, mRNA, and methods for expressing or preparing antibodies

[0104] This disclosure also provides polynucleotide or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs disclosed herein. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of antigen-binding peptides, variants or derivatives thereof, on the same or different polynucleotide molecules. Furthermore, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of antigen-binding peptides, variants or derivatives thereof, on the same or separate polynucleotide molecules.

[0105] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA may be introduced into target cells to express an antibody or a fragment thereof.

[0106] mRNA can be synthesized using any of a variety of known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). In short, IVT typically uses a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0107] In some embodiments, to prepare mRNA encoding an antibody, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter (e.g., a T3, T7, or SP6 promoter) for in vitro transcription, followed by the desired nucleotide sequence for encoding the desired antibody (e.g., encoding a heavy or light chain) and a termination signal.

[0108] The mRNA sequence encoding the desired antibody (e.g., encoding the heavy or light chain) can be determined using standard methods and incorporated into a DNA template. For example, virtual reverse translation based on the degenerate genetic code can be performed, starting with the desired amino acid sequence (e.g., the desired heavy or light chain sequence). Optimization algorithms can then be used to select appropriate codons. Typically, the G / C ratio can be optimized to achieve the highest possible G / C ratio, while the frequency of the tRNA can be considered as much as possible based on codon usage. The optimized RNA sequence can be constructed and displayed, for example, using a suitable display device, and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate the stability and instability properties of the RNA, or its regions can be calculated separately.

[0109] mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance its stability. Modifications to mRNA can include, for example, modifications to the nucleotides of the RNA. Therefore, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives as purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, etc. Glycoside, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil 5-Methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, huaistin, as well as amide phosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-denitroguanine, 5-methylcytosine, and inosine. The preparation of such analogues 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 entirety.

[0110] In some embodiments, mRNA (e.g., mRNA encoding both heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are modifications that chemically modify the phosphate groups 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 methylphosphonates, methylaminophosphates, aminophosphates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), borophosphates, positively charged guanidine groups, etc., which means replacing the phosphodiester bonds with other anionic, cationic, or neutral groups.

[0111] In some embodiments, mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications to the sugars of the nucleotides it contains, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), and 2'-deoxy-2'-deamine-oligonucleotides (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'-cytarabine 5'-triphosphate, 2'-cytarabine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0112] In some embodiments, mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine ribonucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deadenosine 5'-triphosphate, 7-deadenosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthine nucleoside 5'-triphosphate.

[0113] Typically, mRNA synthesis involves adding a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). 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 degradation by exonucleases.

[0114] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding both the heavy and light chains) 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 phosphates via a guanylate transferase, creating a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is methylated via 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.

[0115] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 3' poly(A) tail. The poly(A) tail at the 3' end of the mRNA typically comprises 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 the heavy and light chains) includes a 3' poly(C) tail. A suitable poly-C tail at the 3' end of mRNA typically comprises about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to or may replace the poly-A tail.

[0116] Therefore, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) 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-responsive element. In some embodiments, the length of the 5' untranslated region can be about 50 to 500 nucleotides (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

[0117] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is directly or indirectly linked at the N-terminus to a sequence encoding the heavy or light chain.

[0118] The technology of this 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. This invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or compositions, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0119] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide disclosed herein are fully human. Fully human antibodies can be prepared 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 an antigen challenge, but whose endogenous loci have been deactivated. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584; 6,458,592; 6,420,140, ​​the contents of which are incorporated herein by reference in their entirety.

[0120] Treatment and Uses

[0121] As described herein, the antibodies, variants, or derivatives disclosed herein may be used in certain therapeutic and diagnostic approaches.

[0122] This disclosure further relates to antibody-based therapies involving the administration of the disclosed antibodies or fragments to patients (e.g., animals, mammals, and humans) to treat one or more of the disorders or conditions described herein. Therapeutic compounds disclosed herein include, but are not limited to, the antibodies disclosed (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the antibodies disclosed herein (including variants and derivatives thereof as described herein).

[0123] The disclosed antibody can also be used to treat or inhibit cancer. As mentioned above, MerTK is expressed very little in normal adult tissues, but is highly expressed in the placenta and most of the most common tumors, typically showing high expression in more than 80% of renal cell carcinoma, breast cancer, colon cancer, prostate cancer, and ovarian cancer.

[0124] Therefore, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody or fragment disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses MerTK.

[0125] This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. Suitable cells can be used, transduced with or contacted with a CAR encoding a CAR (or alternatively engineered to express the anti-MerTK antibody disclosed herein), the CAR comprising the anti-MerTK antibody disclosed herein. After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer as disclosed herein. The cells (e.g., T cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0126] 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 from a cell bank. Isolating cells from a cancer patient can minimize adverse immune responses.

[0127] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of stomach cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0128] Other diseases or conditions associated with increased cell survival (which can be treated, prevented, diagnosed, and / or predicted by the antibodies disclosed herein or their variants or derivatives) 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 myeloblastic, promyelocytic, myelomonocytic, monocytic, 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 macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, and chondrosarcoma). Osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystadenoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma.

[0129] In some embodiments, the antibodies, polynucleotides, or compositions disclosed herein may be used to treat inflammatory diseases or conditions. In some embodiments, the inflammatory diseases or conditions to be treated using the disclosed antibodies, fragments, and compositions include one or more of the following: Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

[0130] In some embodiments, the autoimmune diseases or conditions for treatment using the disclosed antibodies, fragments, and compositions include one or more of the following: alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus type 1, celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatous polyangiitis (Wegener's granulomatosis).

[0131] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. RA typically causes hot, swollen, and painful joints. Pain and stiffness usually worsen after rest. Most commonly, it affects the wrists and hands, and often the same joints on both sides of the body. The disease can also affect other parts of the body. While the exact cause of rheumatoid arthritis is not fully understood, it is believed to be related to a combination of genetic and environmental factors. Its underlying mechanism involves the body's immune system attacking these joints. This leads to inflammation and thickening of the joint capsule. The goal of treatment is to reduce pain, reduce inflammation, and improve overall bodily function. Painkillers, steroids, and NSAIDs are commonly 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.

[0132] Osteoarthritis (OA) is a group of joint diseases caused by the destruction and degeneration of articular cartilage and underlying bone. The most common symptoms are joint pain and stiffness. Initially, symptoms may only appear after exercise, but over time they may become persistent. Other symptoms may include joint swelling, reduced range of motion, and, when the spine is involved, 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, or work in jobs that put high pressure on their joints are at higher risk. Osteoarthritis is thought to be caused by mechanical stress on the joints and low-grade inflammatory processes. Treatment includes exercise, minimizing stress on the joints, support groups, and pain medication.

[0133] 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 communication between different parts of the nervous system, leading to a range of signs and symptoms, including physical, psychological, and sometimes psychiatric problems. Specific symptoms may include diplopia, monocular blindness, muscle weakness, sensory disturbances, or difficulty coordinating. Although the cause is not fully understood, the underlying mechanisms are thought to be either the disruptive effects of the immune system or dysfunction of myelin-producing cells. There is currently no known cure for multiple sclerosis. Treatment aims to improve bodily function after an attack and prevent new attacks.

[0134] Asthma is a common, chronic inflammatory disease of both lung airways. It is characterized by variable and recurrent symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include wheezing attacks, cough, chest tightness, and shortness of breath. Asthma is believed to be caused by a combination of genetic and environmental factors. Environmental factors include exposure to air pollution and allergens. Asthma is classified according to the frequency of symptom occurrence, forced expiratory volume in one second (FEV1), and peak expiratory flow. Asthma can also be classified as atopic or non-atopic, with atopic referring to a predisposition to 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, long-acting beta-agonists (LABAs) or leukotriene inhibitors may be used in addition to inhaled corticosteroids. For rapidly worsening symptoms, treatment typically involves 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.

[0135] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by chronic insufficient airflow. COPD can include two main conditions: emphysema and chronic bronchitis. In emphysema, the walls between many alveoli are damaged. As a result, these alveoli lose their shape and become loose. This damage also destroys the alveolar walls, leading to a reduction in the number of alveoli and an increase in their size; they are no longer numerous tiny alveoli. When this happens, the amount of gas exchange in both lungs decreases. In chronic bronchitis, the lining of the airways is constantly irritated and inflamed, which causes the lining to swell. A large amount of thick mucus forms in the airways, leading to difficulty breathing. There is currently no known cure for COPD, but its symptoms can be treated and its progression can be slowed.

[0136] Pain is a distressing sensation, usually triggered by strong or harmful stimuli, such as a bruised toe, a burned finger, an alcohol-soaked wound, or a bump on a numb spot. Pain is a complex subjective phenomenon, and defining it has always been a challenge. Pain is also known as an uncomfortable sensory and emotional experience associated with actual or potential tissue damage. Pain is sometimes considered a symptom of an underlying condition, such as inflammation.

[0137] The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, diet and timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of healthcare professionals regarding these factors is within the realm of general technical skill in this field. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0138] Methods of administering antibodies or fragments include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through the epithelial or mucosal layers of the skin (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides disclosed herein can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as oral or nasal sprays.

[0139] As used in this article, the term “parenteral” refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0140] Administration can be systemic or local. Alternatively, it is preferable to introduce the disclosed antibodies into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations containing nebulizing agents.

[0141] Preferably, the antigen-binding peptides or compositions disclosed herein are applied topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, topical application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implantation, said implant being a porous, non-porous, or gel-like material, including membranes such as sialastic membranes or fibers. Preferably, when administering the proteins (including antibodies) disclosed herein, care must be taken to use materials that are not absorbed by the protein.

[0142] The amount of the disclosed antibody or fragment that is effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined using standard clinical techniques. Additionally, in vitro assays may be optionally used to help determine the ideal 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 based on the physician's judgment and the individual patient's situation. The effective dose can be deduced from dose-response curves derived from in vitro or animal model testing systems.

[0143] As a general recommendation, the dosage of the antibodies or fragments disclosed herein administered to patients is typically 0.001 mg / kg to 100 mg / kg of patient body weight, 0.01 mg / kg to 20 mg / kg of patient body weight, or 0.5 mg / kg to 10 mg / kg of patient body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, it is generally possible to reduce the dosage and frequency of administration of human antibodies. Furthermore, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by modifications (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

[0144] In another embodiment, the disclosed compositions are administered in combination with cytokines. Cytokines that can be administered with the disclosed compositions 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.

[0145] In another embodiment, the disclosed composition is administered in combination with other therapeutic or preventative regimens (e.g., radiotherapy).

[0146] Composition

[0147] This 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 anticancer agent (e.g., an immune checkpoint inhibitor).

[0148] In certain embodiments, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. Furthermore, "pharmaceuticalally acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0149] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for regulating tension, such as sodium chloride or dextrose, are also considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories with conventional binders and carriers (such as triglycerides). Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EWMartin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) and an appropriate carrier to provide a suitable form of administration to the patient. The formulation should be suitable for the mode of administration. Parenteral preparations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0150] In one embodiment, the composition is formulated according to standard procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Generally, the ingredients are provided individually or in combination in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers (such as ampoules or pouches), with the amount of active agent indicated. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a single ampoule of sterile water for injection or saline can be provided to mix the ingredients prior to administration.

[0151] The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts that form with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts that form with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0152] Example

[0153] Example 1: Generation of mouse monoclonal antibody (mAb) against human MerTK

[0154] This example describes the preparation of anti-human MerTK mouse monoclonal antibodies using hybridoma technology.

[0155] 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 the his tag at the C-terminus.

[0156] Immunization: To generate mouse monoclonal antibodies against human MerTK, Balb / c, SJL, C57BL / 6, and SD mice were immunized intraperitoneally and subcutaneously every two weeks using human MerTK-Fc or MerTK-his protein. Serum titers in immunized mice were monitored using ELISA against human MerTK-his and cynomolgus monkey MerTK-his proteins and FACS against human MerTK overexpressed on the CHO-K1 cell line (CHO-K1-hMerTK), with the CHO-K1 parental cell line serving as a negative control. After 2–4 rounds of immunization, mice with sufficient titers were boosted with 25 μg of human MerTK-his protein, and these mice were then selected for fusion.

[0157] Cell fusion and hybridoma screening: Selected mouse spleen cells were fused with the mouse myeloma cell line Sp2 / 0 via electrofusion. The supernatants of these hybridoma cells were first screened using an ELISA targeting the human MerTK-his protein. Then, the supernatants of positive clones were screened using an ELISA targeting the cynomolgus monkey MerTK-his protein, and their ability to block MerTK binding to its ligand, growth arrest-specific protein 6 (Gas6), was assessed using an ELISA-based receptor blocking assay. Subcloning was performed on the positive primary clones resulting from each fusion, and the subclones were further confirmed by an ELISA-based receptor blocking assay and affinity grading. Hybridoma clones exhibiting high binding and blocking abilities were sequenced and selected for further analysis. The amino acid sequences of the variable regions of these clones are provided in Table 1 below.

[0158] Table 1. Sequences of the variable regions of the selected clones (underlined / bold indicates CDR)

[0159]

[0160]

[0161]

[0162] Table 1A. CDR sequences of selected clones

[0163]

[0164]

[0165]

[0166] Example 2: Antigen binding properties of anti-MerTK chimeric monoclonal antibodies

[0167] The variable region of a mouse MerTK antibody was fused to the constant region of human IgG1 containing the L234A / L235A / P329G (LALAPG) mutation to generate a chimeric monoclonal antibody without Fc binding ability. The baseline antibodies (including M6, Ab2000-A7, and h13B4.v16) were prepared individually using the sequences described in patent applications WO 2019084307A1 / WO 2016106221A1 / WO2020214995A1 according to the method described above. This example tested the binding properties of the anti-MerTK chimeric monoclonal antibody.

[0168] ELISA binding activity of anti-MerTK chimeric antibody against human and cynomolgus monkey MerTK protein

[0169] To evaluate binding activity, these chimeric mAbs were subjected to ELISA assays. Briefly, 96-well plates were coated overnight at 4°C with 100 μL / well of 2 μg / mL human or cynomolgus monkey MerTK-his protein in PBS, followed by blocking with 150 μL / well of 1% BSA. A five-fold dilution of the MerTK antibody (starting at 20 nM) was added to each well and incubated at room temperature for 1 hour. The plates were washed with PBS / Tween-20 and then incubated for 30 minutes at room temperature with goat anti-human IgG Fc antibody conjugated with horseradish peroxidase (HRP). After washing, the plates were developed using TMB substrate, followed by the addition of stop solution to complete the reaction. The signal from each well was read at OD 450 nm using a spectrophotometer. Figure 1 , Figure 2 As shown in Table 2, most of the tested MerTK antibodies exhibited binding activity to the human or cynomolgus monkey MerTK-his protein comparable to that of the benchmark Ab2000-7, M6, and h13B4.v16 antibodies.

[0170] Table 2. Cross-species activity of anti-MerTK chimeric monoclonal antibodies

[0171]

[0172] Use Biacore TM Affinity fractionation of anti-MerTK chimeric antibodies

[0173] Using the capture method, with Biacroe TM The T200 assay was used to test the binding affinity of the MerTK chimeric antibody to human MerTK-his protein. In short, the antibody was captured using a Pro-A chip. Two doses (12.5 nM and 50 nM, or 25 nM and 50 nM) of human MerTK-his protein were injected onto the captured antibody at a flow rate of 30 μL / min for 180 s. Antigen dissociation was allowed for 420–800 s. Biacore was used. TM Data analysis was performed using the T200 evaluation software. The results are shown in Table 3 below.

[0174] Table 3. Binding affinity of antibodies to hMerTK protein

[0175]

[0176] Binding with CHO-K1 cells overexpressing human MerTK

[0177] To evaluate the binding properties to 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 cells overexpressing human MerTK (CHO-K1-MerTK) were first incubated with 5-fold serial dilutions (starting at 20 nM) of mAb at 4°C for 1 hour. After washing with FACS buffer, PE-conjugated anti-human IgG-Fc secondary antibody was added to each well and incubated at 4°C for 30 min. The mean fluorescence intensity (MFI) of PE was evaluated using QuantAnalyzer 16. Figure 3 As shown, most of the tested MerTK chimeric antibodies exhibited higher maximum binding capacity (Top) and potency (EC50) to human MerTK expressed on CHOK1 cells than the baseline Ab2000-A7, M6, and h13B4.v16 antibodies.

[0178] Binding to tumor cells overexpressing human MerTK

[0179] To evaluate the binding properties to MerTK expressed on cells, FACS analysis was used to analyze the binding of the chimeric monoclonal antibody to the MerTK-expressing human melanoma cell line SK-MEL-5. Briefly, SK-MEL-5 cells were first incubated with 5-fold serial dilutions (starting at 20 nM) of the chimeric mAb at 4°C for 30 min. After washing with FACS buffer, the cell-antibody complexes conjugated with PE were incubated with these wells at 4°C for 30 min to detect antibody binding to these cells. The MFI of PE was evaluated using QuantAnalyzer 16. Figure 4 As shown, most of the tested MerTK chimeric antibodies exhibited 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.

[0180] Example 3. Blocking activity of anti-MerTK chimeric monoclonal antibody

[0181] Blocking MerTK from binding to its ligand Gas6

[0182] 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 repeat sequences, and two C-terminal laminin G domains. The N-terminal GLA domain can bind to PtdSer exposed on the plasma membrane under various conditions, including apoptosis, immune activation, and coagulation. At the C-terminus, one of the laminin G domains interacts with the Ig-like domain of MerTK to form a heterotetrameric complex, leading to downstream signaling activation.

[0183] To evaluate the inhibitory effect of anti-hMerTK mAb on the binding of hMerTK to its ligand hGas6, a receptor blocking assay was performed. In short, Jurkat cells engineered to overexpress human Gas6 were used at a concentration of 5 × 10⁻⁶ cells / year. 4 Cells / well were added to 96-well microplates at a density of [number] 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 at 120 nM, were added to the 96-well plates and incubated at 4 °C for 30 min. After washing with FACS buffer, the plates were incubated with diluted Alex Fluor 488-conjugated goat anti-mouse IgG antibody at 4 °C for 30 min. After washing, the plates were analyzed using QuantAnalyzer 16. Figure 5 As shown, all antibodies can effectively inhibit human MerTK from binding to human Gas6 expressed on cells.

[0184] Example 4: Functional characteristics of anti-MerTK antibodies

[0185] In this example, a cytotoxicity assay was performed to evaluate the inhibitory activity of the anti-MerTK antibody against macrophage-mediated apoptotic phagocytosis in vitro.

[0186] In short, Jurkat cells were induced to undergo apoptosis by treating them with 1 μM cruciferine for 4 hours. The cells were then washed twice with DPBS and cultured at 1.0 × 10⁻⁶ ppm. 6 The cells were resuspended in DPBS at a density of 100 cells / mL. Apoptotic cells were then labeled with 0.5 μM CFSE at 37°C in the dark for 5 min. After labeling, the cells were washed three times with culture medium and resuspended. CD14+ monocytes were isolated from the erythrocyte sedimentation rate (ESR) amber layer of healthy donors using human CD14 microbeads (Miltenyi Biotec). The CD14+ monocytes were cultured for 7 days in 100 ng / ml M-CSF to differentiate into M0 macrophages exhibiting upregulated MerTK expression. The macrophages were then cultured at a density of 4.0 × 10⁶ cells / mL.4 Macrophages were seeded at a density of 10 cells / well in 96-well microplates. Serial dilutions of the antibody were incubated with macrophages in the 96-well plates for 20 min. Then, freshly prepared CFSE-labeled apoptotic Jurkat cells were seeded at 2.0 × 10⁶ cells / well. 5 Cells were added to microwells at a density of [number] cells / well and co-cultured at 37°C for 90 min to allow macrophages to phagocytose apoptotic cells. After incubation, macrophages in the co-culture system were labeled with APC anti-human CD14 antibody. Phagocytic events were quantified using QuantAnalyzer 16. CD14 + CFSE + The cells represent macrophages that have engulfed apoptotic Jurkat cells. Cell burial assays demonstrated that the anti-MerTK antibody inhibited the phagocytosis of apoptotic cells by human macrophages. Figure 6 The results showed that some of the tested MerTK antibodies exhibited more effective blocking efficacy in terms of cell closure compared to h13B4.v16.

[0187] Example 5: Humanization of chimeric MerTK antibodies

[0188] The variable regions of the 10F7D9, 265F11B5, and 254B4D9 chimeric antibodies were selected for humanization. In short, the amino acid sequences of VH and VL were compared with existing human Ig gene sequence databases to determine the best-matching human germline Ig gene sequence overall. For each clone, the CDRs of the light and heavy chains were transplanted into the candidate germline. A 3D model was then generated to determine if any key mouse amino acids were present in the frame region, and replacing them with human amino acids might affect binding and / or CDR conformation. To maintain the structure and function of the humanized antibody, key amino acids were selected for reversion mutations. The humanized variable region of the antibody was then fused into the constant region of human IgG1 LALAPG for antibody production.

[0189] For the 10F7D9 light chain, the candidate lineage is the IGKV6-21*01 gene. For the heavy chain, the candidate lineage is the IGHV7-4-1*02 gene. In the case of the light chain, L45P, L46W, K48Y, and Y86F in the frame region involve reversion mutations. In the case of the heavy chain, V2I, V20I, R38K, E46K, S84N, S85N, Y95F, and R98T in the frame region involve reversion mutations.

[0190] For the light chain 265F11B5, the candidate lineage is the IGKV6-21*01 gene. For the heavy chain, the candidate lineage is the IGHV1-18*01 gene. In the case of the light chain, K50Y and F72Y in the frame region involve reversion mutations. In the case of the heavy chain, M48I, V68A, M70L, T71A, T72V, Y95F, and A97T in the frame region involve reversion mutations.

[0191] For the light chain 254B4D9, the candidate lineage is the IGKV1-33*01 gene, and for the heavy chain, the candidate lineages are the IGKV7-4-1*02 or IGKV7-81*01 genes. In the case of the light chain, Y49H, T69R, and Y87F in the frame region involve reversion mutations. In the case of the heavy chain, R38K, P38K, E46Q, M72L, Y80F, and Y95F in the frame region are included as reversion mutations (Tables 4 and 5).

[0192] Table 4. Humanized antibody sequences (underline / bold indicates CDR; bold / italic indicates reversion mutation)

[0193]

[0194]

[0195]

[0196] Table 5. Pairing of humanized antibodies VH and VL

[0197]

[0198]

[0199] Example 6: Antigen-binding properties of humanized antibodies

[0200] Binding with recombinant human MerTK

[0201] To evaluate antigen-binding activity, as previously described, the humanized antibody was tested using an ELISA assay. Figure 7 As shown, the humanized antibody exhibits binding efficacy with human MerTK comparable to that of its parental chimeric antibody.

[0202] Use Biacore TM Affinity grading of humanized antibodies

[0203] To explore whether humanized antibodies can maintain their binding kinetics, Biacore was used. TMAffinity fractionation was performed. Antibody capture was achieved using a protein A microarray. Human MerTK-his protein at 50 nM was injected onto the captured antibody at a flow rate of 30 μL / min for 180 s. Antigen dissociation was allowed for 600 s. (Biacore) TM Experiments were conducted on 8K using Biacore. TM Data analysis was performed using 8K evaluation software. The results are shown in Table 6. 265F11B5Hu-4, 265F11B5Hu-7, 265F11B5Hu-8, 254B4D9Hu-1, and 254B4D9Hu-2 showed affinity comparable to their chimeric antibodies.

[0204] Table 6. Affinity ranking results of humanized antibodies

[0205]

[0206] Binding with CHO-K1 cells overexpressing human MerTK

[0207] To evaluate the binding properties to antigens expressed on cells, as previously described, FACS was used to analyze humanized antibodies. Figure 8 As shown, humanized antibodies 265F11B5 and 254B4D9 exhibited cell-binding efficacy comparable to their chimeric counterparts. Meanwhile, some humanized antibodies against 10F7D9 also showed cell-binding efficacy comparable to their chimeric counterparts.

[0208] ***

[0209] The scope of this disclosure is not limited to the particular embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this 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 this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover such modifications and variations, provided they fall within the scope of the appended claims and their equivalents.

[0210] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof being specific for human Mer proto-oncogene tyrosine protein kinase (MerTK) protein and comprising 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 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are respectively the following amino acid sequences: SEQ ID NO:65-70; SEQ ID NO: 65, 75, 67, 76, 69, 77; or SEQ ID NO:65-66, 80-81, 69-70.

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

70.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID 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.

4. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises the amino acid sequence of SEQ ID NO:95 and the VL comprises the amino acid sequence of SEQ ID NO:99, or 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.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:19 and the VL comprises the amino acid sequence of SEQ ID NO:20; or the VH comprises the amino acid sequence of SEQ ID NO:23 and the VL comprises the amino acid sequence of SEQ ID NO:

24.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

7. One or more polynucleotides encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-6.

8. A cell comprising one or more polynucleotides according to claim 7.

9. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-6 and a pharmaceutically acceptable carrier.

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