Monoclonal antibodies specific to FAS ligands and their use

Abstract

This disclosure describes a monoclonal antibody that specifically binds to Fas ligand (FasL) and blocks its function. FasL-specific antibodies can be used in the development of therapeutic agents for the treatment of diseases, disorders, and conditions associated with the Fas / FasL signaling pathway, such as cancer, sepsis, ischemia-reperfusion injury, and coronavirus disease 2019 (COVID-19). This disclosure describes a monoclonal antibody that specifically binds to Fas ligand (FasL) and blocks its function. Furthermore, this specification describes the use of the disclosed antibody in the development of therapeutic agents for the treatment of diseases and conditions associated with the Fas / FasL signaling pathway.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 381,796, filed on November 1, 2022 (which is incorporated herein by reference in its entirety).

[0002] field This disclosure relates to monoclonal antibodies that specifically bind to Fas ligand (FasL), and to methods of using these antibodies, for example, to inhibit Fas / FasL signaling in a subject.

[0003] Embedding of electronic sequence lists The electronic sequence listing, prepared on October 23, 2023, and submitted with this application as an XML file (24,326 bytes) named 6727-109081-02.xml, is incorporated herein by reference in its entirety. [Background technology]

[0004] background Fas ligand (also known as FasL or CD95L) is a ligand for the Fas(CD95) receptor. When FasL binds to its receptor, it can induce either apoptotic or non-apoptotic signaling. Apoptotic signaling leads to cell death in Fas receptor-expressing cells. Non-apoptotic signaling via FasL / Fas receptor interaction can lead to neutrophil chemotaxis, increased proliferation and invasion of cancer cells, and premature T cell differentiation. In addition, FasL has been shown to contribute to the pathogenesis of many human diseases, including cancer, myocardial infarction, stroke, hepatic ischemia-reperfusion injury and renal ischemia-reperfusion injury, sepsis, interstitial lung disease, coronavirus disease 2019 (COVID-19), and autoimmune diseases. In patients with cancer, FasL contributes to disease progression by inducing apoptosis of tumor antigen-reactive invasive lymphocytes, premature T cell differentiation, and increased proliferation and invasiveness of tumor cells. [Overview of the project]

Means for Solving the Problem

[0005] Despite the role of FasL in multiple human diseases and disorders, there are currently no clinically approved FasL-targeted therapeutic agents available. Thus, there is a demonstrated need for potent FasL inhibitors.

[0006] Overview The present disclosure describes monoclonal antibodies that specifically bind to Fas ligand (FasL) and block its function. Also described herein is the use of the disclosed antibodies in the development of therapeutic agents for the treatment of diseases and conditions associated with the Fas / FasL signaling pathway. The antibodies of the present disclosure satisfy an unmet need by binding to FasL with higher affinity and blocking FasL with greater potency than previously described drugs that target FasL.

[0007] Monoclonal antibodies that specifically bind to FasL are provided herein. In some embodiments, the FasL-specific monoclonal antibody comprises the complementarity-determining region (CDR) sequences of antibody M3T01, M3T02 or M3T03. In other embodiments, the FasL-specific monoclonal antibody is an antibody that binds to the same epitope as M3T01, M3T02 or M3T03.

[0008] Conjugates comprising the disclosed FasL-specific monoclonal antibodies are also provided herein. In some embodiments, fusion proteins, bispecific antibodies, chimeric antigen receptors (CARs), CAR-expressing immune cells, immunoconjugates, antibody-drug conjugates (ADCs), and antibody nanoparticle conjugates comprising the monoclonal antibodies disclosed herein are provided.

[0009] Furthermore, nucleic acid molecules and vectors encoding monoclonal antibodies, fusion proteins (e.g., Fc fusions), polyspecific antibodies (e.g., bispecific antibodies), CARs, and immunoconjugates (e.g., immunotoxins) are provided herein.

[0010] Furthermore, this disclosure provides compositions and kits comprising a pharmaceutically acceptable carrier and a FasL-specific monoclonal antibody, fusion protein, polyspecific antibody, CAR, immunoconjugate, ADC, antibody nanoparticle conjugate, isolated nucleic acid molecule, or vector as disclosed herein.

[0011] Furthermore, the present invention provides a method for inhibiting Fas ligand in a target that requires it. In some embodiments, the method involves administering a therapeutically effective amount of a monoclonal antibody, fusion protein, polyspecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein to a target.

[0012] The present invention further provides methods for treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or coronavirus disease 2019 (COVID-19) in subjects. In some embodiments, the methods include administering a therapeutically effective amount of a monoclonal antibody, fusion protein, multispecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein to a subject.

[0013] The aforementioned and other purposes and features of this disclosure will become more apparent from the detailed description below, which proceeds with reference to the attached drawings. [Brief explanation of the drawing]

[0014] [Figure 1]Figures 1A and 1B show the binding affinity of M3T01 to soluble human Fas ligand (FasL), as measured using BIACORE® 8K. The sensorgram of the antibody against the antigen (Figure 1A) and the measured affinity (Figure 1B) are shown.

[0015] [Figure 2] Figure 2A shows the binding of M3T01 to soluble FasL as measured by ELISA.

[0016] Figure 2B shows the binding of M3T01 to cell surface FasL across multiple species (human, cynomolgus macaque, rat, and mouse). M3T01 staining of HEK293T cells transfected with vectors expressing FasL from various species was measured by flow cytometry.

[0017] [Figure 3] Figure 3 shows the M3T01 epitope on recombinant soluble FasL. The M3T01 epitope is a conformational epitope in human FasL, extending from R144 to Y189 (numbered with reference to SEQ ID NO: 17). Residues from human, cynomolgus monkey, mouse, rat, rabbit, and pig FasL that are part of the conformational epitope are enclosed in squares. Partial sequences of human FasL (residues 141-192 of SEQ ID NO: 17), cynomolgus monkey FasL (residues 140-191 of SEQ ID NO: 18), mouse FasL (residues 139-190 of SEQ ID NO: 19), rat FasL (residues 138-189 of SEQ ID NO: 20), rabbit FasL (residues 145-196 of SEQ ID NO: 21), and pig FasL (residues 142-193 of SEQ ID NO: 22) are shown.

[0018] [Figure 4]Figure 4 shows the inhibition of FasL-mediated apoptosis. HEK293T cells (effector cells) overexpressing human FasL were co-cultured with Jurcutt T cells (target cells). M3T01 or soluble CD95-Fc was added to the cell culture at varying concentrations. Apoptosis of Jurcutt cells was measured by flow cytometry after annexin V externalization. The data are reported as a percentage of apoptosis blockade. [Modes for carrying out the invention]

[0019] Sequence List The amino acid sequences listed in the attached sequence listing are shown using the standard single-letter amino acid codes specified in 37 CFR 1.822.

[0020] Sequence ID 1 is the amino acid sequence of the M3T01 VH domain.

[0021] Sequence ID 2 is the amino acid sequence of the M3T01 VL domain.

[0022] Sequence ID 3 is the amino acid sequence of the M3T02 VH domain.

[0023] Sequence ID 4 is the amino acid sequence of the M3T02 VL domain.

[0024] Sequence ID 5 is the amino acid sequence of the M3T03 VH domain.

[0025] Sequence ID 6 is the amino acid sequence of the M3T03 VL domain.

[0026] Sequence ID 7 is the amino acid sequence of the constant region of modified (S228P) human IgG4.

[0027] Sequence ID 8 is the amino acid sequence of the constant region of the human kappa light chain.

[0028] Sequence ID 9 is the amino acid sequence of the M3T01 heavy chain.

[0029] Sequence ID 10 is the amino acid sequence of the M3T01 light chain.

[0030] Sequence ID 11 is the amino acid sequence of the M3T02 heavy chain.

[0031] Sequence ID 12 is the amino acid sequence of the M3T02 light chain.

[0032] Sequence IDs 13-15 are the amino acid sequences of the variant VH domain.

[0033] Sequence ID 16 is the amino acid sequence of the variant VL domain.

[0034] Sequence ID 17 is the amino acid sequence of human FasL. [ka]

[0035] Sequence ID 18 is the amino acid sequence of the cynomolgus monkey FasL. [ka]

[0036] Sequence ID 19 is the amino acid sequence of mouse FasL. [ka] [ka]

[0037] Sequence ID 20 is the amino acid sequence of rat FasL. [ka]

[0038] Sequence ID 21 is the amino acid sequence of rabbit FasL. [ka]

[0039] Sequence ID 22 is the amino acid sequence of porcine FasL. [ka]

[0040] Detailed explanation I. Abbreviations ADC Antibody-drug conjugate CAR chimeric antigen receptor CDR Complementarity Determination Region ELISA enzyme-linked immunosorbent assay FR Framework FasL Fas ligand VH (Variable Heavy Chain) VL Variable Light Chain PBMC peripheral blood mononuclear cells SPR Surface Plasmon Resonance TCR (T cell receptor)

[0041] II. Terminology and Methods Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. Where used herein, the singular forms "a," "an," and "the" refer to both singular and plural unless the context clearly indicates otherwise. For example, the term "an antigen" can be considered equivalent to the phrase "at least one antigen," including singular or plural antigens. Where used herein, the term "comprise" means "include." Any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate and presented for descriptive purposes unless otherwise noted. Many methods and materials similar or equivalent to those described herein are available, but specific suitable methods and materials are described herein. Any conflicts are governed by this specification, including the definitions of terms. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope of this work.

[0042] To facilitate the consideration of various aspects, the following explanations of terms are provided.

[0043] Administration: Providing or administering a drug, such as a monoclonal antibody provided herein (e.g., a Fas ligand-specific monoclonal antibody), to a subject via any effective route. Examples of administration routes include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumor), sublingual, rectal, percutaneous, intranasal, vaginal, and inhalation routes.

[0044] Antibody: A polypeptide ligand containing at least one variable region that recognizes and binds to an antigen, e.g., an epitope of a Fas ligand (e.g., specifically recognizes and specifically binds to it). Mammalian immunoglobulin molecules consist of a heavy (H) chain and a light (L) chain, each of which has a heavy chain variable (V) H ) region and light chain variable (V L It has a variable region named after the ) region. In summary, V H Region and V L The region is responsible for the binding of antigens recognized by antibodies. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the major antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0045] The antibody variable region includes a framework region (FR) and a hypervariable (HV) region, also known as the "complementarity-determining region" or "CDR." The CDR is primarily responsible for binding to the antigen's epitope. The antibody's framework region acts to position and align the CDR in three-dimensional space. The boundaries of the given CDR amino acid sequence are as follows: Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; "Kabat" numbering scheme), Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; "Chothia" numbering scheme), Kunik et al. (Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40 (web server publication):W521-524), This can be easily determined using one of several well-known numbering schemes, including those described in the Kabat, Paratome, and IMGT databases (see 2012; “Paratome CDR”), and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; “IMGT” numbering scheme). The Kabat, Paratome, and IMGT databases are maintained online.

[0046] A "single-domain antibody" refers to an antibody that has a single domain (variable domain) capable of specifically binding to an antigen or an antigenic epitope in the absence of further antibody domains. A single-domain antibody is, for example, V H Domain antibody, V NAR Antibody, camel (camelid) V H H antibody, and V L Contains domain antibodies. VNAR Antibodies are produced by chondrichthyans, such as sharks, wobbegong sharks, spiny dogfish, and bamboo sharks. Shark V NAR is composed of the following regions (from N-terminus to C-terminus): FR1-CDR1-FR2-HV2-FR3a-HV4-FR3b-CDR3-FR4. V NAR The positions of CDR1 and CDR3 of the antibody can be determined, for example, using IMGT. HV2 and HV4 can be determined, for example, using the annotations described in Stanfield et al. (Science 305:1770-1773, 2004) and Fennell et al. (J Mol Biol 400:155-170, 2010). Camelids V H Heavy chain antibodies (H antibodies) are produced by several species, including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy chain antibodies that do not naturally have a light chain. Camel V H H is composed of the following regions (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Camel V H The H CDR residues can be determined, for example, according to IMGT, Kabat or the paratome.

[0047] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by cells transfected with the coding sequence of a single antibody. The term "monoclonal antibody" refers to both complete immunoglobulin molecules (e.g., IgG1, IgG4, IgA, IgM, etc.), antigen-binding fragments of IgG molecules (e.g., scFv and Fab), and / or single domain antibodies (e.g., VH single domain antibodies or camelid V H H nanobodies).

[0048] A "chimeric antibody" has framework residues from one species, e.g., human, and CDRs (which generally confer antigen binding) from another species.

[0049] A “humanized” antibody is an immunoglobulin containing a human framework region and one or more CDRs derived from non-human (e.g., camel, llama, mouse, rabbit, rat, shark, or synthetic) immunoglobulins. The non-human immunoglobulin providing the CDRs is designated “donor,” and the human immunoglobulin providing the framework is designated “acceptor.” In one embodiment, all CDRs of the humanized immunoglobulin are derived from the donor immunoglobulin. A constant region is not necessarily present, but if present, it must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85–90%, e.g., about 95% or higher. Thus, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except possibly the CDRs. The humanized antibody binds to the same antigen as the donor antibody providing the CDRs. Humanized or other monoclonal antibodies may have further conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0050] An antibody that "binds to the same epitope as the reference antibody" refers to an antibody that blocks 50% or more of the binding of the reference antibody to the antigen in a competitive assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to the antigen in a competitive assay. Methods for measuring antibody competition are known and include, for example, ELISA and biolayer interference.

[0051] Antibody-drug conjugates (ADCs) are molecules containing a drug, such as an antibody (or an antigen-binding fragment of an antibody) conjugated to a cytotoxic agent. ADCs can be used to specifically target drugs to specific cells by specifically binding the antibody to a target antigen expressed on the cell surface. Examples of drugs used with ADCs include microtubule inhibitors (e.g., mytansinoids, auristatin E, and auristatin F) and interchain crosslinkers (e.g., pyrrolobenzodiazepines; PBDs).

[0052] Autoimmune diseases: Disorders in which the immune system generates an immune response (e.g., B-cell or T-cell response) to endogenous antigens, resulting in tissue damage. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Graves' disease.

[0053] Binding affinity: The affinity of an antibody to an antigen. In one embodiment, affinity is calculated by a modified scatchard method. In another embodiment, binding affinity is measured by the antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In some embodiments, binding affinity is measured using the Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In other embodiments, Kd is measured using a surface plasmon resonance (SPR) assay using BIACORE® 8K, BIACORES-2000, or BIACORES-3000 (BIAcore Inc., Piscataway, NJ). In other embodiments, antibody affinity is measured by flow cytometry or SPR. An antibody that "specifically binds" to an antigen (e.g., Fas ligand) is an antibody that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens. In some cases, the monoclonal antibody (e.g., the anti-Fas ligand antibody provided herein) is 10 nM or less, for example, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2.5 nM or less, 2.4 nM or less, 2.3 nM or less, 2.1 nM or less, 2 nM or less, 1. It specifically binds to its target at equilibrium constants (Kd) of 9 nM or less, 1.8 nM or less, 1.7 nM or less, 1.6 nM or less, 1.5 nM or less, 1.4 nM or less, 1.3 nM or less, 1.2 nM or less, 1.1 nM or less, 1 nM or less, 975 pM or less, 950 pM or less, 925 pM or less, or 900 nM or less.

[0054] A bispecific antibody is a recombinant protein that contains antigen-binding fragments of two different monoclonal antibodies, thereby enabling it to bind to two different antigens or two different epitopes of the same antigen (e.g., Fas ligand). Similarly, a polyspecific antibody is a recombinant protein that contains antigen-binding fragments of at least two different monoclonal antibodies, e.g., two, three, or four different monoclonal antibodies.

[0055] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell proliferation. Other features often associated with cancer include metastasis, interference with the normal function of adjacent cells, abnormal levels of cytokine or other secretory releases, and suppression or exacerbation of inflammation or immune responses, and invasion of surrounding or distant tissues or organs, such as lymph nodes. "Metastatic cancer" refers to cancer cells that leave their original tumor site (e.g., lungs) and travel to other parts of the body, for example, via the bloodstream or lymphatic system (e.g., lung cancer cells that travel to the liver, brain, or bones).

[0056] Examples of cancers include solid tumors, such as breast cancer (e.g., lobular carcinoma and ductal carcinoma), sarcomas, lung cancer (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), pulmonary mesothelioma, colorectal adenocarcinoma, gastric cancer, prostate adenocarcinoma, ovarian cancer (e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular cancer and germ cell tumors, pancreatic adenocarcinoma, cholangiocarcinoma, hepatocellular carcinoma, bladder cancer (e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (e.g., mixed adenocarcinoma and Müllerian tumor (carcinosarcoma)), endocervical endometrium (endocervix), ectocervix, and vaginal cancer (e.g., adenocarcinoma and squamous cell carcinoma, respectively), skin tumors (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin adnexal tumors). This includes, but is not limited to, appendage tumors, Kaposi's sarcoma, cutaneous lymphoma, skin adnexal tumor, and various types of sarcomas and Merkel cell carcinomas, esophageal cancer, nasopharyngeal and oropharyngeal cancers (including their squamous cell carcinoma and adenocarcinoma), salivary gland cancer, tumors of the brain and central nervous system (including, for example, tumors of glial, neuronal, and meningeal origin), peripheral nerve tumors, soft tissue sarcomas, bone and cartilage sarcomas, head and neck cancers, and lymphoid tumors (including B-cell and T-cell lymphomas).

[0057] Examples of hematological malignancies include, for example, lymphoid malignancies, leukocyte malignancies, and other types of leukemia. In specific cases, the tumors are leukemias (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prelymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia) or lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma) as well as myelomas.

[0058] Chimeric antigen receptors (CARs): Chimeric molecules comprising an antigen-binding moiety (e.g., a monoclonal antibody or its antigen-binding fragment) and a signaling domain, e.g., a signaling domain derived from a T cell receptor (e.g., CD3ζ). Typically, a CAR consists of an antigen-binding moiety, a transmembrane domain, and an endodomain. The endodomain typically contains a signaling chain with an immunoreceptor-activating tyrosine motif (ITAM), e.g., CD3ζ or FcεRIγ. In some examples, the endodomain further includes at least one additional co-stimulatory domain, e.g., the intracellular portion of CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, MYD88-CD40, KIR2DS2, and / or DAP10.

[0059] Complementarity-determining regions (CDRs): Regions of hypervariable amino acid sequences that determine the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, named L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Camel (V H H) Single-domain antibodies, VH single-domain antibodies, and VL single-domain antibodies contain three CDRs called CDR1, CDR2, and CDR3.

[0060] Conservative variants: Proteins containing conservative amino acid substitutions that substantially do not affect or reduce the affinity of an antibody to a Fas ligand. For example, a monoclonal antibody that specifically binds to a Fas ligand may contain up to approximately 1, up to approximately 2, up to approximately 5, and up to approximately 10 or up to approximately 15 conservative substitutions and be able to specifically bind to the Fas ligand. The term “conservative variant” also includes the use of substituted amino acids instead of unsubstituted parent amino acids, provided that the antibody specifically binds to the Fas ligand. Non-conservative substitutions are substitutions that may reduce activity or binding to the Fas ligand.

[0061] A well-known table of conserved amino acid substitutions presents functionally similar amino acids. The following six groups are examples of amino acids that are considered to be mutually conserved substitutions: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0062] Contact: Arrangement in a direct physical meeting, including both solid and liquid forms.

[0063] Coronavirus disease 2019 (COVID-19): A disease caused by the human beta coronavirus SARS-CoV-2. Symptoms of COVID-19 include, for example, fever, chills, dry cough, shortness of breath, fatigue, muscle / generalized pain, headache, new loss of taste or smell, sore throat, nausea or vomiting, and diarrhea. Patients with severe COVID-19 may develop pneumonia, multiple organ failure, and may be fatal.

[0064] Cytotoxic agent: Any drug or compound that causes cell death.

[0065] Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to a cell that is intended to be targeted, in contrast to other cells of an organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than those that are intended to be targeted by the targeting portion of the immunotoxin, while the term "animal toxicity" refers to the toxicity of an immunotoxin to an animal due to the toxicity of the immunotoxin to cells other than those that are intended to be targeted by the immunotoxin.

[0066] Degenerate variants: Polynucleotides that encode polypeptides containing degenerate sequences as a result of genetic coding. There are 20 native amino acids, most of which are identified by more than one codon. Therefore, as long as the amino acid sequence of the polypeptide remains unchanged, it can contain all degenerate nucleotide sequences.

[0067] Drug: Any compound used to treat, ameliorate, or prevent a disease or condition in a subject. In some embodiments herein, a drug is an anticancer agent.

[0068] Effector molecule: A portion of a chimeric molecule intended to have a desired effect on cells that are targeted. Effector molecules are also known as effector moieties (EMs), therapeutic agents, diagnostic agents, or similar terms. Therapeutic agents (or drugs) include compounds, e.g., nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Therapeutic and diagnostic portions of nucleic acids include antisense nucleic acids, derivatized oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA, and triplicating oligonucleotides. Alternatively, the molecule linked to the targeting moiety (e.g., an anti-Fas ligand antibody) may be a therapeutic composition, e.g., a drug, a nucleic acid (e.g., an antisense nucleic acid), or an encapsulation system (such as a liposome or micelle) containing another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means for preparing antibody-binding liposomes are known. Diagnostic agents or diagnostic moieties include radioisotopes and other detectable labels. A useful detectable label for this purpose is a radioactive isotope, for example, 35 S, 11 C, 13 N, 15 O, 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C, 15 N, 90 Y, 99 Tc,111 In, and 125 It includes I, fluorophores, chemiluminescent agents, and enzymes.

[0069] Fas ligand (FasL): A transmembrane protein that is a member of the tumor necrosis factor superfamily. Fas ligand, primarily expressed by activated T cells and natural killer cells, is ubiquitously expressed throughout the body, but when it binds to Fas, which is particularly abundant in the thymus, liver, heart, and kidneys, it induces apoptosis (Peter et al., Cell Death Differ 22(4):549-559, 2015). The Fas / FasL signaling pathway plays a crucial role in regulating the immune system, including activation-induced cell death of T cells and cytotoxic T lymphocyte (CTL)-induced cell death. However, these proteins are also involved in tumorigenic activity. In particular, they have been found to be important survival factors for cancer cells and capable of protecting and promoting cancer stem cells (Peter et al., Cell Death Differ 22(4):549-559, 2015). Fas ligand is also known as CD95 ligand (CD95L) and tumor necrosis factor ligand superfamily member 6 (TNFSF6). The nucleotide and amino acid sequences of FasL are publicly available under NCBI gene ID 356, among others. An example human FasL amino acid sequence is described herein as SEQ ID NO: 17.

[0070] Framework region: The amino acid sequence inserted between CDRs (and / or hypervariable regions).

[0071] Fusion protein: A protein that contains at least a portion of two different (heterogeneous) proteins.

[0072] Different species: Derived from different gene sources or species.

[0073] Host cell: A cell capable of growing a vector and expressing its DNA. This cell may be a prokaryotic or eukaryotic cell. In some cases, the prokaryotic cell is an E. coli cell. In some cases, the eukaryotic cell is a human cell, such as a human embryonic kidney (HEK) cell or HEK293 T cell. This term also includes any offspring of the host cell in question. It is understood that all offspring may not be identical to the parent cell because mutations may occur during replication. However, when using the term “host cell,” such offspring are included.

[0074] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one aspect, this response is specific to a particular antigen ("antigen-specific response"). In one aspect, the immune response is a T cell response, such as CD4 + Response or CD8 + This is a response. In another aspect, this response is a B-cell response, which results in the production of specific antibodies.

[0075] Immunoconjugate: Covalent bonding of an effector molecule to an antibody (e.g., a monoclonal antibody specific to FasL) or this functional fragment. The effector molecule may be, for example, a detectable label, a photon absorber (e.g., IR700), or a toxin (an immunotoxin, e.g., forming an immunotoxin containing Pseudomonas exotoxin or a variant thereof). Certain non-limiting examples of toxins include, but are not limited to, abrin, lysine, Pseudomonas exotoxins (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin or a modified version thereof, or other toxic agents that directly or indirectly inhibit cell proliferation or kill cells. For example, PE and DT are highly toxic compounds that typically result in death due to hepatotoxicity. However, PE and DT can be modified into forms for use as immunotoxins by removing the toxin's natural targeting components (e.g., domain Ia of PE and chain B of DT) and substituting them with different targeting moieties, such as antibodies. In one embodiment, the antibody is conjugated to an effector molecule. In another embodiment, the antibody conjugated to the effector molecule is further conjugated to lipids or other molecules, for example, to extend its half-life in the body. The conjugation may be by either chemical or recombinant means. In one embodiment, the conjugation is chemical, and a reaction between the antibody moiety and the effector molecule generates a covalent bond between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may be included between the antibody and the effector molecule as needed. The terms “conjugated” or “conjugated” refer to two polypeptides being made into one consecutive polypeptide molecule.

[0076] Immunoliposomes: Liposomes conjugated on their surface with an antigen-binding monoclonal antibody (e.g., an antibody specific to FasL). Immunoliposomes can deliver cytotoxic agents or other drugs to antibody-targeted cells, such as tumor cells.

[0077] Interstitial lung disease: A group of chronic lung disorders characterized by inflammation and scarring that prevent lung tissue from receiving sufficient oxygen.

[0078] Ischemia: A vascular phenomenon resulting in reduced blood supply to an organ, tissue, or part of the body, for example, due to narrowing or blockage of one or more blood vessels. Ischemia can result from vasoconstriction, thrombosis, or embolism. Ischemia can cause direct ischemic injury, such as tissue damage, through cell death resulting from reduced oxygen supply.

[0079] Ischemia-reperfusion injury: Tissue damage that occurs after blood flow is restored to an ischemic area. The absence of oxygen and nutrients in the blood during an ischemic event leads to a state in which inflammation and oxidative damage are generated by oxidative stress induced by the restoration of circulation.

[0080] Isolated: A “isolated” biological component, e.g., nucleic acid, protein (including antibodies), or organelle, that has been substantially separated or purified from other biological components in the environment in which it arose (e.g., a cell), e.g., other chromosomes, extrachromosomal DNA and RNA, proteins, and organelles. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. This term also includes nucleic acids and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. In some examples, isolated biological components are at least 90% pure, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, or 100% pure.

[0081] Labeling: A detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or protein, to facilitate its detection. Certain non-limiting examples of labeling include fluorescent tags, enzyme conjugations, and radioisotopes. For example, "labeled antibody" refers to the incorporation of another molecule in an antibody. For instance, labeling is a detectable marker, such as incorporating a radiolabeled amino acid or conjugating a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or colorimetric methods). Various methods for labeling polypeptides and glycoproteins are known and available. Examples of labeling for polypeptides include: radioisotopes or radionuclides (radionucleotides) (e.g., 35 S, 11 C, 13 N, 15 O, 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C, 15 N, 90 Y, 99 Tc, 111 In, and 125 I) Includes, but is not limited to, fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), or magnetic materials, such as gadolinium chelates. In some embodiments, the labels are attached by spacer arms of varying lengths to reduce the potential for steric hindrance.

[0082] Linker: In some cases, a linker is a peptide within an antibody-binding fragment (e.g., an Fv fragment) that helps indirectly bind a variable heavy chain to a variable light chain. Alternatively, “linker” can refer to a peptide that functions to link a targeting moiety, e.g., an antibody, to an effector molecule, e.g., a cytotoxin or a detectable label. The terms “conjugating,” “joining,” “bonding,” or “linking” refer to combining two polypeptides into a single, continuous polypeptide molecule, or covalently binding a radionuclide or other molecule to a polypeptide, e.g., an antibody. Bonding can be either chemical or recombinant. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that a covalent bond is formed between the two molecules to form a single molecule.

[0083] Myocardial infarction: A medical condition that occurs when one or more areas of the heart do not receive enough oxygen. Also known as a "heart attack."

[0084] Functionally linked: When a first nucleic acid sequence is positioned in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, when a promoter affects the transcription or expression of a coding sequence, the promoter is functionally linked to the coding sequence. Generally, functionally linked DNA sequences are contiguous and reside within the same reading frame if necessary for the linking of two protein-coding regions.

[0085] Pharmacologically acceptable carriers: Useful pharmaceutically acceptable carriers are known to those skilled in the art. (Remington: The Science and Practice of Pharmacy, 22) ndThe ed., London, UK: Pharmaceutical Press, 2013 describes compositions and formulations suitable for the pharmaceutically active delivery of polypeptides, antibodies, and other compositions disclosed herein. Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically include injectable fluids containing a pharmaceutically and physiologically acceptable fluid, such as water, saline, equilibrium salt solutions, aqueous dextrose, glycerol, etc., as a vehicle. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic adjuncts, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0086] Preventing, treating, or improving a disease: “Preventing” a disease means preventing the full onset of the disease. “Treating” refers to therapeutic interventions that improve the signs or symptoms of a disease or condition after the onset of the disease. “Improving” refers to a reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0087] Sepsis: An extreme immune response to infection or injury. Most cases of sepsis result from bacterial infection, but sepsis can also result from viral infections (e.g., SARS-CoV-2 and influenza virus), fungal infections, or traumatic injuries. If left untreated, sepsis can lead to tissue damage, organ failure, septic shock, and death.

[0088] Stroke: A medical condition that occurs when the blood supply to the brain (or part of the brain) is interrupted or reduced, preventing brain tissue from receiving sufficient oxygen and nutrients.

[0089] Subjects: A category of living multicellular vertebrates, including both humans and non-human animals (e.g., veterinary subjects or wild animals), such as birds, pigs, mice, rats, rabbits, sheep, horses, cattle, dogs, cattle, ferrets, deer, otters, bank voles, raccoons, tree shrews, fruit bats, hamsters, minks, and non-human primates (e.g., rhesus macaques, crab-eating macaques, baboons, grivet monkeys, and common marmosets). In some cases, subjects have cancer, sepsis, or another condition suitable for treatment with Fas ligand signaling inhibitors. In some cases, subjects have glioblastoma multiforme.

[0090] Synthetic: Synthetic nucleic acids or proteins (e.g., antibodies) produced by artificial means in a laboratory, for example, those that can be chemically synthesized in a laboratory.

[0091] Therapeutic effective dose: A sufficient amount of a single (or combined with other therapeutic agents) agent, such as a monoclonal antibody, to prevent, treat (including preventive measures), reduce, and / or improve the symptoms and / or underlying causes of a disease or disorder, such as cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19. In some embodiments, a therapeutic effective dose is a sufficient amount to reduce or eliminate the symptoms of a disease, such as reducing tumor volume or tumor metastasis.

[0092] For example, a therapeutically effective dose may be the amount necessary to inhibit or suppress tumor growth. In one embodiment, a therapeutically effective dose is the amount necessary to eliminate cancer, reduce its size, or prevent metastasis, for example, by reducing the size and / or volume of the tumor by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100% compared to (or, for example, another treatment) the size / volume / number before treatment, and / or by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100% of the number and / or size / volume of metastases. When administered to a subject, a dose is generally used that achieves the target tissue concentration (e.g., within the tumor) that has been shown to achieve the desired effect in vitro.

[0093] The therapeutically effective dose of the drug may be administered in single or multiple doses, for example, daily, during the course of treatment. However, the therapeutically effective dose may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. The unit dosage form of the drug may be packaged in therapeutic doses or multiple therapeutic doses, for example, in vials containing sterile components (e.g., with perforated lids) or syringes.

[0094] A vector is a nucleic acid molecule that, when introduced into a host cell, produces a transformed host cell. A vector may contain a nucleic acid sequence that can replicate within the host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements. In some embodiments, the vector is a viral vector, such as a lentiviral vector, adenovirus vector, or adeno-associated virus (AAV) vector.

[0095] III. Monoclonal antibodies specific to Fas ligands This specification discloses a monoclonal antibody that specifically binds to a Fas ligand (FasL or CD95L) and blocks its function. When FasL binds to a Fas receptor (Fas or CD95), either apoptotic or non-apoptotic signaling may be induced in cells expressing the Fas receptor. Apoptotic signaling leads to cell death in Fas receptor-expressing cells. Non-apoptotic signaling through FasL / Fas receptor interaction may lead to neutrophil chemotaxis, increased proliferation and invasion of cancer cells, and premature differentiation of T cells.

[0096] FasL contributes to the pathogenesis of many human diseases, including, but not limited to, cancer, myocardial infarction, stroke, hepatic ischemia-reperfusion injury and renal ischemia-reperfusion injury, sepsis, interstitial lung disease, COVID-19, and autoimmune diseases. In cancer, FasL contributes to disease progression by inducing apoptosis of tumor antigen-reactive infiltrating lymphocytes, premature differentiation of T cells, and increased proliferation and invasiveness of tumor cells. Therefore, the use of the antibodies of this disclosure for the treatment of FasL-related diseases and disorders is described.

[0097] Currently, there are no approved drugs that target FasL for the treatment of FasL-related diseases. Asunercept (APG101; Apogenix, Heidelberg, Germany), a fusion protein containing the human Fas receptor and human IgG Fc, has been examined in clinical trials, but this drug has a short half-life and requires weekly administration. In addition, the monoclonal antibody disclosed herein binds to FasL with a considerably higher binding affinity than the soluble CD95-Fc fusion protein having the amino acid sequence of asunercept, and blocks FasL function with much stronger efficacy.

[0098] The amino acid sequences of the VH and VL domains of M3T01, M3T02, and M3T03 are presented below and are referred to herein as Sequence IDs 1-6. The positions of each CDR as determined by Kabat are also shown below. Other numbering schemes, such as IMGT or Chothia, can also be used to determine the boundaries of each CDR. The amino acid sequences of the human IgG4 heavy chain constant region (SEQ ID NO: 7) and the human kappa light chain constant region (SEQ ID NO: 8), which have an S228P substitution that prevents Fab arm exchange, are also shown below. The amino acid sequences of the heavy and light chains of M3T01 and M3T02 are also presented below (and referred to herein as Sequence IDs 9-12). M3T01 VH domain (SEQ ID NO: 1) [ka] M3T01 VL domain (SEQ ID NO: 2) [ka] M3T02 VH domain (SEQ ID NO: 3) [ka] M3T02 VL domain (SEQ ID NO: 4) [ka] M3T03 VH domain (SEQ ID NO: 5) [ka] M3T03 VL domain (SEQ ID NO: 6) [ka] Table 1. Location of heavy chain CDRs and light chain CDRs [Table 1] Human IgG quadruplex constant region (SEQ ID NO: 7) [ka] Human kappa light chain constant region (SEQ ID NO: 8) [ka] M3T01 heavy chain (SEQ ID NO: 9) [ka] VH domain: residues 1-119 IgG4 constant region: residues 120-446 M3T01 Light Chain (Sequence ID 10) [ka] VL domain: residues 1-106 Kappa constant region: residues 107-213 M3T02 double chain (SEQ ID NO: 11) [ka] [ka] VH domain: residues 1-119 IgG4 constant region: residues 120-446 M3T02 Light Chain (Sequence ID 12) [ka] VL domain: residues 1-106 Kappa constant region: residues 107-213

[0099] The present invention provides monoclonal antibodies that specifically bind to FasL. In some embodiments, the FasL-specific antibody has a binding affinity for FasL of 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1.9 nM or less, 1.8 nM or less, 1.7 nM or less, 1.6 nM or less, 1.5 nM or less, 1.4 nM or less, 1.3 nM or less, 1.2 nM or less, 1.1 nM or less, 1 nM or less, 975 pM or less, 950 pM or less, 925 pM or less, or 900 nM or less. In some embodiments, the monoclonal antibody includes a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some examples, the monoclonal antibody includes at least a portion of the amino acid sequences described herein as SEQ ID NO: 1 and / or SEQ ID NO: 2, as determined by any numbering scheme, e.g., IMGT, Kabat, or Chothia, e.g., one or more (e.g., all three) CDR sequences from SEQ ID NO: 1 and / or one or more (e.g., all three) CDR sequences from SEQ ID NO: 2, or any combination thereof. In other examples, the monoclonal antibody includes at least a portion of the amino acid sequences described herein as SEQ ID NO: 3 and / or SEQ ID NO: 4, as determined by any numbering scheme, e.g., IMGT, Kabat, or Chothia, e.g., one or more (e.g., all three) CDR sequences from SEQ ID NO: 3 and / or one or more (e.g., all three) CDR sequences from SEQ ID NO: 4, or any combination thereof.In other examples, the monoclonal antibody comprises at least a portion of the amino acid sequences described herein as SEQ ID NO: 5 and / or SEQ ID NO: 6, as determined by any numbering scheme, e.g., IMGT, Kabat, or Chothia, e.g., one or more (e.g., all three) CDR sequences derived from SEQ ID NO: 5 and / or one or more (e.g., all three) CDR sequences derived from SEQ ID NO: 6, or any combination thereof.

[0100] In some embodiments, the VH domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1, and / or the VL domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 2. In other embodiments, the VH domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, and / or the VL domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4. In other embodiments, the VH domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 5, and / or the VL domain of the monoclonal antibody includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 6. In some examples, the CDR sequences are determined using the Kabat, IMGT, or Chothia numbering scheme, or a combination thereof. In certain cases, the CDR sequence is determined using Kabat.

[0101] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody include residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1, respectively, and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody include residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2, respectively. In some cases, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 1 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1, and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 2 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2. In certain cases, the amino acid sequence of the VH domain contains or is derived from SEQ ID NO: 1, and / or the amino acid sequence of the VL domain contains or is derived from SEQ ID NO: 2.

[0102] In other examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2, SEQ ID NO: 14, or SEQ ID NO: 15. In specific examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 13, and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 14. In other specific examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1, and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 15. In other specific examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 16, and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2. In other specific examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 17, and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2. In other specific cases, the amino acid sequence of the VH domain contains or is derived from SEQ ID NO: 18, and the amino acid sequence of the VL domain contains or is derived from SEQ ID NO: 2.

[0103] In other embodiments, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody include residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3, respectively, and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody include residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4, respectively. In some cases, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 3 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3, and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 4 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4. In certain cases, the amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 3, and / or the amino acid sequence of the VL domain includes or is derived from SEQ ID NO: 4.

[0104] In other embodiments, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody include residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5, and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody include residues 23-36, 52-58, and 91-99 of SEQ ID NO: 6, respectively. In some cases, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 5 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5, and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 6 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), and includes residues 23-36, 52-58, and 91-99 of SEQ ID NO: 6. In certain cases, the amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 5, and / or the amino acid sequence of the VL domain includes or is derived from SEQ ID NO: 6.

[0105] In some embodiments, the monoclonal antibody is an antigen-binding fragment selected from Fab fragments, Fab' fragments, F(ab)'2 fragments, single-stranded variable fragments (scFv), and disulfide-stabilized variable fragments (dsFv). In other embodiments, the monoclonal antibody is IgG, for example, IgG4 or IgG1.

[0106] In some embodiments, the monoclonal antibody includes or further includes a heavy chain constant region and / or a light chain constant region. In some examples, the heavy chain constant region is the human IgG4 heavy chain constant region. In some examples, the heavy chain constant region has one or more modifications compared to the wild-type heavy chain, e.g., an S228P substitution in the human IgG4 heavy chain that prevents Fab arm exchange, in order to improve the half-life, stability, and / or function of the monoclonal antibody. In certain examples, the amino acid sequence of the heavy chain constant region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In certain non-limiting examples, the amino acid sequence of the heavy chain constant region includes or is derived from SEQ ID NO: 7.

[0107] In some cases, the light chain constant region is the human kappa light chain constant region. In some cases, the light chain constant region has one or more modifications compared to the wild-type light chain constant region to improve the half-life, stability, and / or function of the monoclonal antibody. In certain cases, the amino acid sequence of the light chain constant region is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In some cases, the amino acid sequence of the light chain constant region contains or is derived from SEQ ID NO: 8.

[0108] In some embodiments, the monoclonal antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain comprising or derived from SEQ ID NO: 9, and / or the amino acid sequence of the light chain comprising or derived from SEQ ID NO: 10.

[0109] In other embodiments, the monoclonal antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain comprising or derived from SEQ ID NO: 11, and / or the amino acid sequence of the light chain comprising or derived from SEQ ID NO: 12.

[0110] In some embodiments, the monoclonal antibody is a human antibody. In other embodiments, the monoclonal antibody is a humanized antibody. In yet another embodiment, the monoclonal antibody is a chimeric antibody.

[0111] Furthermore, monoclonal antibodies that bind to the same epitope on the Fas ligand to which the M3T01, M3T02, or M3T03 antibodies bind are also provided herein (see also Section IV). In some embodiments, the monoclonal antibody binds to the same epitope as the Fas ligand-specific monoclonal antibody, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10. In some examples, the epitope is a conformational epitope of the Fas ligand described as SEQ ID NO: 17, ranging from R144 to Y189.

[0112] Furthermore, fusion proteins comprising a Fas ligand-specific monoclonal antibody and a heterologous protein are provided herein. In some examples, the heterologous protein is an Fc protein, e.g., human Fc. In other examples, the heterologous protein is a protein tag, e.g., a myc tag, a His tag, an HA tag, or a FLAG tag. In other examples, the heterologous protein is an affinity tag, e.g., a chitin-binding protein, a maltose-binding protein, or glutathione S-transferase (GST).

[0113] Further disclosure herein provides a polyspecific antibody comprising a Fas ligand-specific monoclonal antibody and at least one further monoclonal antibody. In some embodiments, the polyspecific monoclonal antibody is a bispecific or trispecific monoclonal antibody. At least one further monoclonal antibody is capable of binding to different epitopes on the Fas ligand or to different antigens. The polyspecific antibody is further described in Section V.

[0114] Furthermore, chimeric antigen receptors (CARs) comprising monoclonal antibodies disclosed herein are provided herein. In some embodiments, the CAR further comprises a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof. Cells expressing FasL-specific CARs, e.g., immune cells, are further provided. In some examples, the immune cells are T lymphocytes, e.g., CTLs, B cells, natural killer (NK) cells, or macrophages. In some examples, the cells are allogeneic cells, e.g., allogeneic cells obtained from a healthy donor. CARs and CAR-expressing cells are further described in Section VI.

[0115] Further immunoconjugates comprising the FasL-specific monoclonal antibody and effector molecule disclosed herein are provided. In some embodiments, the effector molecule is a toxin, a detectable label, or a photon absorber. The immunoconjugates are further described in Section VII.

[0116] Furthermore, antibody-drug conjugates (ADCs) comprising a drug conjugated to a FasL-specific monoclonal antibody disclosed herein are also provided. In some embodiments, the drug is a small molecule, such as a microtubule inhibitor, an anti-mitotic agent, and / or a cytotoxic agent. ADCs are further described in Section VIII.

[0117] Further details are provided herein of antibody nanoparticle conjugates comprising nanoparticles conjugated to a FasL-specific monoclonal antibody as disclosed herein. In some embodiments, the nanoparticles include polymer nanoparticles, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, or niosomes. Antibody nanoparticle conjugates are further described in Section IX.

[0118] Furthermore, nucleic acid molecules encoding monoclonal antibodies, fusion proteins, conjugates, or polyspecific antibodies as disclosed herein are provided herein. In some embodiments, the nucleic acid molecules are operably ligated to a promoter. Vectors containing nucleic acid molecules as disclosed herein and cells containing nucleic acid molecules or vectors as disclosed herein are further provided. The host cell may be, for example, a mammalian cell, a bacterial cell, or an insect cell. Nucleic acid molecules and vectors are further described in Section X.

[0119] Further compositions are provided, comprising a pharmaceutically acceptable carrier, a monoclonal antibody as disclosed herein, a fusion protein, a conjugate, a multispecific antibody, a nucleic acid molecule, or a vector. These compositions are further described in Section XI.

[0120] Furthermore, methods for inhibiting Fas ligand in subjects requiring such inhibition are provided herein. In some embodiments, the methods involve administering a therapeutically effective amount of a monoclonal antibody, fusion protein, conjugate, multispecific antibody, nucleic acid molecule, vector, or composition disclosed herein to a subject. In some examples, the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19. In specific examples, cancer may be glioblastoma multiforme or myelodysplastic syndrome.

[0121] The present invention further provides methods for treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19 in subjects. In some embodiments, the methods involve administering a therapeutically effective amount of a monoclonal antibody, fusion protein, conjugate, multispecific antibody, nucleic acid molecule, vector, or composition disclosed herein to a subject. In some examples, cancer is glioblastoma multiforme or myelodysplastic syndrome. The methods are further described in Section XII.

[0122] IV. Identification of antibodies that bind to the same epitope on Fas ligand. Furthermore, monoclonal antibodies that bind to the same epitope on the Fas ligand to which the M3T01, M3T02, or M3T03 antibodies bind are provided herein. Antibodies that bind to such epitopes can be identified based on their ability to cross-compete (e.g., competitively inhibit the binding of these antibodies in a statistically significant manner) with the M3T01, M3T02, or M3T03 antibodies provided herein in a Fas ligand binding assay (e.g., the assay described in the Examples). The antibodies are 10 6 ×K DA competing antibody is considered "competitive" in terms of binding if, in the presence of a higher concentration of the competing antibody, it inhibits the binding of the M3T01, M3T02, or M3T03 antibody to the Fas ligand by more than 50%. In certain embodiments, an antibody that binds to the same epitope on the Fas ligand as the M3T01, M3T02, or M3T03 antibody is a human monoclonal antibody. Human antibodies that bind to the same epitope on the Fas ligand to which the M3T01, M3T02, or M3T03 antibody binds can be produced using various known techniques. Such antibodies can be prepared, for example, by administering an immunogen to a genetically modified animal that has been modified to produce an intact human antibody or an intact antibody having a human variable region in response to antigen loading. Such animals typically contain all or part of a human immunoglobulin locus that has been substituted at the endogenous immunoglobulin locus, or that is located outside the animal's chromosome or randomly incorporated into the chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the XENOMOUSE® technology, U.S. Patent No. 5,770,429 describing the HUMAB® technology, U.S. Patent No. 7,041,870 describing the KM MOUSE® technology, and U.S. Patent Application Publication 2007 / 0061900 describing the VELOCIMOUSE® technology. The human variable region derived from intact antibodies produced by such animals can be further modified, for example, by combining it with a different human constant region.

[0123] Furthermore, human antibodies that bind to the same epitope on the Fas ligand to which M3T01, M3T02, or M3T03 antibodies bind can be generated by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the generation of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133:3001, 1984 and Boerner et al., J. Immunol. 147:86, 1991). In addition, human antibodies generated by human B-cell hybridoma technology are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, the methods described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains.

[0124] Furthermore, monoclonal antibodies that specifically bind to the same Fas ligand epitopes as M3T01, M3T02, or M3T03 can be isolated by screening a combinatorial library for antibodies with the desired binding characteristics. For example, various methods are known for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom, Methods Mol Biol 178:1-37, 2002.

[0125] V. Multispecific antibodies A polyspecific antibody is a recombinant protein composed of two or more different monoclonal antibodies (or their antigen-binding fragments). For example, a bispecific antibody may consist of antigen-binding fragments of two different monoclonal antibodies. Thus, a bispecific antibody binds to two different antigens (or two different epitopes of the same antigen), while a trispecific antibody binds to three different antigens or epitopes.

[0126] This specification provides multispecific, e.g., trispecific or dispecific monoclonal antibodies, including FasL-specific monoclonal antibodies. In some embodiments, the multispecific monoclonal antibody further comprises a monoclonal antibody that specifically binds to another protein. In other embodiments, the multispecific monoclonal antibody further comprises a second monoclonal antibody that specifically binds to a different epitope on FasL. Also provided are isolated nucleic acid molecules and vectors encoding the multispecific antibodies, as well as host cells containing the nucleic acid molecules or vectors. Thus, this specification provides a method for treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19 (or any other disease or disorder enhanced by Fas / FasL signaling) in a subject by administering a therapeutically effective dose of a FasL-targeted multispecific (e.g., dispecific) antibody to the subject.

[0127] VI. Chimeric Antigen Receptors (CARs) The monoclonal antibodies of this disclosure can be used to generate CARs and / or immune cells (e.g., T cells, B cells, natural killer (NK) cells, or macrophages) engineered to express CARs. In some embodiments, the CAR includes a binding moiety, an extracellular hinge and spacer element, a transmembrane region, and an endodomain that performs signaling function (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In some examples, the binding moiety is an antigen-binding fragment of the monoclonal antibody, e.g., scFv. The spacer / hinge region typically includes sequences derived from IgG subclasses, e.g., IgG1, IgG4, IgD, CD8, or CD28 domains. The transmembrane domain may be derived from various different T cell proteins, e.g., CD3ζ, CD4, CD8, CD28, or inducible T cell costimulator (ICOS). Several different endodomains have been used for CAR generation. For example, the endodomain may consist of a signaling chain with ITAM, e.g., CD3ζ or FcεRIγ. In some examples, the endodomain further includes at least one additional costimulatory domain, e.g., CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), CD30, ICOS, CD27, MYD88-CD40, killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2), and / or the intracellular portion of DAP10.

[0128] Immune cells expressing CARs (e.g., T cells, B cells, NK cells, or macrophages) can be used to target specific cell types, such as cells expressing FasL. Therefore, the monoclonal antibodies disclosed herein can be used to target manipulated immune cells to FasL-expressing cells by manipulating immune cells expressing CARs containing FasL-specific monoclonal antibodies.

[0129] Furthermore, multispecific (e.g., bispecific) or bicistronic CARs are contemplated in this disclosure. In some embodiments, the multispecific or bispecific CAR comprises an antibody specific to FasL and an antibody specific to a second protein. Similarly, a bicistronic CAR comprises two CAR molecules expressed from the same construct, one of which is a CAR targeted to FasL and the second CAR targets a different protein. See, for example, Qin et al., Blood 130:810, 2017 and International Publication No. 2018 / 213337.

[0130] Accordingly, CARs comprising FasL-specific antibodies, for example, one of the antibodies disclosed herein, are provided herein. Also provided are isolated nucleic acid molecules and vectors encoding CARs (including bispecific CARs and bicistronic CARs), as well as host cells expressing CARs, bispecific CARs, or bicistronic CARs, for example, immune cells (e.g., T cells, B cells, NK cells, or macrophages). Immune cells expressing CARs composed of FasL-specific monoclonal antibodies can be used to treat diseases, disorders, or conditions associated with Fas / FasL signaling.

[0131] Furthermore, we provide herein modified FasL-specific monoclonal antibodies that enable their use in universal CAR systems. Universal CAR systems improve the flexibility of CARs and expand their use against further antigens. Currently, for each patient receiving CAR-T cell therapy, autologous T cells are cultured, proliferated, and modified to express antigen-specific CARs. This process is redundant and expensive, limiting its use. Universal CARs are based on a system that separates the signaling components of a CAR from the antigen-binding portion of the molecule but reassembles them using a "lock-key" system. For example, the biotin-conjugated immune receptor (BBIR) CAR consists of an intracellular T cell signaling domain fused with an extracellular domain containing avidin. A biotinylated antigen-specific (e.g., FasL-specific) monoclonal antibody can then bind to the BBIR and direct immune cells to FasL-expressing cells. Another example is the segmented, universal, and programmable (SUPRA) CAR system. In the SUPRA system, the CAR contains an intracellular signaling domain fused to an extracellular leucine zipper, which pairs with an antigen-specific monoclonal antibody fused to a congeneral leucine zipper. For a review of the universal CAR system, see, for example, Zhao et al., J Hematol Oncol 11(1):132, 2018 and Cho et al., Cell 173:1426-1438, 2018. In some embodiments herein, the FasL-specific antibody is fused to one component of the specific binding pair. In some examples, the antibody is fused to a leucine zipper or biotin.

[0132] Another type of universal CAR can be generated using the saltase enzyme. Saltase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving carboxyl-terminal localization signals. Saltase catalyzes peptide transfer between the saltase recognition motif and the saltase acceptor motif. Thus, antigen-specific CARs can be generated by contacting an antigen-specific antibody fused to a saltase recognition motif with a portion of the CAR molecule that includes an extracellular component containing an intracellular signaling domain(s), a transmembrane region, and a saltase acceptor motif. In the presence of the saltase enzyme, the two components covalently bond to form a complete antigen-specific CAR. Therefore, in some embodiments herein, the FasL-specific antibody is modified to include a saltase recognition motif (see, for example, PCT International Publication 2016 / 014553).

[0133] In some embodiments, FasL-specific CARs are expressed in allogeneic immune cells (e.g., T cells, B cells, NK cells, or macrophages), for example, allogeneic immune cells derived from a healthy donor(s). In some cases, allogeneic cells are genetically engineered to express FasL-specific CARs, for example, by disrupting the expression of endogenous T cell receptors through CAR insertion (see, e.g., MacLeod et al., Mol Ther 25(4): 949-961, 2017). Gene editing can be performed using any suitable gene editing system, such as CRISPR / Cas9, zinc finger nucleases, or transcription activator-like effector nucleases (TALENs).

[0134] VII. Immunoconjugate The monoclonal antibodies of this disclosure can be conjugated to therapeutic agents or effector molecules. The immunoconjugate includes, but is not limited to, molecules in which a covalent bond exists between the therapeutic agent and the antibody. A therapeutic agent is a drug having specific biological activity directed towards a specific target molecule or cells having the target molecule. Those skilled in the art will know that therapeutic agents include various drugs, e.g., vinblastine, daunomycin, etc., cytotoxins, e.g., natural or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (e.g., liposomes) containing pharmacological compositions, radioactive materials, e.g., 125 I, 32 P, 14 C, 3 H, and 35 It is understood that this may include S, a photon absorber, such as IR700, as well as other labels, target moieties, and ligands.

[0135] The selection of a particular therapeutic agent depends on the specific target molecule or cell and the desired biological effect. For example, a therapeutic agent could be a cytotoxin used to induce the death of a specific target cell (e.g., cancer cells). Conversely, if it is desirable to induce a non-lethal biological response, the therapeutic agent can be a non-lethal pharmacological agent or conjugated into liposomes containing a non-lethal pharmacological agent.

[0136] The therapeutic agents and monoclonal antibodies described herein allow those skilled in the art to easily construct a variety of clones containing functionally equivalent nucleic acids, for example, nucleic acids that have different sequences but encode the same effector moiety or antibody sequence. Accordingly, this disclosure provides antibodies and conjugates, as well as nucleic acids encoding fusion proteins thereof.

[0137] Effector molecules can be conjugated to a monoclonal antibody of interest using various means known to those skilled in the art. Both covalent and non-covalent methods may be used. The procedure for conjugating the effector molecule to the antibody varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to produce the conjugation of the effector molecule. Alternatively, the antibody may be derivatized to expose or conjugate to further reactive functional groups. Derivatization may involve conjugation with one of several known linker molecules. The linker can be any molecule used to conjugate the antibody to the effector molecule. The linker can form a covalent bond to both the antibody and the effector molecule. Suitable linkers are well known and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecules are polypeptides, the linker can be attached to the constituent amino acids by side chains (e.g., by disulfide bonds to cysteine) or to the alpha-carbon amino group and carboxyl group of the terminal amino acids.

[0138] Generally, monoclonal antibodies are derivatized or labeled in a way that their binding to the target antigen is not adversely affected. For example, an antibody can be functionally bound to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a photon absorber, a pharmaceutical agent, and / or a protein or peptide capable of mediating the association of the antibody or antibody moiety with another molecule (e.g., a streptavidin core region or polyhistidine tag) (by chemical coupling, gene fusion, non-covalent bonding, or other methods).

[0139] A certain type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type, or different types, for example, to produce a bispecific antibody). Suitable crosslinkers include heterobifunctional crosslinkers (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide) or homobifunctional crosslinkers (e.g., disuccinimidyl suberate) having two separately reactive groups separated by a suitable spacer. Such linkers are commercially available.

[0140] In some situations, it is desirable to release effector molecules from the antibody once the immunoconjugate reaches the target site. Therefore, in such situations, the immunoconjugate contains cleavable linkages near the target site. Cleavage of the linker to release effector molecules from the antibody may be facilitated by the enzyme activity or conditions to which the immunoconjugate is exposed, either within the target cell or near the target site.

[0141] Furthermore, the monoclonal antibodies provided herein can be conjugated with detectable markers, such as those detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optics, and laparoscopy). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also useful. Antibodies can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When an antibody or antigen-binding fragment is conjugated with a detectable enzyme, detection can be achieved by adding further reagents used by the enzyme to produce a recognizable reaction product. For example, in the presence of the active substance horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. Antibodies or antigen-binding fragments can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. Avidin itself can also be conjugated with enzymes or fluorescent labels.

[0142] The antibodies provided herein may be labeled with magnetic materials, such as gadolinium. Antibodies may also be labeled with lanthanides (e.g., europium and dysprosium) and manganese. Paramagnetic particles, such as superparamagnetic iron oxide, are also useful as labels. Furthermore, antibodies may be labeled with predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags) recognized by a secondary reporter. In some embodiments, the label is attached by spacer arms of varying lengths to reduce the possibility of steric hindrance.

[0143] Furthermore, the antibodies provided herein can be labeled with radiolabeled amino acids. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect the expression of a target antigen by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of labeling for polypeptides include the following radioisotopes or radionuclides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 Including, but not limited to, I, a person skilled in the art can determine a suitable method for conjugating a given active substance to an antibody or other polypeptide, considering the numerous reported methods for conjugating a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (e.g., enzymes or fluorescent molecules), drugs, toxins, and other active substances to antibodies.

[0144] Furthermore, the antibodies disclosed herein can be conjugated to a photon absorber. In some embodiments, the photon absorber is, but is not limited to, a phthalocyanine dye, such as IRDye® 700DX (also known as "IR700"). Antibody photon absorber conjugates can be used in photoimmunotherapy (for example, to kill tumor cells).

[0145] Furthermore, antibodies can be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. Such groups may be useful in improving the biological properties of antibodies, for example, by extending the serum half-life or improving tissue binding.

[0146] The toxins can be used in conjunction with the monoclonal antibodies described herein to produce immunotoxins. Examples of toxins include lysine, abrin, diphtheria toxin, and their subunits, as well as botulinum toxins A–F. Such toxins are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). The toxins intended also include variants of the toxins described herein (see, for example, U.S. Patents 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent 5,602,095). As used herein, “Pseudomonas exotoxin” refers to full-length, unmodified (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, the removal of domain Ia, the deletion of various amino acids within domains Ib, II, and III, the substitution of single amino acids, and the addition of one or more sequences at the carboxyl terminus (see, for example, Siegall et al., J. Biol. Chem. 264:14256-14261, 1989).

[0147] The PEs used in conjunction with the monoclonal antibodies described herein may include natural sequences, cytotoxic fragments of natural sequences, and conserved modified variants of natural PEs and their cytotoxic fragments. Cytotoxic fragments of PEs include fragments that are cytotoxic with or without subsequent proteolysis or other processing in target cells. Cytotoxic fragments of PEs include PE40, PE38, and PE35. For further descriptions of PE and this variant, see, for example, U.S. Patents Nos. 4,892,827, 5,512,658, 5,602,095, 5,608,039, 5,821,238, and 5,854,044, U.S. Patent Application Publication No. 2015 / 0099707, PCT International Publication No. 99 / 51643, International Publication No. 2007 / 016150, International Publication No. 2009 / 032954, International Publication No. 2011 / 032022, and International Publication No. 2014 / 052064, Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991, Kondo et al., J. Biol. Chem. See 263:9470-9475, 1988, Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997, Weldon et al., Blood 113(16):3792-3800, 2009, and Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008.

[0148] VIII. Antibody-drug conjugates (ADCs) ADCs are compounds composed of antigen-specific antibodies (e.g., FasL-specific antibodies) and drugs, such as cytotoxic agents (e.g., microtubule inhibitors or crosslinking agents). Because ADCs can specifically target cells expressing a particular antigen, the drugs can be far more potent than those used in standard systemic therapy. For example, the most common cytotoxic drugs currently used with ADCs are ICs that are 100 to 1000 times more potent than conventional chemotherapeutic agents. 50Examples of cytotoxic drugs include microtubule inhibitors, e.g., mytansinoids and auristatins (e.g., auristatin E and auristatin F). Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PBDs), which covalently bind to the minor grooves of DNA to form interstrand crosslinks. In some examples, the ADC contains the antibodies and drugs provided herein in a ratio of 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0149] Antibodies and drugs can be conjugated by cleavable or incleavable linkers. However, in some cases, it is desirable to have a circulatingly stable linker to prevent the systemic release of cytotoxic drugs that may cause significant off-target toxicity. Incleavable linkers prevent the release of cytotoxic agents before the ADC can be internalized by target cells. Once inside the lysosome, the release of cytotoxic agents occurs through digestion of the antibody by lysosomal proteases (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0150] One method for site-specific and stable drug conjugation to monoclonal antibodies (or antibody-Fc fusion proteins) is a glycan manipulation method. Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue within the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant β1,4-galactosyltransferase enzyme (Y289L-Gal-T1; U.S. Patent Application Publications 2007 / 0258986 and 2006 / 0084162), 2-keto-galactose is transferred to a free GlcNAc residue on the antibody heavy chain to generate a chemical handle for conjugation.

[0151] Oligosaccharide chains that bind to monoclonal antibodies can be classified into three groups based on their terminal galactose residues: fully galactosylated (two galactose residues; IgG-G2), with one galactose residue (IgG-G1), or completely degalactosylated (IgG-G0). Monoclonal antibodies are converted to the IgG-G0 glycoform by treatment with β1,4-galactosidase. Mutant β1,4-galactosyltransferase enzymes can transfer 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to GlcNAc residues on the IgG-G1 and IgG-G0 glycoforms. Chemical handles on the transferred sugars enable diverse molecular conjugations to monoclonal antibodies via these glycan residues (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0152] This specification provides ADCs comprising a drug (e.g., an anticancer agent) conjugated to a monoclonal antibody that specifically binds to FasL. In some embodiments, the drug is a small molecule. In some examples, the drug is a crosslinking agent, a microtubule inhibitor, and / or a mitotic inhibitor, or a cytotoxic agent suitable for mediating tumor cell death. Examples of cytotoxic agents include, but are not limited to, PBD, auristatin, mytansinoids, drastatin, calicheamycin, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, erinafide, combretastatin, drastatin, duocalmycin, engine, geldanamycin, indolino-benzodiazepine dimers, puromycin, tubulysin, hemiasterin, spliceostatin or pladienolide, as well as their stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity.

[0153] In some embodiments, the ADC may further include a linker. In some examples, the linker is a bifunctional or polyfunctional moiety that can be used to conjugate one or more drug moieties to an antibody to form an ADC. In some embodiments, the ADC is prepared using a linker having a reactive functional group for covalent bonding to the drug and the antibody. For example, the cysteine ​​thiol of an antibody can form a bond with the reactive functional group of a linker or drug linker intermediate for making an ADC.

[0154] In some cases, the linker has a functional group capable of reacting with free cysteine ​​present on the antibody to form a covalent bond. Examples of linkers having such reactive functional groups include maleimides, haloacetamides, α-haloacetyls, activated esters such as succinimides, 4-nitrophenyls, pentafluorophenyls, tetrafluorophenyls, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0155] In some cases, the linker has a functional group that can react with electrophiles present on the antibody. Examples of such electrophiles include, but are not limited to, aldehyde groups and ketone carbonyl groups. In some cases, the heteroatom of the linker's reactive functional group can react with an electrophile on the antibody to form a covalent bond to the antibody unit. Non-limiting examples include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0156] In some cases, the linker is a cleavable linker that facilitates the release of the drug. Examples of cleavable linkers include acid-unstable linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive), photosensitive linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020).

[0157] The ADCs disclosed herein may be used alone or in combination with other therapeutic agents and / or in combination with any standard treatment for the treatment of diseases, disorders or conditions associated with Fas / FasL signaling (e.g., cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19).

[0158] IX. Antibody Nanoparticle Conjugates The monoclonal antibodies disclosed herein can be conjugated into a variety of different types of nanoparticles to directly deliver cytotoxic agents or other therapeutic agents to FasL-expressing cells. The use of nanoparticles can reduce off-target side effects and improve drug bioavailability, potentially decreasing the dose of drug required to achieve therapeutic efficacy. Nanoparticle formulations can be tailored to suit the drug to be held or encapsulated within the nanoparticle. For example, hydrophobic molecules can be incorporated into the core of the nanoparticle, while hydrophilic drugs can be held within an aqueous core protected by a polymer or lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles or niosomes, and polymer nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).

[0159] Liposomes are a common type of nanoparticle used for drug delivery. Antibodies conjugated to liposomes are often called "immunoliposomes." The liposomal components of immunoliposomes are typically lipid vesicles consisting of one or more concentric phospholipid bilayers. In some cases, the phospholipids consist of a hydrophilic head group and two hydrophobic chains, allowing for the encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from circulation by macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size, and charge of liposomes can be modified. Additionally, the surface of liposomes can be modified with glycolipids or sialic acid, for example. For instance, the inclusion of polyethylene glycol (PEG) significantly extends the circulating half-life. Liposomes for use as drug delivery agents, including those for the preparation of immunoliposomes, have been described in the Art (see, for example, Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655 and 2010 / 00329981).

[0160] Niosomes are vesicles based on nonionic surfactants, possessing a structure similar to liposomes. The niosome membrane is composed solely of nonionic surfactants, such as polyglyceryl alkyl ethers or N-palmitoylglucosamine. Niosomes range from small monolayer particles to large multilayer particles. Such nanoparticles are monodisperse, water-soluble, chemically stable, low-toxicity, biodegradable, and non-immunogenic, increasing the bioavailability of encapsulated drugs.

[0161] Dendrimers comprise a variety of branched polymer complexes. Such nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Generally, a dendrimer consists of an initiating core surrounded by a layer of selected polymer grafted onto the core, forming a branched polymer complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers are used in a diverse range of therapeutic and diagnostic applications, including applications for the delivery of DNA, RNA, bioimaging contrast agents, chemotherapeutic agents, and other drugs.

[0162] Polymer micelles consist of aggregates of amphiphilic copolymers (composed of both hydrophilic and hydrophobic monomer units) assembled around a hydrophobic core and surrounded by a corona of hydrophilic polymer chains exposed to an aqueous environment. In many cases, the polymers used in the preparation of polymer micelles are heterobifunctional copolymers, consisting of a hydrophilic block of PEG, poly(vinylpyrrolidone), and hydrophobic poly(L-lactide) or poly(L-lysine) forming the particle core. Polymer micelles can be used to hold poorly soluble drugs. Such nanoparticles have been used to encapsulate several drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed for the holding of DNA or RNA molecules.

[0163] Polymer nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid polymer matrix, while nanocapsules contain an aqueous core. The selected formulation typically depends on the solubility of the therapeutic agent to be held / encapsulated; therefore, drugs that are poorly soluble in water are more readily encapsulated within nanospheres, while water-soluble and unstable drugs, such as DNA and proteins, are more readily encapsulated within nanocapsules. Polymers used to produce such nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkylcyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic acid-co-glycolic acid) (PLGA).

[0164] The antibodies provided herein can be conjugated to suitable nanoparticles according to standard methods known in the art. For example, the conjugation may be covalent or non-covalent. In some embodiments where the nanoparticles are liposomes, the antibody is conjugated to sterically stabilized, long-circulating liposomes via a PEG chain. The coupling of antibodies or antibody fragments to liposomes may also involve thioester bonds, for example, through the reaction of a thiol group with a maleimide group. Crosslinking agents can be used to create sulfhydryl groups to conjugate antibodies to nanoparticles (Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012).

[0165] X. Nucleic acid molecules The disclosure provides nucleic acid molecules (e.g., DNA, cDNA, mRNA, or RNA molecules) encoding amino acid sequences of monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates that specifically bind to FasL. Nucleic acid molecules encoding such molecules can be readily generated using amino acid sequences provided herein (e.g., CDR sequences), sequences available in the art (e.g., framework or constant region sequences), and genetic codes. In some embodiments, the nucleic acid molecules can be expressed in host cells (e.g., mammalian cells, yeast cells, or bacterial cells) to generate the monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, or immunoconjugates of the disclosure.

[0166] Genetic codes can be used to construct diverse functionally equivalent nucleic acid sequences, for example, nucleic acids that encode different but identical antibody sequences.

[0167] Nucleic acid molecules encoding monoclonal antibodies, fusion proteins, polyspecific antibodies, CARs, and immunoconjugates that specifically bind to FasL can be prepared by any suitable method, including, for example, cloning of a suitable sequence, or by direct chemical synthesis using standard methods. Chemical synthesis produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using DNA polymerase with a single-stranded oligonucleotide as a template.

[0168] Example nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques include, for example, Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4) th This can be found in (ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements).

[0169] Furthermore, nucleic acids can be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification (TAS), and autologous persistent sequence replication (3SR).

[0170] Nucleic acid molecules can be expressed in recombinantly engineered cells, such as bacterial, plant, yeast, insect, or mammalian cells. Monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates can be expressed as individual proteins containing monoclonal antibodies (which may, as appropriate, bind to effector molecules or detectable markers) or as fusion proteins. Any suitable method may be used to express and purify antibodies and antigen-binding fragments; non-limiting examples are presented in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011.

[0171] One or more DNA sequences encoding monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates can be expressed in vitro by DNA introduction into suitable host cells. The cells may be prokaryotic or eukaryotic. Numerous expression systems available for protein expression, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, such as mammalian cells (e.g., COS cell lines, CHO cell lines, HeLa cell lines, and myeloma cell lines), can be used for the expression of the disclosed antibodies. Methods for stable introduction, i.e., sustained maintenance of foreign DNA within the host, may be used.

[0172] The expression of nucleic acids encoding monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates described herein can be achieved by operably ligating DNA or cDNA to a promoter (either constitutive or inductive) and then incorporating it into an expression cassette. The promoter may be any promoter of interest, e.g., a cytomegalovirus promoter. Optionally, an enhancer, e.g., a cytomegalovirus enhancer, may be included in the construct. The cassette may be suitable for replication and incorporation in either prokaryotes or eukaryotes. A typical expression cassette contains specific sequences useful for regulating the expression of protein-coding DNA. For example, an expression cassette may include a suitable promoter, enhancer, transcriptional and translational terminators, a start sequence, a start codon (i.e., ATG) preceding the protein-coding gene, a splicing signal for an intron, a sequence for maintaining the correct reading frame of the gene to enable proper translation of mRNA, and a stop codon. The vector can encode selectable markers, such as drug resistance (e.g., ampicillin or tetracycline resistance).

[0173] To obtain high levels of expression of the cloned gene, the expression cassette may include, for example, a potent promoter to induce transcription, a ribosome binding site for translation initiation (e.g., an internal ribosome binding sequence), and a transcription / translation terminator. In E. coli, this may include a promoter, e.g., T7, trp, lac, or lambda promoter, a ribosome binding site, and a transcription termination signal. With respect to eukaryotic cells, the regulatory sequence may include, for example, an immunoglobulin gene, a promoter and / or enhancer derived from HTLV, SV40, or cytomegalovirus, as well as a polyadenylation sequence, and may further include a splice donor sequence and / or splice acceptor sequence (e.g., splice acceptor and splice donor sequences of CMV and / or HTLV). The cassette can be introduced into selected host cells by any suitable method, e.g., transformation or electroporation in E. coli, or calcium phosphate treatment, electroporation, or lipofection in mammalian cells. Cells transformed by the cassette can be selected based on their resistance to antibiotics, which is conferred by genes included in the cassette, such as the amp gene, gpt gene, neo gene, and hyg gene.

[0174] Nucleic acids encoding antibodies or conjugates described herein can be modified without reducing their biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the antibody into a fusion protein. Such modifications include, for example, termination codons, sequences that create conveniently located restriction sites, and sequences that add methionine to the amino terminus to generate an initiation site, or additional amino acids (e.g., polyHis) that are useful in purification steps.

[0175] If expressed, antibodies, fusion proteins, multispecific antibodies, and other conjugates can be purified using standard procedures including ammonium sulfate precipitation, affinity columns, and column chromatography (generally, refer to Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). Monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates do not need to be 100% pure. When used prophylactically, proteins should not substantially contain endotoxins, provided they are partially or homogeneously purified as required.

[0176] XI. Composition The present disclosure provides compositions comprising one or more monoclonal antibodies that specifically bind to FasL in a carrier. It also provides compositions comprising fusion proteins, multispecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, or antibody nanoparticle conjugates, as well as nucleic acid molecules and vectors encoding such molecules. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the physician performing the procedure to achieve the desired outcome. Monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, antibody nanoparticle conjugates, isolated nucleic acid molecules, vectors, or compositions can be formulated for systemic or topical administration. In one example, the composition is formulated for intravenous administration. In another example, the composition is formulated for intramuscular or intraperitoneal administration. In yet another example, the composition is formulated for intratumoral administration. In yet another example, the composition is formulated for subcutaneous administration.

[0177] In some embodiments, the composition comprises one or more FasL-specific monoclonal antibodies disclosed herein, e.g., two or three different antibodies (or multiple fusion proteins, polyspecific antibodies, CARs, CAR-expressing immune cells, immunoconjugates, ADCs, antibody nanoparticle conjugates, isolated nucleic acid molecules, or vectors). Alternatively, a kit comprising one or more FasL-specific monoclonal antibodies disclosed herein, e.g., two or three different antibodies (or multiple fusion proteins, polyspecific antibodies, CARs, CAR-expressing immune cells, immunoconjugates, ADCs, antibody nanoparticle conjugates, isolated nucleic acid molecules, or vectors) is provided. Such a kit may comprise one or more other therapeutic agents, e.g., therapeutic agents provided herein (e.g., other mAbs, chemotherapeutic agents, or combinations thereof).

[0178] The administration composition may comprise a pharmaceutically acceptable carrier, such as a solution of monoclonal antibodies, fusion proteins, polyspecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, antibody nanoparticle conjugates, isolated nucleic acid molecules, and / or vectors in an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. Such solutions are sterile and generally free of undesirable substances. Such compositions can be sterilized by conventional, well-known sterilization techniques. The composition may also comprise pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffering agents, toxicity adjusting agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentrations of monoclonal antibodies, fusion proteins, polyspecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, antibody nanoparticle conjugates, nucleic acids, and / or vectors in such formulations may vary and may be selected based on the fluid volume, viscosity, body weight, etc., according to the needs of the chosen specific administration method and target.

[0179] A pharmaceutical composition for intravenous administration may contain approximately 0.1 to 10 mg of antibody (or fusion protein, multispecific antibody, etc.) per subject per day. In particular, when administering the drug to an isolated site rather than within the circulatory or lymphatic system, such as a body cavity or organ cavity, the dosage may range from 0.1 to a maximum of approximately 100 mg per subject per day. In some embodiments, the composition may be a liquid formulation containing one or more antibodies in a concentration range of approximately 0.1 mg / ml to approximately 20 mg / ml, or approximately 0.5 mg / ml to approximately 20 mg / ml, or approximately 1 mg / ml to approximately 20 mg / ml, or approximately 0.1 mg / ml to approximately 10 mg / ml, or approximately 0.5 mg / ml to approximately 10 mg / ml, or approximately 1 mg / ml to approximately 10 mg / ml. Practical methods for preparing administerable compositions are publicly known or apparent to those skilled in the art, as described in Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Further details can be found in publications such as Edition (2005).

[0180] Furthermore, the compositions disclosed herein can be administered by other routes, including inhalation or oral routes, for example, by oral administration of yeast or bacteria (e.g., Lactococcus lactis) engineered to express the disclosed antibody or conjugate (see, for example, Vandenbroucke et al., Mucosal Immunol 3(1):49-56, 2010).

[0181] The disclosed compositions are also provided in sterile solutions of known concentrations, but are provided in lyophilized form and can be rehydrated with sterile water before administration. The antibody solution can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases can be administered at doses of 0.5 to 15 mg per kg of body weight. There is considerable experience in the art with the administration of antibody drugs, and they have been commercially available in the United States since the approval of RITUXAN® in 1997. The disclosed compositions can be administered by slow infusion rather than intravenous push or intravenous bolus. In one example, a higher loading dose is administered followed by a lower maintenance dose.

[0182] Controlled-release parenteral formulations can be manufactured as implants, oily injections, or particle systems. For an overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed within the particles. Particles smaller than approximately 1 μm, microspheres, and microcapsules are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries have a diameter of approximately 5 μm, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0183] Polymers can be used for the ion-controlled release of antibody-based compositions disclosed herein. Various degradable and non-degradable polymer matrices for use in controlled drug delivery are known (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous further systems for the controlled delivery of therapeutic proteins are known (see U.S. Patents No. 5,055,303; No. 5,188,837; No. 4,235,871; No. 4,501,728; No. 4,837,028; No. 4,957,735; No. 5,019,369; No. 5,055,303; No. 5,514,670; No. 5,413,797; No. 5,268,164; No. 5,004,697; No. 4,902,505; No. 5,506,206; No. 5,271,961; No. 5,254,342, and No. 5,534,496).

[0184] XII. Treatment Methods Furthermore, methods for inhibiting Fas ligand in subjects requiring such inhibition are provided herein. In some embodiments, the methods involve administering a therapeutically effective amount of a monoclonal antibody, fusion protein, multispecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein to a subject. In some embodiments, the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19.

[0185] Furthermore, this specification provides methods for treating diseases, disorders, or conditions associated with Fas / FasL signaling in subjects, such as, but not limited to, cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, myelodysplastic syndrome (MDS), or COVID-19. In some embodiments, the methods involve administering a therapeutically effective amount of a monoclonal antibody, fusion protein, multispecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein to a subject.

[0186] In some examples of the methods disclosed, the methods reduce Fas / FasL signaling by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to, for example, Fas / FasL signaling before treatment.

[0187] In some cases, the method increases the subject's survival by, for example, at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to survival in the absence of the treatment. In some cases, the method extends the subject's survival time by, for example, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months compared to survival time in the absence of the treatment.

[0188] In some cases, the method reduces inflammation in the subject by, for example, at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to the inflammation before treatment.

[0189] In some cases where the subject has cancer, the method reduces the size, volume, and / or weight of the tumor by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to, for example, the size, volume, and / or weight of the tumor before treatment. In some cases where the subject has cancer, the method reduces the size, volume, and / or weight of metastases by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to, for example, the size, volume, and / or weight of metastases before treatment.

[0190] In some embodiments, the second therapy or treatment agent is administered to the target. For example, a subject with cancer may be treated with a second anti-cancer therapy, such as chemotherapy, biological therapy (e.g., different monoclonal antibodies), radiotherapy, surgical resection, cryosurgery, laser therapy, and / or checkpoint inhibitors.

[0191] Examples of anticancer agents include, but are not limited to, chemotherapeutic agents such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, antihormone agents (e.g., antiandrogens), and anti-angiogenic agents. Other anticancer treatments include radiotherapy and antibodies (e.g., mAbs) that specifically target cancer cells or other cells (e.g., anti-PD-1, anti-PD-L1, anti-CLTA4, anti-EGFR, or anti-VEGF). For example, cancer is treated by administering one or more of the polypeptides, antibodies, fusion proteins, CARs, CAR-expressing cells, immunoconjugates, ADCs, multispecific antibodies, antibody nanoparticle conjugates or compositions disclosed herein, and one or more therapeutic mAbs, such as PD-L1 antibodies (e.g., durvalumab, KN035, cosibelimab, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737), anti-PD-1 antibodies (e.g., pembrolizumab, semiprimab, or nivolumab), anti-EGFR antibodies (e.g., cetuximab or panitumumab), anti-VEGF antibodies (e.g., bevacizumab or ramucizumab), or CLTA-4 antibodies (e.g., ipilimumab or tremelimumab). For example, cancer is treated with the compositions disclosed herein and one or more monoclonal antibodies, such as 3F8, avagovomab, adecatumumab, aftuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab, alsitumomab, bavituximab, bectumomab, belimumab, becylesomab, bevacizumab, and bivatuzumab meltansine.Mertansine), Blinatumomab, Brentuximab Vedotin, Cantuzumab Meltansine, Capromab Pendetide, Katumakisomab, CC49, Cetuximab, Citatuzumab Bogatox, Cizutumumab, Clivatuzumab Tetraxetan tetraxetan), conatumumab, dasetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etalacizumab, faretuzumab, figtumumab, galiximab, gemtuzumab ozogamicin, gilentuximab, glembatumumab vedotin Vedotin, ibritumobab tiuxetan, igobomab, imciromab, intetumumab, inotuzumab ozogamicin, ipilimumab, iratumumab, rabetuzumab, lexatumumab, lintuzumab, rorbotuzumab meltansine, lucatumumab, lumiliximab, mapatuzumab, matuzumab, mepolizumab, metemumab, milatuzumab, mitumomab, morolimumab, nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nivolumab, nofetumomab merpentan Merpentan, ofatumumab, olaratumab, oportuzumab monatox, oregobomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacutuzumab tetraxetan, taplitumomab paptox paptox), Tenatumomab, TGN1412, Tremelimumab, Tigatuzumab, TNX-650, Tisotumab Vedotin-tftv, Trastuzumab, Tremelimumab, Tucotuzumab (Cermolloukin)Treatment involves administering celmoleukin, beltuzumab, boroximab, botumumab, saltumumab, or a combination thereof.

[0192] XIII. Further aspects Embodiment 1. A monoclonal antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, which specifically binds to a Fas ligand, The VH domain contains the complementarity-determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 1, and the VL domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 2, The VH domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, and the VL domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, or A monoclonal antibody in which the VH domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 5, and the VL domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 6.

[0193] Embodiment 2. The CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VH domain contain residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1, respectively. The CDR1, CDR2, and CDR3 sequences of the VL domain contain residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2, respectively. A monoclonal antibody as described in Embodiment 1.

[0194] Embodiment 3. The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 1, and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1. The amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 2, and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2. The monoclonal antibody described in Embodiment 2.

[0195] Apparatus 4. The amino acid sequence of the VH domain contains or is derived from Sequence ID No. 1. The amino acid sequence of the VL domain contains or is derived from SEQ ID NO: 2. A monoclonal antibody according to any one of embodiments 1 to 3.

[0196] Embodiment 5. The CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VH domain contain residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 sequences of the VL domain contain residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4, respectively. A monoclonal antibody as described in Embodiment 1.

[0197] Embodiment 6. The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 3, and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3. The amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 4, and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4. A monoclonal antibody as described in Embodiment 5.

[0198] Embodiment 7. The amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 3. The amino acid sequence of the VL domain contains or is derived from SEQ ID NO: 4. A monoclonal antibody according to any one of Embodiments 1 and Embodiments 5 to 6.

[0199] Embodiment 8. The CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VH domain contain residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5, respectively. The CDR1, CDR2, and CDR3 sequences of the VL domain contain residues 23-36, 52-58, and 91-99 of SEQ ID NO: 6, respectively. A monoclonal antibody as described in Embodiment 1.

[0200] Embodiment 9. The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 5, and includes residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5. The amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 6, and includes residues 23-36, 52-58, and 91-99 of SEQ ID NO: 6. A monoclonal antibody as described in Embodiment 8.

[0201] Appearance 10. The amino acid sequence of the VH domain includes or is derived from Sequence ID No. 5. The amino acid sequence of the VL domain contains or is derived from SEQ ID NO: 6. A monoclonal antibody according to any one of Embodiments 1 and Embodiments 8 to 9.

[0202] Embodiment 11. A monoclonal antibody according to any one of Embodiments 1 to 10, further comprising a heavy chain constant region and a light chain constant region.

[0203] Embodiment 12. The monoclonal antibody according to Embodiment 11, wherein the heavy chain constant region is the human IgG4 heavy chain constant region.

[0204] Embodiment 13. The monoclonal antibody according to Embodiment 12, wherein the amino acid sequence of the human IgG quadri-chain constant region includes or is derived from SEQ ID NO: 7.

[0205] Embodiment 14. A monoclonal antibody according to any one of Embodiments 11 to 13, wherein the light chain constant region is the human kappa light chain constant region.

[0206] Embodiment 15. The monoclonal antibody according to Embodiment 14, wherein the amino acid sequence of the constant region of the human kappa light chain includes or is derived from SEQ ID NO: 8.

[0207] Embodiment 16. A monoclonal antibody according to any one of Embodiments 11 to 15, comprising a heavy chain and a light chain, The amino acid sequence of the heavy chain comprises or consists of SEQ ID NO: 9, and the amino acid sequence of the light chain comprises or consists of SEQ ID NO: 10, or A monoclonal antibody in which the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO: 11, and the amino acid sequence of the light chain comprises or consists of SEQ ID NO: 12.

[0208] Aspect 17. A heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and A light chain comprising the amino acid sequence of SEQ ID NO: 10 A monoclonal antibody that binds to the same epitope as the Fas ligand-specific monoclonal antibody comprising

[0209] Aspect 18. The monoclonal antibody according to aspect 17, wherein the epitope is a conformational epitope extending from R144 to Y189 of the Fas ligand described as SEQ ID NO: 17.

[0210] Aspect 19. The monoclonal antibody according to any one of aspects 1 to 18, wherein the antibody is a human antibody or a humanized antibody.

[0211] Aspect 20. The monoclonal antibody according to any one of aspects 1 to 18, wherein the antibody is a chimeric antibody.

[0212] Aspect 21. A fusion protein comprising the monoclonal antibody according to any one of aspects 1 to 20 and a heterologous protein.

[0213] Aspect 22. The fusion protein according to aspect 21, wherein the heterologous protein is an Fc protein.

[0214] Aspect 23. A multispecific antibody comprising the monoclonal antibody according to any one of aspects 1 to 20 and at least one additional monoclonal antibody or an antigen-binding fragment thereof.

[0215] Aspect 24. The multispecific antibody according to aspect 23, which is a bispecific antibody.

[0216] Aspect 25. A chimeric antigen receptor (CAR) comprising the monoclonal antibody according to any one of Aspects 1 to 20.

[0217] Aspect 26. An isolated cell expressing the CAR according to Aspect 25.

[0218] Aspect 27. The isolated cell according to Aspect 26, wherein the cell is an immune cell.

[0219] Aspect 28. An immunoconjugate comprising the monoclonal antibody according to any one of Aspects 1 to 20 and an effector molecule.

[0220] Aspect 29. The immunoconjugate according to Aspect 28, wherein the effector molecule is a toxin, a detectable label or a photon absorber.

[0221] Aspect 30. An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody according to any one of Aspects 1 to 20.

[0222] Aspect 31. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the monoclonal antibody according to any one of Aspects 1 to 20.

[0223] Aspect 32. The antibody-nanoparticle conjugate according to Aspect 31, wherein the nanoparticle comprises a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle or a niosome.

[0224] Aspect 33. A nucleic acid molecule encoding the monoclonal antibody according to any one of Aspects 1 to 20, the fusion protein according to Aspect 21 or Aspect 22, the multispecific antibody according to Aspect 23 or Aspect 24, the CAR according to Aspect 25, or the immunoconjugate according to Aspect 28 or Aspect 29.

[0225] Aspect 34. The nucleic acid molecule according to Aspect 33, operably linked to a promoter.

[0226] Embodiment 35. A vector comprising the nucleic acid molecule described in Embodiment 33 or Embodiment 34.

[0227] Embodiment 36. An isolated host cell containing a nucleic acid molecule as described in Embodiment 33 or Embodiment 34, or a vector as described in Embodiment 35.

[0228] Embodiment 37. A composition comprising a pharmaceutically acceptable carrier, and a monoclonal antibody according to any one of Embodiments 1 to 20, a fusion protein according to Embodiment 21 or Embodiment 22, a polyspecific antibody according to Embodiment 23 or Embodiment 24, a CAR according to Embodiment 25, isolated cells according to any one of Embodiments 26, 27, and 36, an immunoconjugate according to Embodiment 28 or Embodiment 29, an ADC according to Embodiment 30, an antibody nanoparticle conjugate according to Embodiment 31 or Embodiment 32, a nucleic acid molecule according to Embodiment 33 or Embodiment 34, or a vector according to Embodiment 35.

[0229] Embodiment 38. A method for inhibiting Fas ligand in a target requiring such inhibition, comprising administering to the target a therapeutically effective amount of a monoclonal antibody according to any one of Embodiments 1 to 20, a fusion protein according to Embodiment 21 or Embodiment 22, a polyspecific antibody according to Embodiment 23 or Embodiment 24, a CAR according to Embodiment 25, isolated cells according to any one of Embodiments 26, 27, and 36, an immunoconjugate according to Embodiment 28 or Embodiment 29, an ADC according to Embodiment 30, an antibody nanoparticle conjugate according to Embodiment 31 or Embodiment 32, a nucleic acid molecule according to Embodiment 33 or Embodiment 34, a vector according to Embodiment 35, or a composition according to Embodiment 37, thereby inhibiting Fas ligand in the target.

[0230] Apparatus 39. The method according to Apparatus 38, wherein the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, myelodysplastic syndrome (MDS), or coronavirus disease 2019 (COVID-19).

[0231] Apparatus 40. The method according to Apparatus 39, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

[0232] Apparatus 41. A method for treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, myelodysplastic syndrome (MDS), or coronavirus disease 2019 (COVID-19) in a subject, comprising administering a therapeutically effective amount of a monoclonal antibody according to any one of Apparatus 1 to 20, a fusion protein according to Apparatus 21 or 22, a polyspecific antibody according to Apparatus 23 or 24, a CAR according to Apparatus 25, isolated cells according to any one of Apparatus 26, 27, and 36, an immunoconjugate according to Apparatus 28 or 29, an ADC according to Apparatus 30, an antibody nanoparticle conjugate according to Apparatus 31 or 32, a nucleic acid molecule according to Apparatus 33 or 34, a vector according to Apparatus 35, or a composition according to Apparatus 37 to the subject.

[0233] Apparatus 42. The method according to Apparatus 41, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

[0234] The following embodiments are provided to illustrate certain features and / or embodiments. Such embodiments should not be construed as limiting the scope to the specific features or embodiments described herein. [Examples]

[0235] (Example 1) A monoclonal antibody specific to Fas ligand. In this example, three monoclonal antibodies that bind to human FasL and block its function are described. Two of these antibodies (M3T01 and M3T02) are human monoclonal antibodies isolated from human FasL-immunized transgenic mice (ATX-Gx™, Alloy Therapeutics) having human antibody variable region genes. The third antibody is a mouse antibody (M3T03) selected from human FasL-immunized Balb / c mice. In the tests described below, it is demonstrated that M3T01 binds with high affinity to soluble cell surface FasL, shows cross-reactivity with FasL from several different species, potently inhibits FasL-mediated apoptosis, and is highly stable.

[0236] Antibody sequences M3T01 and M3T02 are full-length human IgG4 / kappa monoclonal antibodies having an S228P modification that prevents Fab arm exchange (see the heavy chain constant region sequence set forth in SEQ ID NO: 7). The VH domain and VL domain of M3T01 are set forth herein as SEQ ID NOs: 1 and SEQ ID NO: 2, respectively, and the heavy chain and light chain of M3T01 are set forth herein as SEQ ID NOs: 9 and SEQ ID NO: 10, respectively. The VH domain and VL domain of M3T02 are set forth herein as SEQ ID NOs: 3 and SEQ ID NO: 4, respectively, and the heavy chain and light chain of M3T02 are set forth herein as SEQ ID NOs: 11 and SEQ ID NO: 12, respectively. M3T03 is a full-length mouse antibody. The VH domain sequence and VL domain sequence are set forth herein as SEQ ID NOs: 5 and SEQ ID NO: 6, respectively.

[0237] Binding assay The binding of M3T01 to human FasL was measured using a BIACORE™ 8K by multiple-dose SPR. As shown in FIGS. 1A-1B, M3T01 bound to human FasL with high affinity (974 pM). In another test, the binding of M3T01 to soluble FasL (sFasL) was measured by ELISA. The results show that M3T01 bound to sFasL with high affinity (FIG. 2A).

[0238] Next, assays were performed to evaluate the binding of M3T01 to membrane-bound FasL of diverse species origins on the cell surface. HEK293T cells were transfected with vectors expressing FasL from mouse, human, rat, or cynomolgus monkey, and binding by M3T01 was measured by flow cytometry. As shown in Figure 2B, M3T01 bound to cell surface FasL from all species examined. These data demonstrate that M3T01 exhibits broad species cross-reactivity.

[0239] Further studies demonstrated that M3T01 binds to cell surface FasL on activated human lymphocytes. In these studies, human peripheral blood mononuclear cells (PBMCs) were activated with PMA / ionomycin, and FasL expression was evaluated using M3T01 and NOK1 by Western blotting, qPCR, and flow cytometry (commercial anti-human FasL antibodies were used as positive controls).

[0240] FasL epitope to which M3T01 binds The epitope for M3T01 was characterized by CovalX. This methodology included high-mass MALDI mass spectrometry of the M3T01 / human FasL complex after chemical crosslinking and protease digestion. The results demonstrate that M3T01 binds to a conformational epitope on FasL extending from R144 to Y189 (see Figure 3; amino acid numbering is based on human FasL described as Sequence ID No. 17). The epitope for M3T01 is a region of FasL that is highly conserved across various species, including cynomolgus monkeys, rats, mice, rabbits, and pigs (Figure 3).

[0241] Functional activation of M3T01 To evaluate the functional activity of M3T01, inhibition of apoptosis was measured using HEK293T cell lines transfected to express high levels of human FasL (hFasL / HEK293T). This cell line was used to induce apoptosis in Jurcut target cells expressing the Fas receptor. Co-culturing these cell lines for 4 hours resulted in apoptosis in Jurcut cells. To evaluate the inhibition of FasL-mediated apoptosis, M3T01 antibody or a soluble CD95-Fc (sCD95-Fc) fusion protein targeting FasL was added to hFasL / HEK293T cells, followed by co-culturing with Jurcut cells. As shown in Figure 4, M3T01 showed stronger efficacy compared to sCD95-Fc in inhibiting apoptosis. Specifically, this test demonstrated that M3T01 had an IC50 of 0.33 nM, which is 310 times potenter than sCD95-Fc (102.3 nM).

[0242] M3T01 Stability To evaluate the stability of M3T01 under high-stress conditions, M3T01 was incubated at 37°C for 3 weeks, and then its binding to cell surface FasL and inhibition of FasL-mediated apoptosis were examined. Incubation of M3T01 at 37°C for 3 weeks did not affect cell surface FasL binding or inhibition of FasL-mediated apoptosis. Similarly, incubation at 40°C for 2 weeks did not result in a decrease in M3T01 activity. In addition, M3T01 was subjected to repeated freeze-thaw cycles (freezing at -80°C followed by thawing at room temperature for 5 cycles). Repeated freeze-thaw cycles did not reduce cell surface FasL binding or inhibition of FasL-mediated apoptosis.

[0243] Tissue cross-reactivity A large-scale tissue cross-reactivity study was conducted. The study evaluated 37 types of human tissue derived from three separate donors. This study demonstrated excellent specificity, with M3T01 binding only to mononuclear cells in lymph nodes and reticular endothelial cells in the spleen (consistent with known physiological tissue expression of FasL). No binding of M3T01 was detected in other normal / healthy organs / tissues.

[0244] Furthermore, M3T01 binds to fresh, frozen human tumor specimens. Because M3T01 recognizes conformational epitopes present only in natural (non-denatured) FasL, it does not bind to FasL in formalin-fixed, paraffin-embedded tissue specimens.

[0245] (Example 2) Variant of M3T01 Several variants of the M3T01 antibody were isolated, and their binding affinity to human FasL was tested using single-dose SPR affinity assays. Each variant clone contained the same CDR sequence as M3T01, but with one or more amino acid substitutions in the framework region (FR) of the VH domain and / or VL domain. The variant sequences, substitutions compared to the VH and VL domains of M3T01, and the measured binding affinities are listed in Table 2. The binding affinities reported in Table 2 are from single-dose SPR assays. The results demonstrate that the binding affinity of each variant is not significantly different from that of M3T01 (represented as M27.2 in Table 2). Table 2. Mutations within the FR region and their effects on binding affinity [Table 2-1] [Table 2-2]

[0246] (Example 3) Clinical trials of M3T01 This example describes the evaluation of M3T01 in patients with advanced cancer resistant to standard treatment in a Phase I dose-escalation clinical trial. Various drug dosages and treatment intervals will be evaluated in this clinical trial. After establishing safe / tolerable doses and treatment intervals in the Phase I clinical trial, a Phase II clinical trial will be conducted in patients with sepsis, severe COVID-19 disease, and myocardial ischemia.

[0247] Given the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the embodiments shown are merely examples of the disclosure and should not be considered as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. Accordingly, the inventors claim all that falls within such claims and spirit.

Claims

1. A monoclonal antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, which specifically binds to a Fas ligand, The VH domain includes the complementarity determination region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 1, and the VL domain includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 2, The VH domain includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 3, and the VL domain includes the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, or A monoclonal antibody wherein the VH domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 5, and the VL domain contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

6.

2. The CDR1, CDR2, and CDR3 sequences of the VH domain each contain residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1, respectively, and the CDR1, CDR2, and CDR3 sequences of the VL domain each contain residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2, The CDR1, CDR2, and CDR3 sequences of the VH domain each contain residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3, respectively, and the CDR1, CDR2, and CDR3 sequences of the VL domain each contain residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4, respectively, or The CDR1, CDR2, and CDR3 sequences of the VH domain each contain residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5, and the CDR1, CDR2, and CDR3 sequences of the VL domain each contain residues 23-36, 52-58, and 91-99 of SEQ ID NO:

6. The monoclonal antibody according to claim 1.

3. The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 1, and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 1; the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 2, and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 2, The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 3, and includes residues 31-35, 50-66, and 99-108 of SEQ ID NO: 3; the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 4, and includes residues 24-34, 50-56, and 89-96 of SEQ ID NO: 4, or The amino acid sequence of the VH domain is at least 90% identical to that of SEQ ID NO: 5, and includes residues 31-35, 50-66, and 99-107 of SEQ ID NO: 5; the amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO: 6, and includes residues 23-36, 52-58, and 91-99 of SEQ ID NO:

6. The monoclonal antibody according to claim 2.

4. The amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 1, and the amino acid sequence of the VL domain includes or is derived from SEQ ID NO:

2. The amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 3, and the amino acid sequence of the VL domain includes or is derived from SEQ ID NO: 4, The amino acid sequence of the VH domain includes or is derived from SEQ ID NO: 5, and the amino acid sequence of the VL domain includes or is derived from SEQ ID NO:

6. The monoclonal antibody according to claim 1.

5. The monoclonal antibody according to claim 1, further comprising a heavy chain constant region and a light chain constant region.

6. The monoclonal antibody according to claim 5, wherein the heavy chain constant region is a human IgG4 heavy chain constant region, and / or the light chain constant region is a human kappa light chain constant region.

7. The monoclonal antibody according to claim 6, wherein the amino acid sequence of the human IgG4 heavy chain constant region includes or is derived from SEQ ID NO: 7, and / or the amino acid sequence of the human kappa light chain constant region includes or is derived from SEQ ID NO:

8.

8. A monoclonal antibody according to claim 1, comprising a heavy chain and a light chain, The amino acid sequence of the heavy chain includes or is derived from SEQ ID NO: 9, and the amino acid sequence of the light chain includes or is derived from SEQ ID NO: 10, A monoclonal antibody in which the amino acid sequence of the heavy chain includes or is derived from SEQ ID NO: 11, and the amino acid sequence of the light chain includes or is derived from SEQ ID NO:

12.

9. A heavy chain containing the amino acid sequence of SEQ ID NO: 9, and Light chain containing the amino acid sequence of SEQ ID NO: 10 A monoclonal antibody that binds to the same epitope as a Fas ligand-specific monoclonal antibody containing [specific compound].

10. The monoclonal antibody according to claim 9, wherein the epitope is a conformational epitope of the Fas ligand described as SEQ ID NO: 17, extending from R144 to Y189.

11. The monoclonal antibody according to claim 1, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

12. A fusion protein comprising a monoclonal antibody and a heterologous protein according to any one of claims 1 to 11.

13. The fusion protein according to claim 12, wherein the heterologous protein is an Fc protein.

14. A polyspecific antibody comprising a monoclonal antibody according to any one of claims 1 to 11, and at least one further monoclonal antibody or its antigen-binding fragment.

15. A polyspecific antibody according to claim 14, which is a bispecific antibody.

16. A chimeric antigen receptor (CAR) comprising a monoclonal antibody according to any one of claims 1 to 11.

17. Isolated cells expressing the CAR described in claim 16.

18. The isolated cells according to claim 17, wherein the cells are immune cells.

19. An immunoconjugate comprising a monoclonal antibody and an effector molecule according to any one of claims 1 to 11.

20. The immunoconjugate according to claim 19, wherein the effector molecule is a toxin, a detectable label, or a photon absorber.

21. An antibody-drug conjugate (ADC) comprising a drug conjugated to a monoclonal antibody according to any one of claims 1 to 11.

22. An antibody nanoparticle conjugate comprising nanoparticles conjugated to a monoclonal antibody according to any one of claims 1 to 11.

23. The antibody nanoparticle conjugate according to claim 22, wherein the nanoparticles include polymer nanoparticles, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, or niosomes.

24. A nucleic acid molecule encoding a monoclonal antibody according to any one of claims 1 to 11.

25. The nucleic acid molecule according to claim 24, which is operably linked to a promoter.

26. A vector comprising the nucleic acid molecule described in claim 25.

27. An isolated host cell containing the vector according to claim 26.

28. A composition comprising a pharmaceutically acceptable carrier and a monoclonal antibody according to any one of claims 1 to 11.

29. A composition for inhibiting a Fas ligand in a target, comprising a monoclonal antibody according to any one of claims 1 to 11.

30. The composition according to claim 29, wherein the subject is cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, myelodysplastic syndrome (MDS), or coronavirus disease 2019 (COVID-19).

31. The composition according to claim 30, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

32. A composition for treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, myelodysplastic syndrome (MDS), or coronavirus disease 2019 (COVID-19), comprising a monoclonal antibody according to any one of claims 1 to 11.

33. The composition according to claim 32, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

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