Monoclonal antibodies specific for FAS ligands and uses thereof

Abstract

Described herein is a monoclonal antibody that specifically binds to and blocks the function of a Fas ligand (FasL). The 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).

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 381,796, filed on November 1, 2022, which is hereby incorporated by reference in its entirety. Technical field

[0003] The present disclosure relates to monoclonal antibodies that specifically bind Fas ligand (FasL) and methods of using them, such as for inhibiting Fas / FasL signaling in a subject.

[0004] Incorporation of electronic sequence listing

[0005] An electronic sequence listing, submitted herein as an XML file named 6727 - 109081 - 02.xml (24,326 bytes), created on October 23, 2023, is hereby incorporated by reference in its entirety. Background art

[0006] Fas ligand (FasL, also known as CD95L) is the ligand of the Fas (CD95) receptor. Binding of FasL to its receptor can induce apoptotic or non - apoptotic signaling. Apoptotic signaling leads to cell death of Fas receptor - expressing cells. Non - apoptotic signaling through the FasL / Fas receptor interaction may result in neutrophil chemotaxis, increased proliferation and invasion of cancer cells, and premature differentiation of T cells. Additionally, FasL has been shown to contribute to the pathogenesis of many human diseases, including cancer, myocardial infarction, stroke, hepatic and renal ischemia - reperfusion injury, sepsis, interstitial lung disease, coronavirus disease 2019 (COVID - 19), and autoimmune diseases. In cancer patients, 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.

[0007] Although FasL plays a role in a variety of human diseases and disorders, there are currently no clinically approved FasL - targeting therapeutic agents. Thus, there is a need for potent inhibitors of FasL. Summary of the invention

[0008] The present disclosure describes monoclonal antibodies that specifically bind and block the function of Fas ligand (FasL). Also described herein are the uses of the disclosed antibodies in the development of therapeutic agents for treating diseases and conditions associated with the Fas / FasL signaling pathway. The antibodies disclosed herein bind FasL with higher affinity and block FasL with greater potency than previously described drugs targeting FasL, thus fulfilling an unmet need.

[0009] Provided herein are monoclonal antibodies that specifically bind FasL. In some aspects, the FasL-specific monoclonal antibodies comprise the complementarity determining region (CDR) sequences of antibody M3T01, M3T02, or M3T03. In other aspects, the FasL-specific monoclonal antibodies are antibodies that bind the same epitope as M3T01, M3T02, or M3T03.

[0010] Also provided herein are conjugates comprising the disclosed FasL-specific monoclonal antibodies. In some aspects, provided are fusion proteins, multispecific antibodies, chimeric antigen receptors (CARs), CAR-expressing immune cells, immunoconjugates, antibody-drug conjugates (ADCs), and antibody-nanoparticle conjugates comprising the monoclonal antibodies disclosed herein.

[0011] Further provided herein are nucleic acid molecules and vectors encoding the disclosed monoclonal antibodies, fusion proteins (such as Fc fusions), multispecific antibodies (such as bispecific antibodies), CARs, and immunoconjugates (such as immunotoxins).

[0012] Compositions and kits comprising a pharmaceutically acceptable carrier and the disclosed FasL-specific monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, isolated nucleic acid molecules, or vectors are also provided by this disclosure.

[0013] Also provided are methods of inhibiting Fas ligand in a subject in need thereof. In some aspects, the methods shown include administering to the subject a therapeutically effective amount of the disclosed monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, antibody-nanoparticle conjugates, isolated nucleic acid molecules, vectors, or compositions.

[0014] Further provided is a method of 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 a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of the disclosed monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, antibody-nanoparticle conjugates, isolated nucleic acid molecules, vectors, or compositions.

[0015] The foregoing and other objects and features of this disclosure will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A-1B : Binding affinity of M3T01 to soluble human Fas ligand (FasL), using BIACORETM 8K measurement. The sensorgrams showing antibody against antigen ( Figure 1A ) and affinity measurement ( Figure 1B ) are shown.

[0017] Figure 2A : Binding of M3T01 to soluble FasL measured by ELISA.

[0018] Figure 2B : Binding of M3T01 to cell surface FasL of multiple species (human, cynomolgus monkey, rat, and mouse). M3T01 staining of HEK293T cells transfected with vectors expressing FasL from various species was measured by flow cytometry.

[0019] Figure 3 : M3T01 epitope on recombinant soluble FasL. The M3T01 epitope is a conformational epitope spanning R144 to Y189 of human FasL (numbering refers to SEQ ID NO: 17).

[0020] Residues of human, cynomolgus monkey, mouse, rat, rabbit, and pig FasL that are part of the conformational epitope are boxed. Shown are 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).

[0021] Figure 4 : Inhibition of FasL-mediated apoptosis. HEK293T cells overexpressing human FasL (effector cells) were co-cultured with Jurkat T cells (target cells). M3T01 or soluble CD95-Fc was added to the cell culture at different concentrations. Apoptosis of Jurkat cells was measured by annexin-V externalization by flow cytometry. Data are reported as the percentage of apoptosis blocked.

[0022] Sequence Listing

[0023] The amino acid sequences listed in the attached sequence listing are shown using the standard single-letter code for amino acids as defined in 37 C.F.R. 1.822. In the attached sequence listing:

[0024] SEQ ID NO: 1 is the amino acid sequence of the VH domain of M3T01.

[0025] SEQ ID NO: 2 is the amino acid sequence of the M3T01 VL domain.

[0026] SEQ ID NO: 3 is the amino acid sequence of the M3T02 VH domain.

[0027] SEQ ID NO: 4 is the amino acid sequence of the M3T02 VL domain.

[0028] SEQ ID NO: 5 is the amino acid sequence of the M3T03 VH domain.

[0029] SEQ ID NO: 6 is the amino acid sequence of the M3T03 VL domain.

[0030] SEQ ID NO: 7 is the amino acid sequence of the modified (S228P) human IgG4 constant domain.

[0031] SEQ ID NO: 8 is the amino acid sequence of the human κ light chain constant region.

[0032] SEQ ID NO: 9 is the amino acid sequence of the M3T01 heavy chain.

[0033] SEQ ID NO: 10 is the amino acid sequence of the M3T01 light chain.

[0034] SEQ ID NO: 11 is the amino acid sequence of the M3T02 heavy chain.

[0035] SEQ ID NO: 12 is the amino acid sequence of the M3T02 light chain.

[0036] SEQ ID NOs: 13 - 15 are the amino acid sequences of the variant VH domains.

[0037] SEQ ID NO: 16 is the amino acid sequence of the variant VL domain.

[0038] SEQ ID NO: 17 is the amino acid sequence of human FasL.

[0039] MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPPPLPPP PPPPPLPPLPLPPLKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRE 5TSQMHTA55LEKQIGHP5PPPEKKEL RKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVK YKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMS YCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL

[0040] SEQ ID NO: 18 is the amino acid sequence of cynomolgus monkey FasL.

[0041] MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPPPLPPPPPSPLPPLPLPPLKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRESTSQKHTASSLEKQIGHPSPPPEKKEQRKVAHLTGKPNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCTNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWAHSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL

[0042] SEQ ID NO: 19 is the amino acid sequence of mouse FasL.

[0043] MQQPMNYPCPQIFWVDSSATSSWAPPGSVFPCPSCGPRGPDQRRPPPPPPPVSPLPPPSQPLPLPPLTPLKKKDHNTNLWLPVVFFMVLVALVGMGLGMYQLFHLQKELAELREFTNQSLKVSSFEKQIANPSTPSEKKEPRSVAHLTGNPHSRSIPLEWEDTYGTALISGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNQPLNHKVYMRNSKYPEDLVLMEEKRLNYCTTGQIWAHSSYLGAVFNLTSADHLYVNISQLSLINFEESKTFFGLYKL

[0044] SEQ ID NO: 20 is the amino acid sequence of rat FasL.

[0045] MQQPVNYPCPQIYWVDSSATSPWAPPGSVFSCPSSGPRGPGQRRPPPPPPPPSPLPPPSQPPPLPPLSPLKKKDNIELWLPVIFFMVLVALVGMGLGMYQLFHLQKELAELREFTKfHSLRVSSFEKQIAKfPSTPSETKKPRSVAHLTGNPRSRSIPLEWEDTYGTALISGVKYKKGGLVINEAGLYFVYSKVYFRGQSCNSQPLSHKVYMRNFKYPGDLVLMEEKKLNYCTTGQIWAHSSYLGAVFNLTVADHLYVNISQLSLINFEESKTFFGLYKL

[0046] SEQ ID NO: 21 is the amino acid sequence of rabbit FasL.

[0047] MQQPFSYPYPQIYWVDSTASSPWAPPGSVLPCPSSVPERPGQRRPPPPLPPPPPPLPPPPLPPLPPLPPLPPPPLKKRKDHSTGLCLLLMFFMVLVAXVGLGWDVQLYHLQXELAELRESFSQRHTASSSMEKQTAHPSPPQEKKETKKVAHLTGKSNSRSNPLEWEDTYGIALVSGLKYKKGNLVINDTGLYFVYSKVYFRGQSCSNQPLTHKVYMKNSKYHQDLMLMEEKMMNYCTTGQMWARSSYLGAVFNLTSADHVYVNVSEISLVNFEESKTFFGLYKL

[0048] SEQ ID NO: 22 is the amino acid sequence of porcine FasL.

[0049] MQQPFNYPPQIFWVDSSATSPWASPGSVFPCPASVPGRPGQRRPPPPPPPPPPPPPPPPTLLPSRPLPPPPPPPPSLKKKRDHNAGLCLLVMFFMVLVALVGLGMFQLFHLQKELTELRESASQRHTESSLEKQIGHPNLPSEKKELRKVAHLTGKPNSRSIPLEWEDTYGIALVSGVKYMKGSLVINDTGLYFVYSKVYFRGQYCNNQPLSHKVYTRNSRYPQDLVLMEGKMMNYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESKTFFGLYCL Detailed implementation manners

[0050] I. Abbreviations

[0051] ADC Antibody-drug conjugate

[0052] CAR Chimeric antigen receptor

[0053] CDR Complementary determining region

[0054] ELISA Enzyme-linked immunosorbent assay

[0055] FR Framework

[0056] FasL Fas ligand

[0057] VH Variable heavy chain

[0058] VL Variable light chain

[0059] PBMC Peripheral blood mononuclear cell

[0060] SPR Surface plasmon resonance

[0061] TCR T cell receptor

[0062] II. Terms and methods

[0063] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of many of the terms commonly used in molecular biology can be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, the term “antigen” includes one or more antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprising...” means “including...”. It should be further understood that 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 are provided for descriptive purposes only, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described herein. In case of conflict, the present specification, including the definitions of the terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0064] For ease of review of the various aspects, the following glossary of terms is provided:

[0065] Administration: The provision or giving of an agent to a subject by any effective route, such as a monoclonal antibody provided herein (e.g., a Fas ligand-specific monoclonal antibody). Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0066] Antibody: A polypeptide ligand that comprises at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen, such as Fas ligand. Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has a variable region, called the variable heavy chain (V H ) region and the variable light chain (V L ) region, respectively. The V H region and the V L region together are responsible for binding the antigen recognized by the antibody. Mammalian immunoglobulins have five major heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary 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.

[0067] An antibody variable region contains framework regions (FRs) and hypervariable (HV) regions, called "complementary determining regions" or "CDRs". The CDRs are primarily responsible for binding to the epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the "Kabat" numbering scheme), Chothia et al (see 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; the "Chothia" numbering scheme), Kunik et al (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; "Paratome CDRs") and those described in the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; the "IMGT" numbering scheme). The Kabat, Paratome, and IMGT databases are maintained online.

[0068] A "single domain antibody" refers to an antibody having a single domain (variable domain) that is capable of specifically binding an antigen or an epitope of an antigen in the absence of additional antibody domains. Single domain antibodies include, for example, V H domain antibodies, V NAR antibodies, camel V H H antibodies, and V L domain antibodies. V NAR antibodies are produced by chondrichthyans, such as nurse sharks, wobbegong sharks, spiny dogfish sharks, and bamboo sharks. Shark V NAR is composed of the following regions (N-terminus to C-terminus): FR1-CDR1-FR2-HV2-FR3a-HV4-FR3b-CDR3-FR4. V NARThe positions of CDR1 and CDR3 of an 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). Camel V H H antibodies are produced by multiple species, including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy-chain antibodies that are naturally lacking in light chains. Camel V H H consists of the following regions (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Camel V H H CDR residues can be determined, for example, according to IMGT, Kabat, or Paratome.

[0069] "Monoclonal antibody" refers to an antibody produced by a single clone of lymphocytes or by cells in which the coding sequence of a single antibody has been transfected. The term "monoclonal antibody" refers to whole immunoglobulin molecules (such as IgG1, IgG4, IgA, IgM, etc.), antigen-binding fragments of IgG molecules (such as scFv and Fab), and / or single-domain antibodies (such as VH single-domain antibodies or camel V H H nanobodies).

[0070] "Chimeric antibody" has framework residues from one species such as a human and CDRs from another species, which generally confer antigen binding.

[0071] A "humanized" antibody is an immunoglobulin that includes human framework regions and one or more CDRs from a non-human (such as camel, llama, mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor", and the human immunoglobulin providing the framework is referred to as the "receptor". In one aspect, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90% such as about 95% or more identical. Thus, all parts of the humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A humanized antibody binds the same antigen as the donor antibody providing the CDRs. A humanized or other monoclonal antibody can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0072] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Methods for measuring antibody competition are known and include, for example, ELISA and biolayer interferometry.

[0073] Antibody-drug conjugate (ADC): A molecule comprising an antibody (or an antigen-binding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. An ADC can be used to specifically target a drug to a particular cell by the specific binding of the antibody to a target antigen expressed on the cell surface. Exemplary drugs used with ADCs include antimicrotubule agents, such as maytansinoids, auristatin E, and auristatin F, and interstrand crosslinkers, such as pyrrolobenzodiazepines; PBDs.

[0074] Autoimmune disease: A disease in which the immune system mounts an immune response (e.g., a B cell or T cell response) against an endogenous antigen, resulting in damage to tissues. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Graves' disease.

[0075] Binding affinity: The affinity of an antibody for an antigen. In one aspect, the affinity is calculated by a modification of the Scatchard method. In another aspect, the binding affinity is measured by the antigen / antibody dissociation rate. In another aspect, high binding affinity is measured by competitive radioimmunoassay. In another aspect, the binding affinity is measured by ELISA. In some aspects, the binding affinity is measured using an Octet system (Creative Biolabs), which is based on biolayer interferometry (BLI) technology. In other aspects, the Kd is measured using surface plasmon resonance (SPR) assays, and SPR uses BIACORE TM8K, BIACORES-2000, or BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.). In other aspects, antibody affinity is measured by flow cytometry or by SPR. An antibody that "specifically binds" an antigen, such as Fas ligand, is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens. In some examples, a monoclonal antibody, such as an anti-Fas ligand antibody provided herein, specifically binds its target with an equilibrium constant (Kd) of 10 nM or less, such as 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, 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.

[0076] Bispecific antibody: A recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies and is thus capable of binding two different antigens or two different epitopes of the same antigen, such as Fas ligand. Similarly, a multispecific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three, or four different monoclonal antibodies.

[0077] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features commonly associated with cancer include metastasis, interference with the normal function of adjacent cells, release of abnormal levels of cytokines or other secreted products, and suppression or exacerbation of the inflammatory or immune response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic cancer" refers to cancer cells that have left the original tumor site (e.g., the lung) and migrated to other parts of the body, such as lung cancer cells that have migrated to the liver, brain, or bone, for example, via the bloodstream or lymphatic system.

[0078] Exemplary cancers include, but are not limited to, solid tumors such as breast cancer (e.g., lobular and ductal carcinoma), sarcoma, lung cancer (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, gastric cancer, prostate cancer, ovarian cancer (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular cancer, and germ cell tumors, pancreatic adenocarcinoma, biliary tract adenocarcinoma, hepatocellular carcinoma, bladder cancer (including, for example, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial cancer (including, for example, adenocarcinoma and müllerian adenosarcoma (carcinosarcoma)), endometrial carcinoma, cervical cancer, and vaginal cancer (such as adenocarcinoma and squamous carcinoma of each of them), skin tumors (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi's sarcoma, cutaneous lymphoma, subcutaneous tumors, and various types of sarcoma and Merkel cell carcinoma), esophageal cancer, nasopharyngeal cancer, and oropharyngeal cancer (including squamous carcinoma and adenocarcinoma thereof), salivary gland adenocarcinoma, brain and central nervous system tumors (including, for example, tumors of glial, neuronal, and meningeal origin), peripheral nerve tumors, soft tissue sarcomas, and bone and cartilage sarcomas, head and neck cancers, and lymphoid tumors (including B-cell and T-cell malignant lymphomas).

[0079] Exemplary tumors of the blood include, for example, lymphoid tumors, leukocyte tumors, and other types of leukemia. In specific instances, the tumor is leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell proliferative leukemia (T-PLL), large granular lymphocyte leukemia, and adult T-cell leukemia), or lymphoma (such as Hodgkin lymphoma and non-Hodgkin lymphoma), and myeloma).

[0080] Chimeric antigen receptor (CAR): A chimeric molecule comprising an antigen-binding portion (such as a monoclonal antibody or an antigen-binding fragment thereof) and a signaling domain, such as a signaling domain from a T-cell receptor (e.g., CD3ζ). Typically, a CAR consists of an antigen-binding portion, a transmembrane domain, and an extracellular domain. The extracellular domain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIζ. In some cases, the extracellular domain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, MYD88-CD40, KIR2DS2, and / or DAP10.

[0081] Complementary determining region (CDR): A region of hypervariable amino acid sequences that defines 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. Camelid (V H H) single-domain antibodies, VH single-domain antibodies, and VL single-domain antibodies contain three CDRs, called CDR1, CDR2, and CDR3.

[0082] Conservative variant: A protein that contains conservative amino acid substitutions that do not substantially affect or reduce the affinity of the protein, such as an antibody against Fas ligand. For example, a monoclonal antibody that specifically binds to Fas ligand may include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and specifically binds to Fas ligand. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parental amino acid, provided that the antibody specifically binds to Fas ligand. Non-conservative substitutions are those that may reduce activity or binding to Fas ligand.

[0083] Conservative amino acid substitution tables that provide functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0084] 1) Alanine (A), serine (S), threonine (T);

[0085] 2) Aspartic acid (D), glutamic acid (E);

[0086] 3) Asparagine (N), glutamine (Q);

[0087] 4) Arginine (R), lysine (K);

[0088] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0089] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0090] Contact: Placed in direct physical association; includes both solid and liquid forms.

[0091] 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 / body aches, headache, new loss of taste or smell, sore throat, nausea or vomiting, and diarrhea. Severe COVID-19 patients can develop pneumonia, multi-organ failure, and death.

[0092] Cytotoxic agent: Any drug or compound that kills cells.

[0093] Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to the targeted cells, rather than to other cells of the organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than the cells intended to be targeted by the targeted portion of the immunotoxin, and the term "animal toxicity" refers to the toxicity of an immunotoxin to an animal through the toxicity of the immunotoxin to cells other than the cells intended to be targeted by the immunotoxin.

[0094] Degenerate variant: A polynucleotide encoding a polypeptide that includes sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide remains the same.

[0095] Drug: Any compound used to treat, ameliorate, or prevent a disease or disorder in a subject. In some aspects herein, the drug is an anti-cancer agent.

[0096] Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on the cells targeted by the chimeric molecule. Effector molecules are also referred to as effector portions (EMs), therapeutic agents, diagnostic agents, or similar terms. Therapeutic agents (or drugs) include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic portions include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking to single- or double-stranded DNA, and triplex-forming oligonucleotides. Alternatively, a molecule linked to a targeting portion, such as an anti-Fas ligand antibody, can be an encapsulation system, such as a liposome or micelle, that contains a therapeutic composition such as a drug, nucleic acid (such as an antisense nucleic acid), or another therapeutic portion that can be shielded from direct exposure to the circulatory system. Means for preparing liposomes attached to antibodies are known. Diagnostic agents or portions include radioisotopes and other detectable labels. Detectable labels useful for such purposes include radioisotopes such as 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, fluorophores, chemiluminescent agents, and enzymes.

[0097] Fas ligand (FasL): A transmembrane protein that is a member of the tumor necrosis factor superfamily. Fas ligand, which is mainly expressed by activated T cells and natural killer cells, induces apoptosis when it binds to Fas. Fas is ubiquitously expressed throughout the body but is particularly abundant in the thymus, liver, heart, and kidneys (Peter et al., Cell Death Differ 22(4):549-559, 2015). The Fas / FasL signaling pathway plays an important role in immune system regulation, including the cell death of activated-induced T cells and the cell death induced by cytotoxic T lymphocytes (CTLs). However, these proteins are also involved in pro-tumor activities. In particular, they have been found to be key survival factors for cancer cells and are 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 for FasL are publicly available, such as under NCBI Gene ID 356. An exemplary human FasL amino acid sequence is shown herein as SEQ ID NO:17.

[0098] Framework region: The amino acid sequence intervening between CDRs (and / or hypervariable regions).

[0099] Fusion protein: A protein that contains at least a portion of two different (heterologous) proteins.

[0100] Heterologous: Derived from a separate genetic source or species.

[0101] Host cell: A cell in which a vector can replicate and express its DNA. The cell can be prokaryotic or eukaryotic. In some instances, the prokaryotic cell is an Escherichia coli cell. In some instances, the eukaryotic cell is a human cell, such as a human embryonic kidney (HEK) cell or a HEK293 T cell. The term also includes any progeny of the subject host cell. It should be understood that all progeny may not be identical to the parental cell because mutations may occur during replication. However, such progeny are included when the term "host cell" is used.

[0102] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one aspect, the response is specific for a particular antigen ("antigen-specific response"). In one aspect, the immune response is a T cell response, such as a CD4 + response or a CD8 + response. In another aspect, the response is a B cell response and results in the production of specific antibodies.

[0103] Immunoconjugate: Covalent linkage of an effector molecule with an antibody (such as a monoclonal antibody specific for FasL) or a functional fragment thereof. The effector molecule can be, for example, a detectable label, a photon absorber (such as IR700), or a toxin (to form an immunotoxin, such as an immunotoxin comprising Pseudomonas exotoxin or a variant thereof). Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or a modified toxin thereof, or other agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death through hepatotoxicity. However, PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE and the B chain of DT) and replacing it with a different targeting moiety such as an antibody. In one aspect, the antibody is linked to the effector molecule. In another aspect, the antibody linked to the effector molecule is further linked to a lipid or other molecule, such as to increase its half-life in vivo. The linkage can be carried out by chemical or recombinant means. In one aspect, the linkage is chemical, where the reaction between the antibody moiety and the effector molecule results in the formation of a covalent bond between the two molecules to form a single molecule. Optionally, a peptide linker (short peptide sequence) can be included between the antibody and the effector molecule. The terms "conjugated" or "linked" mean to make two polypeptide molecules into one continuous polypeptide molecule.

[0104] Immunoliposome: A liposome having an antigen-binding monoclonal antibody (such as an antibody specific for FasL) conjugated to its surface. Immunoliposomes can carry cytotoxic agents or other drugs to antibody-targeted cells such as tumor cells.

[0105] Interstitial lung disease: A group of chronic lung diseases characterized by inflammation and scarring that prevent the lung tissue from receiving sufficient oxygen.

[0106] Ischemia: A vascular phenomenon in which the blood supply to a body organ, tissue, or part is reduced, for example, caused by the constriction or blockage of one or more blood vessels. Ischemia is sometimes caused by vasoconstriction, thrombosis, or embolism. Ischemia can lead to direct ischemic injury, such as tissue damage resulting from cell death due to reduced oxygen supply.

[0107] Ischemia-reperfusion injury: Tissue damage that occurs after blood flow is restored to an ischemic site. The lack of oxygen and nutrients in the blood during an ischemic event results in a condition where the restoration of circulation produces inflammation and oxidative damage through the induction of oxidative stress.

[0108] Isolated: An "isolated" biological component, such as a nucleic acid, protein (including an antibody), or organelle, has been substantially separated or purified away from other biological components in the environment in which the component occurs (such as a cell), such as other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids. In some instances, the isolated biological component is at least 90% pure, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, or 100% pure.

[0109] Tag: A detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or protein, to facilitate the detection of that molecule. Specific, non-limiting examples of tags include fluorescent tags, enzymatic conjugates, and radioisotopes. In one instance, a "tagged antibody" refers to the incorporation of another molecule into the antibody. For example, a tag is a detectable label, such as a polypeptide incorporating a radiolabeled amino acid or attached to a biotinyl moiety detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity detectable by optical or colorimetric methods). A variety of methods for labeling polypeptides and glycoproteins are known and can be used. Examples of tags for polypeptides include, but are not limited to, the following: radioisotopes or radiolabeled nucleosides (such as 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), fluorescent tags (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzyme labels (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some aspects, the tag is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0110] Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) that serves to indirectly bond the variable heavy chain to the variable light chain. "Linker" can also refer to a peptide used to link a targeting moiety such as an antibody to an effector molecule such as a cytotoxin or a detectable label. The terms "conjugation", "joining", "bonding", or "linking" refer to making two polypeptides into a continuous polypeptide molecule, or covalently attaching a radionuclide or other molecule to a polypeptide such as an antibody. The linking can be carried out chemically or recombinantly. "Chemically" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0111] Myocardial infarction: A medical condition that occurs when one or more regions of the heart do not receive enough oxygen. Also known as a "heart attack".

[0112] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, join two protein-coding regions in the same reading frame.

[0113] Pharmaceutically acceptable carrier: The use of pharmaceutically acceptable carriers is known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for drug delivery of the polypeptides, antibodies, and other compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as in the form of powders, pellets, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, etc., such as sodium acetate or sorbitan monolaurate.

[0114] Preventing, treating, or ameliorating a disease: "Preventing" a disease means inhibiting the full development of the disease. "Treating" means a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. "Ameliorating" means a reduction in the number or severity of the signs or symptoms of a disease such as cancer.

[0115] Sepsis: An extreme immune response to infection or injury. Most cases of sepsis are caused by bacterial infections, but sepsis can also be caused by viral infections (such as SARS-CoV-2 and influenza viruses), fungal infections, or traumatic injuries. If left untreated, sepsis can lead to tissue damage, organ failure, septic shock, and death.

[0116] Stroke: A medical condition that occurs when the blood supply to the brain (or a part of the brain) is interrupted or reduced, preventing brain tissue from receiving adequate oxygen and nutrients.

[0117] Subject: A category of living, multicellular vertebrate organisms, including humans and non-human animals (such as veterinary subjects or wild animals), e.g., birds, pigs, mice, rats, rabbits, sheep, horses, cows, dogs, cats, ferrets, deer, otters, bank voles, raccoons, tree shrews, fruit bats, hamsters, minks, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, baboons, green monkeys, and common marmosets). In some instances, the subject has cancer, sepsis, or another condition amenable to treatment with an inhibitor of Fas ligand signaling. In one instance, the subject has glioblastoma multiforme.

[0118] Synthetic: Produced by artificial means in a laboratory, e.g., synthetic nucleic acids or proteins (such as antibodies) can be chemically synthesized in a laboratory.

[0119] Therapeutically effective amount: The amount of an agent such as a monoclonal antibody that, alone (or in combination with other therapeutic agents), is sufficient to prevent, treat (including prevent), reduce, and / or ameliorate the symptoms and / or underlying cause of a disease or disorder, e.g., prevent, inhibit, and / or treat cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune disease, or COVID-19. In some aspects, the therapeutically effective amount is sufficient to reduce or eliminate the symptoms of the disease, such as reducing tumor volume or tumor metastasis.

[0120] For example, this can be an amount necessary to inhibit or suppress tumor growth. In one aspect, a therapeutically effective amount is an amount necessary to eliminate cancer, reduce the size of cancer, or prevent cancer metastasis, such as reducing the size and / or volume of a 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%, and / or reducing the number and / or size / volume of metastases 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%, for example, compared to the size / volume / number before treatment (or for example, compared to another treatment). When administered to a subject, a dose will generally be used that will achieve a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect.

[0121] A therapeutically effective amount of an agent can be administered as a single dose or in several doses, e.g., daily, during a course of treatment. However, a therapeutically effective amount can depend on the subject being treated, the severity and type of the disorder being treated, and the mode of administration. Unit dosage forms of the agent can be packaged in a therapeutically effective amount or in multiples of a therapeutically effective amount, e.g., in vials (e.g., with a pierceable lid) or syringes having sterile components.

[0122] Vector: A nucleic acid molecule is introduced into a host cell to produce a transformed host cell. The vector can include nucleic acid sequences that permit its replication in the host cell, such as an origin of replication. The vector can also include one or more selectable marker genes and other genetic elements. In some aspects, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV).

[0123] III. Monoclonal Antibodies Specific for Fas Ligand

[0124] Disclosed herein are monoclonal antibodies that specifically bind and block the function of Fas ligand (FasL or CD95L). Binding of FasL to the Fas receptor (Fas or CD95) can induce apoptotic or non-apoptotic signaling in cells expressing the Fas receptor. Apoptotic signaling results in cell death of cells expressing the Fas receptor. Non-apoptotic signaling through the FasL / Fas receptor interaction can lead to neutrophil chemotaxis, increased proliferation and invasion of cancer cells, and premature differentiation of T cells.

[0125] FasL contributes to the pathogenesis of many human diseases, including but not limited to cancer, myocardial infarction, stroke, hepatic 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. Accordingly, the use of the disclosed antibodies for the treatment of FasL-related diseases and disorders is described.

[0126] There are currently no approved drugs targeting FasL for the treatment of FasL-related diseases. Although the fusion protein Asunercept (APG101; Apogenix, Heidelberg, Germany), which includes the human Fas receptor and the human IgG Fc, has been tested in clinical trials, this drug has a short half-life and requires weekly administration. In addition, the monoclonal antibodies disclosed herein bind FasL with a much higher binding affinity and block FasL function with a potency far greater than that of the soluble CD95-Fc fusion protein having the amino acid sequence of Asunercept.

[0127] The amino acid sequences of the VH and VL domains of M3t01, M3T02, and M3T03 are provided below and are shown herein as SEQ ID NOs: 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 sequence of the human IgG4 heavy chain constant region with the S228P substitution (SEQ ID NO: 7) that prevents Fab arm exchange, and the amino acid sequence of the human κ light chain constant region (SEQ ID NO: 8) are also shown below. The amino acid sequences of the heavy and light chains of M3T01 and M3T02 are also provided below (and are shown herein as SEQ ID NOs: 9-12).

[0128] M3T01 VH domain (SEQ ID NO: 1)

[0129] QVQLVESGGGVVOPGRSLRLSCAASGFTFS TYGIH WVRQAPGKGLEWVA VIWYDGSDKFYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRDNWNYFDY WGQGTLVTVSS

[0130] M3T01 VL domain (SEQ ID NO: 2)

[0131] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAPKFLIY QASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYIT FGQGTRLEIK

[0132] M3T02 VH domain (SEQ ID NO:3)

[0133] OVOLVDSGGGVVOPGRSLRLSCAASGFTFS SYGMH WVROAPGKGPEWVA VIWYDGSNKYYADSVKG RFTISRDNSKNTLYLOMNSLRAEDTAVYYCAR DRDNWNHFDY WGOGTLVTVSS

[0134] M3T02 VL domain (SEQ ID NO:4)

[0135] ETVMTQSPATLSVSPGERATLSC RASOSFSSNLA WYQQKPGOAPRLLIY GASTRAT GTPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWLT FGGGTKVEIK

[0136] M3T03 VH domain (SEQ ID NO:5)

[0137] QIQLVOSGPDLKKPGETVKISCKASGYTFT NYGMN WVKKAPGKGLKWMG WINTNTGEPSYAEEFKG RFAFSLETSAGTAYLHINNLKNEDTATYFCVK YSRYYAMDF WGQGTSVTVSS

[0138] M3T03 VL domain (SEQ ID NO:6)

[0139] QAVVTQESALTTSPGETVTLTC RSSTGAVTTSNYAN WVQEKPDHLFTGLIG DTNN RAP GVPARFSGSLIGDKAALTITGAQTEDEAMYFC ALWYSNHWV FGGGTKLTVL

[0140] Table 1. Positions of heavy and light chain CDRs

[0141] Antibody domain SEQ ID NO: CDR1 CDR2 CDR3 M3T01 VH domain I 31-35 50-66 99-108 M3T01 VL domain 2 24-34 50-56 89-96 M3T02 VH domain 3 31-35 50-66 99-108 M3T02 VL domain 4 24-34 50-56 89-96 M3T03 VH domain 5 31-35 50-66 99-107 M3T03 VL domain 6 23-36 52-58 91-99

[0142] Human IgG4 heavy chain constant region (SEQ ID NO:7)

[0143] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSrEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0144] Human κ light chain constant region (SEQ ID NO:8)

[0145] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0146] M3T01 heavy chain (SEQ ID NO:9)

[0147] OVOLVESGGGVVOPGRSLRLSCAASGFTFSTYGIHWVROAPGKGLEWVAVIWYDGSDKFYADSVKGRF TISRDNSKNTLYLOMNSLRAEDTAVYYCARDRDNWNYFDYWGOGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMrSRTPEVTCVVVDVSQEDPEVQFIVWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSrEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDrAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0148] VH domain: residues 1 - 119

[0149] IgG4 constant region: residues 120 - 446

[0150] M3T01 light chain (SEQ ID NO:10)

[0151] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKFLIYQASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0152] VL domain: residues 1 - 106

[0153] κ constant region: residues 107 - 213

[0154] M3T02 heavy chain (SEQ ID NO:11)

[0155] QVOLVDSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGPEWVAVIWYDGSNKYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDNWNHFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSYLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTrSKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0156] VH domain: residues 1 - 119

[0157] IgG4 constant region: residues 120 - 446

[0158] M3T02 light chain (SEQ ID NO:12)

[0159] EIVMTOSPATLSVSPGERATLSCRASQSFSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT EFTLTISSLQSEDFAVYYCQQYNNWLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0160] VL domain: residues 1 - 106

[0161] κ constant region: residues 107 - 213

[0162] Provided herein are monoclonal antibodies that specifically bind FasL. In some aspects, 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 aspects, the monoclonal antibody comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain. In some instances, the monoclonal antibody comprises at least a portion of the amino acid sequences shown herein as SEQ ID NO:1 and / or SEQ ID NO:2, such as one or more (such as all three) CDR sequences from SEQ ID NO:1 and / or one or more (such as all three) CDR sequences from SEQ ID NO:2, as determined by any numbering scheme, such as IMGT, Kabat, or Chothia, or any combination thereof. In other instances, the monoclonal antibody comprises at least a portion of the amino acid sequences shown herein as SEQ ID NO:3 and / or SEQ ID NO:4, such as one or more (such as all three) CDR sequences from SEQ ID NO:3 and / or one or more (such as all three) CDR sequences from SEQ ID NO:4, as determined by any numbering scheme, such as IMGT, Kabat, or Chothia, or any combination thereof. In other instances, the monoclonal antibody comprises at least a portion of the amino acid sequences shown herein as SEQ ID NO:5 and / or SEQ ID NO:6, such as one or more (such as all three) CDR sequences from SEQ ID NO:5 and / or one or more (such as all three) CDR sequences from SEQ ID NO:6, as determined by any numbering scheme, such as IMGT, Kabat, or Chothia, or any combination thereof.

[0163] In some aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:1 and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:2. In other aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:3 and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:4. In other aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:5 and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody comprise the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:6. In some instances, the CDR sequences are determined using the Kabat, IMGT, or Chothia numbering scheme or a combination thereof. In certain instances, the CDR sequences are determined using Kabat.

[0164] In some aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise 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 comprise residues 24-34, 50-56, and 89-96 of SEQ ID NO:2, respectively. In some instances, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:1 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises 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 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises residues 24-34, 50-56, and 89-96 of SEQ ID NO:2. In certain instances, the amino acid sequence of the VH domain consists of or comprises SEQ ID NO:1 and / or the amino acid sequence of the VL domain consists of or comprises SEQ ID NO:2.

[0165] In other instances, the amino acid sequence of the VH domain comprises 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 comprises or consists of SEQ ID NO:2, SEQ ID NO:14, or SEQ ID NO:15. In certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:13 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:14. In other certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:1 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:15. In other certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:16 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:2. In other certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:17 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:2. In other certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:18 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:2.

[0166] In other aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise 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 comprise residues 24-34, 50-56, and 89-96 of SEQ ID NO:4, respectively. In some instances, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:3 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises 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 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises residues 24-34, 50-56, and 89-96 of SEQ ID NO:4. In certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:3 and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:4.

[0167] In other aspects, the CDR1, CDR2, and CDR3 sequences of the VH domain of the monoclonal antibody comprise residues 31-35, 50-66, and 99-107 of SEQ ID NO:5, respectively; and / or the CDR1, CDR2, and CDR3 sequences of the VL domain of the monoclonal antibody comprise residues 23-36, 52-58, and 91-99 of SEQ ID NO:6, respectively. In some instances, the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:5 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises 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 (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) and comprises residues 23-36, 52-58, and 91-99 of SEQ ID NO:6. In certain instances, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:5 and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:6.

[0168] In some aspects, the monoclonal antibody is an antigen-binding fragment selected from the group consisting of Fab fragments, Fab' fragments, F(ab)' 2 fragments, single-chain variable fragments (scFv), and disulfide-stabilized variable fragments (dsFv). In other aspects, the monoclonal antibody is an IgG, such as IgG4 or IgG1.

[0169] In some aspects, the monoclonal antibody comprises or further comprises a heavy chain constant region and / or a light chain constant region. In some instances, the heavy chain constant region is a human TgG4 heavy chain constant region. In some instances, the heavy chain constant region has one or more modifications relative to the wild-type heavy chain to increase the half-life, stability, and / or function of the monoclonal antibody, such as the S228P substitution in the human IgG4 heavy chain to prevent Fab arm exchange. In certain instances, 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 a specific non-limiting instance, the amino acid sequence of the heavy chain constant region comprises or consists of SEQ ID NO:7.

[0170] In some instances, the light chain constant region is a human kappa light chain constant region. In some instances, the light chain constant region has one or more modifications relative to the wild-type light chain constant region to increase the half-life, stability, and / or function of the monoclonal antibody. In certain instances, 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 instances, the amino acid sequence of the light chain constant region comprises or consists of SEQ ID NO:8.

[0171] In some aspects, the monoclonal antibody comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO:9 and / or the amino acid sequence of the light chain comprises or consists of SEQ ID NO:10.

[0172] In other aspects, the monoclonal antibody comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO:11 and / or the amino acid sequence of the light chain comprises or consists of SEQ ID NO:12.

[0173] In some aspects, the monoclonal antibody is a human antibody. In other aspects, the monoclonal antibody is a humanized antibody. In still other aspects, the monoclonal antibody is a chimeric antibody.

[0174] Also provided herein are monoclonal antibodies that bind to the same epitope on Fas ligand as the M3T01, M3T02, or M3T03 antibody (see also Section IV). In some aspects, the monoclonal antibody binds to the same epitope as a Fas ligand-specific monoclonal antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO:9 and a light chain having the amino acid sequence of SEQ ID NO:10. In some instances, the epitope is a conformational epitope spanning R144 to Y189 of Fas ligand, as shown in SEQ ID NO:17.

[0175] Further provided herein are fusion proteins comprising the disclosed Fas ligand-specific monoclonal antibody and a heterologous protein. In some instances, the heterologous protein is an Fc protein, such as human Fc. In other instances, the heterologous protein is a protein tag, such as a myc tag, His tag, HA tag, or FLAG tag. In other instances, the heterologous protein is an affinity tag, such as chitin binding protein, maltose binding protein, or glutathione-S-transferase (GST).

[0176] Also provided are multispecific antibodies that include the Fas ligand-specific monoclonal antibodies disclosed herein and at least one additional monoclonal antibody. In some aspects, the multispecific monoclonal antibody is a bispecific or trispecific monoclonal antibody. The at least one additional monoclonal antibody is capable of binding to a different epitope on Fas ligand or is capable of binding to a different antigen. The multispecific antibodies are further described in Section V.

[0177] Also provided herein are chimeric antigen receptors (CARs) that include the monoclonal antibodies disclosed herein. In some aspects, the CAR further includes a hinge region, a transmembrane domain, a co-stimulatory signaling moiety, a signaling domain, or any combination thereof. Further provided are cells that express a FasL-specific CAR, such as immune cells. In some instances, the immune cell is a T lymphocyte, such as a CTL, a B cell, a natural killer (NK) cell, or a macrophage. In some instances, the cell is an allogeneic cell, such as an allogeneic cell obtained from a healthy donor. The CARs and CAR-expressing cells are further described in Section VI.

[0178] Also provided are immunoconjugates that include a FasL-specific monoclonal antibody disclosed herein and an effector molecule. In certain aspects, the effector molecule is a toxin, a detectable label, or a photon absorber. The immunoconjugates are further described in Section VII.

[0179] Also provided are antibody-drug conjugates (ADCs) that include a drug conjugated to a FasL-specific monoclonal antibody disclosed herein. In some aspects, the drug is a small molecule, such as an anti-microtubule agent, an anti-mitotic agent, and / or a cytotoxic agent. The ADCs are further described in Section VIII.

[0180] Also provided are antibody-nanoparticle conjugates that include a nanoparticle conjugated to a FasL-specific monoclonal antibody disclosed herein. In some aspects, the nanoparticle includes a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle, or a nonionic surfactant vesicle. The antibody-nanoparticle conjugates are further described in Section IX.

[0181] Also provided herein are nucleic acid molecules that encode the monoclonal antibodies, fusion proteins, conjugates, or multispecific antibodies disclosed herein. In some aspects, the nucleic acid molecule is operably linked to a promoter. Further provided are vectors that include the nucleic acid molecules disclosed herein, and cells that include the nucleic acid molecules or vectors disclosed herein. The host cell can be, for example, a mammalian cell, a bacterial cell, or an insect cell. The nucleic acid molecules and vectors are further described in Section X.

[0182] Further provided is a composition comprising a pharmaceutically acceptable carrier and a monoclonal antibody, fusion protein, conjugate, multispecific antibody, nucleic acid molecule, or vector disclosed herein. The composition is further described in Section XI.

[0183] Also provided herein is a method of inhibiting Fas ligand in a subject in need thereof. In some aspects, the method comprises administering to the subject a therapeutically effective amount of a monoclonal antibody, fusion protein, conjugate, multispecific antibody, nucleic acid molecule, vector, or composition disclosed herein. In some instances, the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, an autoimmune disease, or COVID-19. In certain instances, the cancer is glioblastoma multiforme or myelodysplastic syndrome.

[0184] Further provided is a method of treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, an autoimmune disease, or COVID-19 in a subject. In some aspects, the method comprises administering to the subject a therapeutically effective amount of a monoclonal antibody, fusion protein, conjugate, multispecific antibody, nucleic acid molecule, vector, or composition disclosed herein. In some instances, the cancer is glioblastoma multiforme or myelodysplastic syndrome. The method is further described in Section XII.

[0185] IV. Identification of Antibodies that Bind to the Same Epitope on Fas Ligand

[0186] Also provided herein are monoclonal antibodies that bind to the same epitope on Fas ligand that binds to the M3T01, M3T02, or M3T03 antibody. Antibodies that bind to such an epitope can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with the M3T01, M3T02, or M3T03 antibody provided herein in a Fas ligand binding assay (such as those described in the examples). When the competing antibody concentration is above 10 6 x K DIn the presence of a competing antibody, an antibody "competes" for binding when the competing antibody inhibits Fas ligand binding by more than 50% of an M3T01, M3T02, or M3T03 antibody. In one aspect, the antibody that binds to the same epitope on Fas ligand as an M3T01, M3T02, or M3T03 antibody is a human monoclonal antibody. Human antibodies that bind to the same epitope on Fas ligand as an M3T01, M3T02, or M3T03 antibody can be generated using a variety of known techniques. Such antibodies can be prepared, for example, by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus has generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe the TM XENOMOUSE technology; U.S. Patent No. 5,770,429, which describes the technology; U.S. Patent No. 7,041,870, which describes K-M technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes the

[0187] Human antibodies that bind to the same epitope on Fas ligand that binds to the M3T01, M3T02, or M3T03 antibodies can also be made by a hybridoma-based method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001, 1984; and Boerner et al., J. Immunol. 147:86, 1991). Human antibodies produced via human B cell hybridoma technology are also described in Li et ai, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Triomatechnology) 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 subsequently be combined with desired human constant domains.

[0188] Monoclonal antibodies that specifically bind to the same epitope on Fas ligand that binds to M3T01, M3T02, or M3T03 can also be isolated by screening combinatorial libraries of antibodies for the desired binding properties. For example, a variety of methods are known for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed in, e.g., Hoogenboom, Methods Mol Biol 178:1-37, 2002.

[0189] V. Multispecific Antibodies

[0190] Multispecific antibodies are recombinant proteins composed of two or more monoclonal antibodies (or antigen-binding fragments thereof) of two or more different monoclonal antibodies. For example, bispecific antibodies can be composed of the antigen-binding fragments of two different monoclonal antibodies. Thus, bispecific antibodies bind two different antigens (or two different epitopes of the same antigen) and trispecific antibodies bind three different antigens or epitopes.

[0191] Provided herein are multispecific such as trispecific or bispecific monoclonal antibodies that comprise a FasL-specific monoclonal antibody. In some aspects, the multispecific monoclonal antibody further comprises a monoclonal antibody that specifically binds to another protein. In other aspects, 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 comprising the nucleic acid molecule or vector. Accordingly, provided herein is a method of 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) by administering to a subject a therapeutically effective amount of a FasL-targeted multispecific (such as bispecific) antibody.

[0192] VI. Chimeric Antigen Receptor (CAR)

[0193] The disclosed monoclonal antibodies can be used to generate CARs and / or immune cells (such as T cells, B cells, natural killer (NK) cells, or macrophages) engineered to express CARs. In some aspects, the CAR comprises a binding portion, an extracellular hinge and spacer element, a transmembrane region, and an intracellular domain that performs a signaling function (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In some instances, the binding portion is an antigen-binding fragment of a monoclonal antibody, such as an scFv. The spacer / hinge region typically comprises sequences from IgG subclasses, such as IgGl, IgG4, IgD, CD8, or CD28 domains. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8, CD28, or inducible T cell co-stimulator (ICOS). Several different intracellular domains have been used to generate CARs. For example, the intracellular domain can consist of a signaling chain having an ITAM such as CD3ζ or FcεRIζ. In some instances, the intracellular domain further comprises the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), CD30, ICOS, CD27, MYD88-CD40, killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2), and / or DAP10.

[0194] Immune cells expressing a CAR (such as T cells, B cells, NK cells or macrophages) can be used to target specific cell types, such as cells expressing FasL. Thus, the monoclonal antibodies disclosed herein can be used to engineer immune cells expressing a CAR containing a FasL-specific monoclonal antibody, thereby targeting the engineered immune cells to cells expressing FasL.

[0195] Multispecific (such as bispecific) or bicistronic CARs are also contemplated by the present disclosure. In some aspects, the multispecific or bispecific CARs include an antibody specific for FasL and an antibody specific for a second protein. Similarly, the bicistronic CAR includes two CAR molecules expressed from the same construct, wherein one CAR molecule is a FasL-targeting CAR and the second CAR targets a different protein. See, for example, Qin et al, Blood 130:810, 2017; and WO / 2018 / 213337.

[0196] Thus, provided herein are CARs comprising a FasL-specific antibody, such as any of the antibodies disclosed herein. Also provided are isolated nucleic acid molecules and vectors encoding the CARs (including bispecific and bicistronic CARs) and host cells, such as immune cells (e.g., T cells, B cells, NK cells or macrophages) expressing the CARs, bispecific CARs and bicistronic CARs. Immune cells expressing a CAR comprising a FasL-specific monoclonal antibody can be used to treat diseases, disorders or conditions associated with Fas / FasL signaling.

[0197] The present disclosure also provides modified FasL-specific monoclonal antibodies to enable their use with universal CAR systems. Universal CAR systems increase CAR flexibility and expand their use to additional antigens. Currently, for each patient receiving CAR-T cell therapy, autologous T cells are cultured, expanded, and modified to express an antigen-specific CAR. This process is lengthy and expensive, limiting its use. Universal CARs are based on a system in which the signaling components of the CAR are split from the antigen-binding portion of the molecule but come together using a "lock-and-key" system. For example, a biotin-binding immunoreceptor (BBIR) CAR comprises an intracellular T cell signaling domain fused to an extracellular domain containing avidin. A biotinylated antigen-specific (such as FasL-specific) monoclonal antibody can then bind to the BBIR to direct immune cells to FasL-expressing cells. Another example is the split, universal, and programmable (SUPRA) CAR system. In the SUPRA system, the CAR comprises an intracellular signaling domain fused to an extracellular leucine zipper that pairs with an antigen-specific monoclonal antibody fused to a cognate leucine zipper. For a review of universal CAR systems, see, e.g., Zhao et al., J Hematol Oncol 11(1):132, 2018; and Cho et al., Cell 173:1426-1438, 2018. In some aspects of the present disclosure, the FasL-specific antibody is fused to one component of a specific binding pair. In some examples, the antibody is fused to a leucine zipper or biotin.

[0198] Another type of universal CAR can be generated using a transpeptidase. A transpeptidase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. The transpeptidase catalyzes a transpeptidation between a transpeptidase recognition motif and a transpeptidase receptor motif. Thus, an antigen-specific CAR can be generated by contacting an antigen-specific antibody fused to a transpeptidase recognition motif with a portion of a CAR molecule comprising an intracellular signaling domain, a transmembrane region, and an extracellular portion containing a transpeptidase receptor motif. In the presence of the transpeptidase, the two components become covalently attached to form a complete antigen-specific CAR. Thus, in some aspects of the present disclosure, the FasL-specific antibody is modified to include a transpeptidase recognition motif (see, e.g., PCT Publication No. WO2016 / 014553).

[0199] In some aspects, the FasL-specific CAR is expressed in allogeneic immune cells such as T cells, B cells, NK cells, or macrophages, such as allogeneic immune cells from a healthy donor. In some instances, the allogeneic cells are genetically engineered to express the FasL-specific CAR, for example, by disrupting the expression of the endogenous T cell receptor via insertion of the CAR (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 (TALEN).

[0200] VII. Immunoconjugates

[0201] The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent with the antibody. A therapeutic agent is a preparation having a specific biological activity against a specific target molecule or a cell carrying the target molecule. Those skilled in the art will appreciate that therapeutic agents can include various drugs such as vinblastine, daunomycin, etc., cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) containing a pharmacological composition, radiopharmaceuticals such as 125 I、 32 P、 14 C、 3 H and 35 S, photon absorbers such as IR700, and other labels, target moieties, and ligands.

[0202] The choice of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin, which is used to cause the death of a particular target cell such as a cancer cell. Conversely, in cases where a non-lethal biological response is desired, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0203] Using the therapeutic agents and monoclonal antibodies described herein, a variety of clones containing functionally equivalent nucleic acids can be readily constructed, such as nucleic acids that differ in sequence but encode the same effector portion or antibody sequence. Accordingly, the present disclosure provides nucleic acids encoding antibodies and their conjugates and fusion proteins.

[0204] The effector molecule can be attached to the monoclonal antibody of interest using any number of means known to those skilled in the art. Both covalent and non-covalent attachment means can be used. The procedure for attaching the effector molecule to the antibody varies depending on the chemical structure of the effector molecule. Polypeptides typically contain multiple functional groups; such as carboxylic acid (COOH), free amine (-NH 2) or a thiol (-SH) group, which can be used to react with a suitable functional group on the antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any of a number of known linker molecules. The linker can be any molecule used to connect the antibody to the effector molecule. The linker is capable of forming covalent bonds with both the antibody and the effector molecule. Suitable linkers are known and include, but are not limited to, straight-chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the effector molecule are polypeptides, the linker can be attached to the alpha-carbon amino and carboxyl groups of the constituent amino acids or terminal amino acids through its side groups (such as through a disulfide bond with cysteine).

[0205] Generally, monoclonal antibodies are derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical conjugation, genetic fusion, non-covalent association, or other means) 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 that can mediate the association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0206] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types, such as to produce a bispecific antibody). Suitable cross-linking agents include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl esters). Such linkers are commercially available.

[0207] In some cases, it is desirable to release the effector molecule from the antibody when the immunoconjugate has reached its target site. Thus, in these cases, the immunoconjugate includes a bond that is cleavable near the target site. Cleavage of the linker to release the effector molecule from the antibody can be promoted by enzymatic activity or conditions that the immunoconjugate undergoes inside the target cell or near the target site.

[0208] The monoclonal antibodies provided herein can also be conjugated with a detectable label; for example, the detectable label can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels 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 labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide fluorophores, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). The antibody can also be conjugated with an enzyme useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody or antigen-binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents for the enzyme to produce a distinguishable reaction product. For example, when horseradish peroxidase is present, the addition of hydrogen peroxide and di-nitroaniline results in a colored reaction product that is visually detectable. The antibody or antigen-binding fragment can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. Avidin itself can also be conjugated with an enzyme or a fluorescent tag.

[0209] The antibodies provided herein can be labeled with magnetic agents such as gadolinium. The antibody can also be labeled with lanthanide elements (e.g., europium and dysprosium) and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also used as labels. The antibody can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter, such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal-binding domain, an epitope tag. In some aspects, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0210] The antibodies provided herein can also be labeled with radiolabeled amino acids. The radiolabeling can be used for both diagnostic and therapeutic purposes. For example, the radiolabeling can be used to detect the expression of a target antigen by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radiolabeled nucleotides: 3 H 14 C 15 N 35 S 90 Y 99 Tc 111 In 125 I 131I. Given the numerous methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of ordinary skill in the art can determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0211] The antibodies disclosed herein can also be conjugated to photon absorbers. In some aspects, the photon absorber is a phthalocyanine dye, such as, but not limited to 700DX (also known as "IR700"). Antibody-photon absorber conjugates can be used in photoimmunotherapy (e.g., to kill tumor cells).

[0212] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or carbohydrate groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life or increasing tissue binding.

[0213] Toxins can be used with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and its subunits, and botulinum toxins A - F. These toxins are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Envisioned toxins also include variants of the toxins described herein (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401). In one aspect, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to the full-length native (naturally occurring) PE or PE that has been modified. Such modifications can include, but are not limited to, the elimination of domain la, various amino acid deletions in domains lb, II, and III, single amino acid substitutions, and the addition of one or more sequences at the carboxyl terminus (see, e.g., Siegall et al., J. Biol. Chem. 264:14256 - 14261, 1989).

[0214] The PE used with the monoclonal antibodies described herein can include native sequences, cytotoxic fragments of native sequences, and conservative modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those that are cytotoxic in target cells with or without subsequent proteolytic or other processing. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional descriptions of PE and its variants, see, for example, U.S. Patent 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 Publication Nos. WO 99 / 51643, WO 2007 / 016150, WO 2009 / 032954, WO 2011 / 032022, and WO 2014 / 052064; Pai etaL, Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et aL, J. Biol. Chem. 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.

[0215] VIII. Antibody-Drug Conjugates (ADCs)

[0216] An ADC is a compound composed of an antigen-specific antibody (such as a FasL-specific antibody) and a drug, such as a cytotoxic agent (such as an anti-microtubule agent or a cross-linking agent). Since ADCs can specifically target cells expressing a specific antigen, the drug is much more effective than drugs used for standard systemic therapy. For example, the IC of the most common cytotoxic drugs currently used with ADCs 50100 to 1000 times more potent than traditional chemotherapeutic agents. Exemplary cytotoxic drugs include anti-microtubule agents such as maytansinoid compounds and auristatins (such as auristatin E and auristatin F). Other cytotoxins used with ADCs include pyrrolobenzodiazepines (PBDs) which covalently bind to the minor groove of DNA to form interstrand crosslinks. In some instances, the ADC comprises an antibody provided herein and a drug in a ratio of 1:2 to 1:4 (Bander, Clinical Advances in Hematology&Oncology 10(8;suppl10):3-7,2012).

[0217] The antibody and the drug can be linked by a cleavable or non-cleavable linker. However, in some cases, it is desirable to have a linker that is stable in the circulation to prevent systemic release of the cytotoxic drug that may lead to significant off-target toxicity. The non-cleavable linker prevents release of the cytotoxic agent until the ADC is internalized by the target cell. Once inside the lysosome, digestion of the antibody by lysosomal proteases results in release of the cytotoxic agent (Bander, Clinical Advances in Hematology&Oncology 10(8;suppl10):3-7,2012).

[0218] One method for site-specific and stable conjugation of a drug to a monoclonal antibody (or antibody-Fc fusion protein) is via glycan engineering. Monoclonal antibodies have a conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526,2008). Using a mutant β1,4-galactosyltransferase (Y289L-Gal-T1; US Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162), 2-keto-galactose is transferred to the free GlcNAc residue on the antibody heavy chain to provide a chemical handle for conjugation.

[0219] Oligosaccharide chains attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residue - fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-G1), or fully degalactosylated (IgG-G0). Treatment of monoclonal antibodies with β1,4-galactosidase converts the antibodies to the IgG-G0 glycoform. Mutant β1,4-galactosyltransferases can transfer 2-ketogalactose or 2-azidogalactose from their corresponding UDP derivatives to the GlcNAc residues on IgG-G1 and IgG-G0 glycoforms. The chemical handles on the transferred sugars enable the conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0220] Provided herein are ADCs that include a drug (such as an anti-cancer agent) conjugated to a monoclonal antibody that specifically binds FasL. In some aspects, the drug is a small molecule. In some instances, the drug is a crosslinker, an anti-microtubule agent and / or an anti-mitotic agent, or any cytotoxic agent suitable for mediating the killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PBD, auristatin, maytansine, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, elinafide, combretastatin, dolastatin, enediyne, geldanamycin, indoline-benzodiazepine dimer, puromycin, tubulysin, hamycin, sprengelastatin, pladienolide, and stereoisomers, isomers, analogs, and derivatives thereof having cytotoxic activity.

[0221] In some aspects, the ADC can further include a linker. In some instances, the linker is a bifunctional or multifunctional moiety that can be used to attach one or more drug moieties to the antibody to form the ADC. In some aspects, the ADC is prepared using a linker having reactive functionalities for covalent attachment to the drug and the antibody. For example, the cysteine thiol of the antibody can form a bond with the reactive functional group of the linker or the drug-linker intermediate to make the ADC.

[0222] In some instances, the linker has a functionality capable of reacting with free cysteine present on the antibody to form a covalent bond. Exemplary linkers having such reactive functionalities include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0223] In some instances, the linker has functionality that can react with electrophilic groups present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyls. In some cases, the heteroatom of the reactive functionality of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody moiety. Non-limiting examples include hydrazine, oxime, amino, hydrazide, thiosemicarbazide, hydrazine carboxylate, and aromatic acylhydrazide.

[0224] In some instances, the linker is a cleavable linker that facilitates the release of the drug. Examples of cleavable linkers include acid-labile linkers (e.g., containing hydrazone), protease-sensitive linkers (e.g., peptidase-sensitive), photo-labile linkers, and disulfide-containing linkers (Chari et aL, Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020).

[0225] The ADCs disclosed herein can be used to treat diseases, disorders, or conditions associated with Fas / FasL signaling, alone or in combination with another therapeutic agent and / or in combination with any standard therapy for treating 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 diseases, or COVID-19).

[0226] IX. Antibody-Nanoparticle Conjugates

[0227] The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic or other therapeutic agents directly to FasL-expressing cells. The use of nanoparticles reduces off-target side effects and can also increase drug bioavailability and reduce the drug dose required to achieve a therapeutic effect. Nanoparticle formulations can be customized to suit the drug to be carried or encapsulated within the nanoparticles. For example, hydrophobic molecules can be incorporated into the inner core of the nanoparticles, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, nonionic surfactant vesicles, and polymeric nanoparticles (Fayand Scott, Immunotherapy 3(3):381-394, 2011).

[0228] Liposomes are a common type of nanoparticles used for drug delivery. Antibodies conjugated to liposomes are commonly referred to as "immunoliposomes". The liposomal component of immunoliposomes is typically a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, the phospholipid consists of a hydrophilic head group and two hydrophobic chains to enable encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from the circulation by macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size, and charge of the liposomes can be modulated. The surface of the liposomes can also be modified, such as with glycolipids or sialic acid. For example, the inclusion of polyethylene glycol (PEG) significantly increases the circulation half-life. Liposomes for use as drug delivery agents, including 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 No. 2011 / 0268655; 2010 / 00329981).

[0229] Nonionic surfactant vesicles are nonionic surfactant-based vesicles with a structure similar to liposomes. The membrane of nonionic surfactant vesicles consists only of nonionic surfactants, such as polyglycerol alkyl ethers or N-palmitoyl glucosamine. Nonionic surfactant vesicles range from small, single-layer to large, multi-layer particles. These nanoparticles are monodisperse, water-soluble, chemically stable, of low toxicity, biodegradable and non-immunogenic, and increase the bioavailability of encapsulated drugs.

[0230] Dendrimers comprise a series of branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Generally, dendrimers consist of an initiator core surrounded by layers of selected polymers grafted to the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used in a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, chemotherapeutic agents, and other drugs.

[0231] Polymeric micelles consist of aggregates of amphiphilic copolymers (composed of both hydrophilic and hydrophobic monomer units), assembled into a hydrophobic core surrounded by a corona of hydrophilic polymer chains exposed to the aqueous environment. In many cases, the polymers used to prepare polymeric micelles are hetero-bifunctional copolymers composed of hydrophilic blocks of PEG, poly(vinylpyrrolidone), and hydrophobic poly(L-lactide) or poly(L-lysine) that form the particle core. Polymeric micelles can be used to carry poorly soluble drugs. These nanoparticles have been used to encapsulate many drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0232] Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The selected formulation typically depends on the solubility of the therapeutic agent to be carried / encapsulated; drugs with poor water solubility are more easily encapsulated within nanospheres, while water-soluble and labile drugs such as DNA and proteins are more easily encapsulated within nanocapsules. Polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkyl cyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0233] The antibodies provided herein can be conjugated to suitable nanoparticles according to standard methods known in the art. For example, the conjugation can be covalent or non-covalent. In some aspects in which the nanoparticles are liposomes, the antibody is attached via a PEG chain to a sterically stabilized, long-circulating liposome. The coupling of an antibody or antibody fragment to a liposome can also involve a thioester bond, for example, through the reaction of a thiol and a maleimide group. Crosslinking agents can be used to generate thiols for attaching the antibody to the nanoparticles (Paszko and Senge, Curr MedChem 19(31)5239-5277, 2012).

[0234] X. Nucleic Acid Molecules

[0235] Nucleic acid molecules (such as DNA, cDNA, mRNA, or RNA molecules) encoding the amino acid sequences of the disclosed monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates that specifically bind FasL are provided. The nucleic acid molecules encoding these molecules can be readily generated using the amino acid sequences provided herein (such as CDR sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In some aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell, yeast cell, or bacterial cell) to produce the disclosed monoclonal antibody, fusion protein, multispecific antibody, CAR, or immunoconjugate.

[0236] The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that differ in sequence but encode the same antibody sequence.

[0237] Nucleic acid molecules encoding monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates that specifically bind FasL can be prepared by any suitable method, including, for example, cloning appropriate sequences or direct chemical synthesis by standard methods. Chemical synthesis produces single-stranded oligonucleotides. This can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization using the single strand as a template with a DNA polymerase.

[0238] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th th 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).

[0239] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR).

[0240] Nucleic acid molecules can be expressed in recombinant engineered cells, such as in bacterial, plant, yeast, insect, or mammalian cells. Monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates can be expressed as individual proteins, including monoclonal antibodies (optionally linked to effector molecules or detectable markers as needed), or can be expressed as fusion proteins. Any suitable method for expressing and purifying antibodies and antigen-binding fragments can be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011).

[0241] One or more DNA sequences encoding monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates can be expressed in vitro by transferring the DNA into a suitable host cell. The cells can be prokaryotic or eukaryotic. Many expression systems available for expressing proteins, including Escherichia coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as mammalian cells such as COS, CHO, HeLa, and myeloma cell lines, can be used to express the disclosed antibodies. Stable transfer methods can be used, which means that the foreign DNA is continuously maintained in the host.

[0242] Expression of the nucleic acids encoding the monoclonal antibodies, fusion proteins, multispecific antibodies, CARs, and immunoconjugates described herein can be achieved by operably linking the DNA or cDNA to a promoter (which can be constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, such as the cytomegalovirus promoter. Optionally, an enhancer such as the cytomegalovirus enhancer is included in the construct. The expression cassette can be suitable for replication and integration in prokaryotes or eukaryotes. A typical expression cassette contains specific sequences useful for regulating the expression of the DNA encoding the protein. For example, the expression cassette can include a suitable promoter, enhancer, transcriptional and translational terminators, initiation sequences, a start codon (i.e., ATG) before the protein-coding gene, splicing signals for introns, sequences for maintaining the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The vector can encode selectable markers, such as markers encoding drug resistance (such as ampicillin or tetracycline resistance).

[0243] To obtain high-level expression of the cloned gene, the expression cassette can include, for example, a strong promoter for directing transcription, a ribosome binding site for translation initiation (such as an internal ribosome entry sequence), and transcriptional / translational terminators. For Escherichia coli, this can include promoters such as the T7, trp, lac, or lambda promoter, ribosome binding sites, and transcriptional termination signals. For eukaryotic cells, the control sequences can include promoters and / or enhancers derived from, for example, immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and can further include splicing donor and acceptor sequences (such as CMV and / or HTLV splice acceptor and donor sequences). The expression cassette can be transferred into the selected host cell by any suitable method such as transformation or electroporation for Escherichia coli and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed with the expression cassette can be selected by antibiotic resistance conferred by the genes (such as the amp, gpt, neo, and hyg genes) contained in the expression cassette.

[0244] The nucleic acids encoding the antibodies or conjugates described herein can be modified without reducing their biological activity. Modifications can be made to facilitate cloning, expression, or incorporation into a fusion protein of the antibody. Such modifications include, for example, stop codons, sequences that create conveniently located restriction sites, and sequences that add methionine at the amino terminus to provide a start site, or additional amino acids (such as polyHis) to assist in purification steps.

[0245] Once expressed, antibodies, fusion proteins, multispecific antibodies, and other conjugates can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, etc. (generally see 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 need not be 100% pure. Once purified, partially purified, or of the desired homogeneity, if to be used prophylactically, the protein should be substantially endotoxin-free.

[0246] XI. Compositions

[0247] Compositions are provided that include one or more of the disclosed monoclonal antibodies that specifically bind FasL in a carrier. Compositions are also provided that include fusion proteins, multispecific antibodies, CARs, CAR-expressing cells, immunoconjugates, ADCs, or antibody-nanoparticle conjugates, as well as nucleic acid molecules and vectors encoding these molecules. These compositions are prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired result. 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 local administration. In one example, the composition is formulated for intravenous administration. In other examples, the composition is formulated for intramuscular or intraperitoneal administration. In other examples, the composition is formulated for intratumoral administration. In further examples, the composition is formulated for subcutaneous administration.

[0248] In some aspects, the composition includes more than one FasL-specific monoclonal antibody disclosed herein, such as 2 or 3 different antibodies (or multiple fusion proteins, multispecific antibodies, CARs, CAR-expressing immune cells, immunoconjugates, ADCs, antibody-nanoparticle conjugates, isolated nucleic acid molecules, or vectors). Kits are also provided that include one or more FasL-specific monoclonal antibodies disclosed herein, such as 2 or 3 different antibodies (or multiple fusion proteins, multispecific antibodies, CARs, CAR-expressing immune cells, immunoconjugates, ADCs, antibody-nanoparticle conjugates, isolated nucleic acid molecules, or vectors). Such kits can include one or more other therapeutic agents, such as those provided herein (e.g., other mAbs, chemotherapeutic agents, or combinations thereof).

[0249] The composition for administration can include a solution of a monoclonal antibody, fusion protein, multispecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody-nanoparticle conjugate, isolated nucleic acid molecule, and / or vector in a pharmaceutically acceptable carrier (such as an aqueous carrier). A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional well-known sterilization techniques. The composition can contain pharmaceutically acceptable adjuvants as needed to approximate physiological conditions, such as pH adjustment and buffering agents, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the monoclonal antibody, fusion protein, multispecific antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, antibody-nanoparticle conjugate, nucleic acid, and / or vector in these formulations can vary and can be selected based on the fluid volume, viscosity, body weight, etc., according to the particular mode of administration chosen and the needs of the subject.

[0250] Exemplary pharmaceutical compositions for intravenous administration include from about 0.1 to 10 mg of antibody (or fusion protein, bispecific antibody, etc.) / subject / day. Doses from 0.1 up to about 100 mg / subject / day can be used, especially if the agent is administered to a secluded site rather than into the circulation or lymphatic system, such as into the lumen of a body cavity or organ. In some aspects, the composition can be a liquid formulation, including a concentration range from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 1 mg / ml to about 10 mg / ml. The actual methods of preparing the administrable compositions are known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005).

[0251] The compositions disclosed herein can also be administered by other routes, including via inhalation or orally, such as by oral administration of yeast or bacteria (e.g., Lactococcus lactis) engineered to express the disclosed antibody or conjugate (see, e.g., Vandenbroucke et al, Mucosal Immunol 3(1):49 - 56, 2010).

[0252] The disclosed compositions can be provided in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided as sterile solutions at known concentrations. The antibody solution can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dose of 0.5 to 15 mg / kg body weight. The field has considerable experience in administering antibody drugs, and since the approval of RITUXAN in 1997 TM the drug has been on the market in the United States. The disclosed compositions can be administered by slow infusion rather than by intravenous bolus or push. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level.

[0253] Controlled release parenteral formulations can be made as implants, oily injections or as particulate systems. For a general overview of protein delivery systems, see Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres and microcapsules less than about 1 μm in size are generally referred to as nanoparticles, nanospheres and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so that nanoparticles can only be administered intravenously. Microparticles typically have a diameter of about 100 μm 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)

[0254] Polymers can be used for the ion-controlled release of the antibody-based compositions disclosed herein. A variety of degradable and non-degradable polymeric 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 flowing 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.PharmA 12:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of lipid-encapsulated drugs ((Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many other systems for controlling the delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496)

[0255] XII. Methods of Treatment

[0256] Also provided herein are methods of inhibiting Fas ligand in a subject in need thereof. In some aspects, the methods include administering to the subject 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. In some aspects, the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, an autoimmune disease, or COVID-19.

[0257] The present disclosure further provides methods of treating diseases, disorders or conditions associated with Fas / FasL signaling in a subject such as, but not limited to, cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, autoimmune diseases or COVID-19. In some aspects, the methods include administering to the subject 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.

[0258] In some examples of the disclosed methods, 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% relative to Fas / FasL signaling prior to treatment.

[0259] In some examples, the methods increase the survival of the subject, such as 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 survival without treatment. In some examples, the methods increase the survival time of the subject, such as by 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 relative to the survival time without treatment.

[0260] In some examples, the methods reduce inflammation in the subject, such as 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% relative to inflammation prior to treatment.

[0261] In some examples in which the subject has cancer, the methods reduce 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% relative to the size, volume and / or weight of the tumor prior to treatment. In some examples in which the subject has cancer, the methods reduce the size, volume and / or weight of the metastasis 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% relative to the size, volume and / or weight of the metastasis prior to treatment.

[0262] In some aspects, a subject is administered a second therapy or therapeutic agent. For example, a subject with cancer can be treated with a second anti-cancer therapy, such as chemotherapy, biotherapy (e.g., different monoclonal antibodies), radiotherapy, surgical resection, cryosurgery, laser therapy, and / or administration of a checkpoint inhibitor.

[0263] Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g., anti-androgens) and anti-angiogenic agents. Other anti-cancer therapies 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). In one instance, cancer is treated by administering a polypeptide, antibody, fusion protein, CAR, CAR-expressing cell, immunoconjugate, ADC, multispecific antibody, antibody-nanoparticle conjugate or composition disclosed herein and one or more therapeutic mAbs such as a PD-L1 antibody (e.g., durvalumab, KN035, cosibelimab, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A or MEDI-4737), an anti-PD-1 antibody (e.g., pembrolizumab, cemiplimab, or nivolumab), an anti-EGFR antibody (e.g., cetuximab or panitumumab), an anti-VEGF antibody (e.g., bevacizumab or ramucirumab) or a CLTA-4 antibody (e.g., ipilimumab or tremelimumab).In one instance, cancer is treated by administering the compositions disclosed herein and one or more monoclonal antibodies, such as: 3F8, abagovomab, adalimumab, obinutuzumab, perakizumab, alemtuzumab, pentixaforatumomab, mapatumumab, apolizumab, asimozumab, batoximab, betumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, mepolizumab, carotuximab pendetide, catumaxomab, CC49, cetuximab, pascolizumab, cixutumumab, tucotuzumab celmoleukin, canakinumab, dacizumab, dimab, emibetuzumab, eculizumab, evorpacept, epratuzumab, ertumaxomab, edrumab, farletuzumab, fontolizumab, galiximab, gemtuzumab ozogamicin, gemtuzumab, vintafolide - glembatumumab, ibritumomab tiuxetan, igovomab, inotuzumab ozogamicin, ipilimumab, itolizumab, labretuzumab, lesinurad, lintuzumab, moxetumomab pasudotox, lucatumumab, luxizumab, mapatumumab, matuzumab, mepolizumab, metelimumab, mirvetuximab soravtansine, mitumomab, molgramostim, tanakizumab, tanatumumab, necitumumab, nimotuzumab, nivolumab, nofetumomab, ofatumumab, olaratumab, moxetumomab pasudotox, ogovomab, panitumumab, pembrolizumab, pertuzumab, panitumumab, primatumumab, ramucirumab, rituximab, rituxan, rotumomab, satumomab pendetide, sirukumab, sonepcizumab, tefibazumab, patritumab, tigatuzumab, TNX - 650, tesetumab, trastuzumab, tremelimumab, simtuzumab, vetelizumab, vorloximab, votumumab, zalutumumab or a combination thereof.

[0264] XIII. Further aspects

[0265] Aspect 1. A monoclonal antibody that specifically binds Fas ligand, comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein:

[0266] the VH domain comprises the complementarity - determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO:1, and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:2;

[0267] The VH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:3, and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:4; or

[0268] The VH domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:5, and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6.

[0269] Aspect 2. The monoclonal antibody according to aspect 1, wherein:

[0270] The CDR1, CDR2 and CDR3 sequences of the VH domain respectively comprise residues 31-35, 50-66 and 99-108 of SEQ ID NO:1; and

[0271] The CDR1, CDR2 and CDR3 sequences of the VL domain respectively comprise residues 24-34, 50-56 and 89-96 of SEQ ID NO:2.

[0272] Aspect 3. The monoclonal antibody according to aspect 2, wherein:

[0273] The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:1 and comprises residues 31-35, 50-66 and 99-108 of SEQ ID NO:1; and

[0274] The amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:2 and comprises residues 24-34, 50-56 and 89-96 of SEQ ID NO:2.

[0275] Aspect 4. The monoclonal antibody according to any one of aspects 1 to 3, wherein:

[0276] The amino acid sequence of the VH domain comprises or consists of SEQ ID NO:1; and

[0277] The amino acid sequence of the VL domain comprises or consists of SEQ ID NO:2.

[0278] Aspect 5. The monoclonal antibody according to aspect 1, wherein:

[0279] The CDR1, CDR2 and CDR3 sequences of the VH domain respectively comprise residues 31-35, 50-66 and 99-108 of SEQ ID NO:3; and

[0280] The CDR1, CDR2, and CDR3 sequences of the VH domain respectively contain residues 24-34, 50-56, and 89-96 of SEQ ID NO:4.

[0281] Aspect 6. The monoclonal antibody according to aspect 5, wherein:

[0282] The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:3 and contains residues 31-35, 50-66, and 99-108 of SEQ ID NO:3; and

[0283] The amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:4 and contains residues 24-34, 50-56, and 89-96 of SEQ ID NO:4.

[0284] Aspect 7. The monoclonal antibody according to aspect 1 and any one of aspects 5-6, wherein:

[0285] The amino acid sequence of the VH domain contains or consists of SEQ ID NO:3; and

[0286] The amino acid sequence of the VL domain contains or consists of SEQ ID NO:4.

[0287] Aspect 8. The monoclonal antibody according to aspect 1, wherein:

[0288] The CDR1, CDR2, and CDR3 sequences of the VH domain respectively contain residues 31-35, 50-66, and 99-107 of SEQ ID NO:5; and

[0289] The CDR1, CDR2, and CDR3 sequences of the VL domain respectively contain residues 23-36, 52-58, and 91-99 of SEQ ID NO:6.

[0290] Aspect 9. The monoclonal antibody according to aspect 8, wherein:

[0291] The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:5 and contains residues 31-35, 50-66, and 99-107 of SEQ ID NO:5; and

[0292] The amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:6 and contains residues 23-36, 52-58, and 91-99 of SEQ ID NO:6.

[0293] Aspect 10. The monoclonal antibody according to aspect 1 and any one of aspects 8-9, wherein:

[0294] The amino acid sequence of the VH domain comprises or consists of SEQ ID NO:5; and

[0295] the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:6.

[0296] Aspect 11. The monoclonal antibody according to any one of Aspects 1-10, further comprising a heavy chain constant region and a light chain constant region.

[0297] Aspect 12. The monoclonal antibody according to Aspect 11, wherein the heavy chain constant region is a human IgG4 heavy chain constant region.

[0298] Aspect 13. The monoclonal antibody according to Aspect 12, wherein the amino acid sequence of the human IgG4 heavy chain constant region comprises or consists of SEQ ID NO:7.

[0299] Aspect 14. The monoclonal antibody according to any one of Aspects 11-13, wherein the light chain constant region is a human kappa light chain constant region.

[0300] Aspect 15. The monoclonal antibody according to Aspect 14, wherein the amino acid sequence of the human kappa light chain constant region comprises or consists of SEQ ID NO:8.

[0301] Aspect 16. The monoclonal antibody according to any one of Aspects 11-15, which comprises a heavy chain and a light chain, wherein:

[0302] 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

[0303] 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.

[0304] Aspect 17. A monoclonal antibody that binds to the same epitope as a Fas ligand-specific monoclonal antibody, comprising:

[0305] a heavy chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9; and

[0306] a light chain, the light chain comprising the amino acid sequence of SEQ ID NO:10.

[0307] Aspect 18. The monoclonal antibody according to Aspect 17, wherein the epitope is a conformational epitope spanning R144 to Y189 of Fas ligand, as shown in SEQ ID NO:17.

[0308] Aspect 19. The monoclonal antibody according to any one of Aspects 1-18, wherein the antibody is a human antibody or a humanized antibody.

[0309] Aspect 20. The monoclonal antibody according to any one of Aspects 1-18, wherein the antibody is a chimeric antibody.

[0310] Aspect 21. A fusion protein comprising the monoclonal antibody according to any one of Aspects 1-20 and a heterologous protein.

[0311] Aspect 22. The fusion protein according to Aspect 21, wherein the heterologous protein is an Fc protein.

[0312] Aspect 23. A multispecific antibody comprising the monoclonal antibody according to any one of Aspects 1-20 and at least one additional monoclonal antibody or an antigen-binding fragment thereof.

[0313] Aspect 24. The multispecific antibody according to Aspect 23, which is a bispecific antibody.

[0314] Aspect 25. A chimeric antigen receptor (CAR) comprising the monoclonal antibody according to any one of Aspects 1-20.

[0315] Aspect 26. An isolated cell expressing the CAR according to Aspect 25.

[0316] Aspect 27. The isolated cell according to Aspect 26, wherein the cell is an immune cell.

[0317] Aspect 28. An immunoconjugate comprising the monoclonal antibody according to any one of Aspects 1-20 and an effector molecule.

[0318] Aspect 29. The immunoconjugate according to Aspect 28, wherein the effector molecule is a toxin, a detectable label, or a photon absorber.

[0319] Aspect 30. An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody according to any one of Aspects 1-20.

[0320] Aspect 31. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the monoclonal antibody according to any one of Aspects 1-20.

[0321] 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 nonionic surfactant vesicle.

[0322] Aspect 33. A nucleic acid molecule encoding a monoclonal antibody according to any one of aspects 1-20, a fusion protein according to aspect 21 or aspect 22, a multispecific antibody according to aspect 23 or aspect 24, a CAR according to aspect 25, or an immunoconjugate according to aspect 28 or aspect 29.

[0323] Aspect 34. The nucleic acid molecule according to aspect 33, which is operably linked to a promoter.

[0324] Aspect 35. A vector comprising the nucleic acid molecule according to aspect 33 or aspect 34.

[0325] Aspect 36. An isolated host cell comprising the nucleic acid molecule according to aspect 33 or aspect 34, or the vector according to aspect 35.

[0326] Aspect 37. A composition comprising a pharmaceutically acceptable carrier and a monoclonal antibody according to any one of aspects 1-20, a fusion protein according to aspect 21 or aspect 22, a multispecific antibody according to aspect 23 or aspect 24, a CAR according to aspect 25, an isolated cell according to any one of aspects 26, 27, and 36, an immunoconjugate according to aspect 28 or aspect 29, an ADC according to aspect 30, an antibody-nanoparticle conjugate according to aspect 31 or aspect 32, the nucleic acid molecule according to aspect 33 or aspect 34, or the vector according to aspect 35.

[0327] Aspect 38. A method of inhibiting Fas ligand in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody according to any one of aspects 1-20, a fusion protein according to aspect 21 or aspect 22, a multispecific antibody according to aspect 23 or aspect 24, a CAR according to aspect 25, an isolated cell according to any one of aspects 26, 27, and 36, an immunoconjugate according to aspect 28 or aspect 29, an ADC according to aspect 30, an antibody-nanoparticle conjugate according to aspect 31 or aspect 32, the nucleic acid molecule according to aspect 33 or aspect 34, the vector according to aspect 35, or the composition according to aspect 37, thereby inhibiting Fas ligand in the subject.

[0328] Aspect 39. The method according to aspect 38, wherein the subject has cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, an autoimmune disease, myelodysplastic syndrome (MDS), or coronavirus disease 2019 (COVID-19).

[0329] Aspect 40. The method according to aspect 39, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

[0330] Aspect 41. A method of 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 to the subject a therapeutically effective amount of the monoclonal antibody according to any one of aspects 1-20, 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, the isolated cell according to any one of aspects 26, 27 and 36, the immunoconjugate according to aspect 28 or aspect 29, the ADC according to aspect 30, the antibody-nanoparticle conjugate according to aspect 31 or aspect 32, the nucleic acid molecule according to aspect 33 or aspect 34, the vector according to aspect 35 or the composition according to aspect 37.

[0331] Aspect 42. The method according to aspect 41, wherein the cancer is glioblastoma multiforme or myelodysplastic syndrome.

[0332] The following examples are provided to illustrate certain specific features and / or aspects. These examples should not be construed as limiting the disclosure to the specific features or aspects described.

[0333] Examples

[0334] Example 1: Monoclonal Antibodies Specific for Fas Ligand

[0335] This example describes three monoclonal antibodies that bind and block human FasL function. Two of the antibodies (M3T01 and M3T02) are human monoclonal antibodies isolated from transgenic mice immunized with human FasL having human antibody variable region genes (ATX-Gx TM , Alloy Therapeutics). The third antibody is a murine antibody (M3T03) selected from Balb / c mice immunized with human FasL. The studies described below show that M3T01 binds soluble and cell surface FasL with high affinity, exhibits cross-reactivity with FasL from several different species, effectively inhibits FasL-mediated apoptosis, and is highly stable.

[0336] Antibody Sequences

[0337] M3T01 and M3T02 are fully human IgG4 / κ monoclonal antibodies with an S228P alteration to prevent Fab arm exchange (see the heavy chain constant region sequence as shown in SEQ ID NO:7). The VH and VL domains of M3T01 are shown herein as SEQ ID NO:1 and 2, respectively; and the heavy and light chains of M3T01 are shown herein as SEQ ID NO:9 and 10, respectively. The VH and VL domains of M3T02 are shown herein as SEQ ID NO:3 and 4, respectively; and the heavy and light chains of M3T02 are shown herein as SEQ ID NO:11 and 12, respectively. M3T03 is a fully murine antibody. The VH and VL domain sequences are shown herein as SEQ ID NO:5 and 6, respectively.

[0338] Binding assay

[0339] Using BIACORE TM 8K measured the binding of M3T01 to human FasL by multi-dose SPR. As Figure 1A-1B shown, M3T01 binds to human FasL with high affinity (974 pM). In another study, the binding of M3T01 to soluble FasL (sFasL) was determined by ELISA. The results showed that M3T01 binds to sFasL with high affinity ( Figure 2A ).

[0340] Next, assays were performed to evaluate the binding of M3T01 to cell surface, membrane-bound FasL from multiple different species. HEK293T cells were transfected with a vector expressing either murine, human, rat or cynomolgus monkey FasL, and binding by M3T01 was measured by flow cytometry. As Figure 2B shown, M3T01 binds to cell-surface FasL from all species tested. These data indicate that M3T01 has broad species cross-reactivity.

[0341] Additional studies showed that M3T01 binds to cell surface FasL on activated human lymphocytes. In this study, human peripheral blood mononuclear cells (PBMCs) were activated with PMA / ionomycin and FasL expression was evaluated by western blot, qPCR and flow cytometry using M3T01 and NOK1 (a commercially available anti-human FasL antibody used as a positive control).

[0342] FasL epitope bound by M3T01

[0343] The epitope of M3T01 was characterized by CovalX. The method included high-quality MALDI mass spectrometry analysis of the M3T01 / human FasL complex after chemical cross-linking and protease digestion. The results showed that M3T01 bound to a conformational epitope on FasL from R144 to Y189 (see Figure 3 ; based on the amino acid numbering of human FasL as shown in SEQ ID NO:17). The epitope of M3T01 is a highly conserved region of FasL across various species including cynomolgus monkey, mouse, rat, rabbit, and pig ( Figure 3 ).

[0344] Functional activity of M3T01

[0345] To evaluate the functional activity of M3T01, the inhibition of apoptosis was measured using the HEK293T cell line transfected to express high levels of human FasL (hFasL / HEK293T). This cell line was used to induce apoptosis of Jurkat target cells expressing the Fas receptor. Co-culture of these cell lines for 4 hours led to apoptosis of Jurkat cells. To evaluate the inhibition of FasL-mediated apoptosis, the M3T01 antibody or the soluble CD95-Fc (sCD95-Fc) fusion protein targeting FasL was added to the hFasL / HEK293T cells before co-culture with Jurkat cells. As Figure 4 shown, M3T01 exhibited greater potency in inhibiting apoptosis relative to sCD95-Fc. Specifically, this study showed that M3T01 had an IC50 of 0.33 nM, which was 310-fold more potent than sCD95-Fc (102.3 nM).

[0346] Stability of M3T01

[0347] To evaluate the stability of M3T01 under high-stress conditions, M3T01 was incubated at 37 °C for 3 weeks and then tested for its binding to cell surface FasL and inhibition of FasL-mediated apoptosis. 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 resulted in no loss of M3T01 activity. Additionally, M3T01 was subjected to repeated freeze-thaw cycles (5 cycles of freezing at -80 °C followed by thawing at room temperature). The repeated freeze-thaw cycles did not reduce cell surface FasL binding or inhibition of FasL-mediated apoptosis.

[0348] Tissue cross-reactivity

[0349] Extensive tissue cross-reactivity studies were performed. The studies evaluated 37 human tissues from 3 separate donors. The studies showed excellent specificity, with M3T01 showing binding only to monocytes in lymph nodes and reticuloendothelial cells in the spleen (consistent with the known physiological tissue expression of FasL). No binding of M3T01 was detected in other normal / healthy organs / tissues.

[0350] M3T01 also binds to fresh frozen human tumor specimens. Since M3T01 recognizes a conformational epitope present only in native (non-denatured) state FasL, it does not bind to FasL in formalin-fixed paraffin-embedded tissue specimens.

[0351] Example 2: Variants of M3T01

[0352] Several variants of the M3T01 antibody were isolated and tested for binding affinity to human FasL using single-dose SPR affinity measurements. Each variant clone included the same CDR sequences as M3T01 but included one or more amino acid substitutions in the framework regions (FRs) of the VH domain and / or VL domain. The variant sequences, the substitutions relative to the M3T01 VH and VL domains, and the measured binding affinities are listed in Table 2. The binding affinities reported in Table 2 were from single-dose SPR studies. The results showed that the binding affinity of each variant was not significantly different from that of M3T01 (represented by M27.2 in Table 2).

[0353] Table 2. Mutations in the FR regions and their effects on binding affinity

[0354]

[0355]

[0356] Example 3: Clinical testing of M3T01

[0357] This example describes the evaluation of M3T01 in a Phase I dose-escalation clinical trial in patients with advanced cancer refractory to standard therapies. Various drug doses and treatment intervals were evaluated in the clinical trial. After establishing the safe / tolerable dose and treatment interval in the Phase I clinical trial, a Phase II clinical trial was conducted in patients with sepsis, severe COVID-19 disease, and myocardial ischemia.

[0358] Given the many possible aspects to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated aspects are merely examples of the disclosure and should not be regarded as limiting the scope of the disclosure. On the contrary, the scope of the disclosure is defined by the following claims. Accordingly, we claim all that comes within the scope and spirit of these claims.

Claims

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

6.

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

6.

3. The monoclonal antibody according to claim 2, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:1 and comprises residues 31-35, 50-66, and 99-108 of SEQ ID NO:1; and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:2 and comprises 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 comprises residues 31-35, 50-66, and 99-108 of SEQ ID NO:3; and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:4 and comprises 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 SEQ ID NO:5 and comprises residues 31-35, 50-66, and 99-107 of SEQ ID NO:5; and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO:6 and comprises residues 23-36, 52-58, and 91-99 of SEQ ID NO:

6.

4. The monoclonal antibody according to claim 1, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:1 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:2; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:3 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:4; or the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:5 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:

6.

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 κ 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 comprises or consists of SEQ ID NO:7 and / or the amino acid sequence of the human κ light chain constant region comprises or consists of SEQ ID NO:

8.

8. The monoclonal antibody according to claim 1, comprising a heavy chain and a light chain, wherein: 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 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.

9. A monoclonal antibody that binds to the same epitope as the Fas ligand-specific monoclonal antibody, comprising: a heavy chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9; and a light chain, the light chain comprising the amino acid sequence of SEQ ID NO:

10.

10. The monoclonal antibody according to claim 9, wherein the epitope is a conformational epitope spanning R144 to Y189 of the Fas ligand shown in SEQ ID NO:

17.

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 the monoclonal antibody according to claim 1 and a heterologous protein.

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

14. A multispecific antibody that comprises the monoclonal antibody according to claim 1 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

15. The multispecific antibody according to claim 14, which is a bispecific antibody.

16. A chimeric antigen receptor (CAR) comprising the monoclonal antibody according to claim 1.

17. An isolated cell expressing the CAR according to claim 16.

18. The isolated cell according to claim 17, wherein the cell is an immune cell.

19. An immunoconjugate comprising the monoclonal antibody according to claim 1 and an effector molecule.

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 the monoclonal antibody according to claim 1.

22. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the monoclonal antibody according to claim 1.

23. The antibody-nanoparticle conjugate according to claim 22, wherein the nanoparticle comprises a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle or a nonionic surfactant vesicle.

24. A nucleic acid molecule encoding the monoclonal antibody according to claim 1.

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

26. A vector comprising the nucleic acid molecule according to claim 25.

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

28. A composition comprising a pharmaceutically acceptable carrier and the monoclonal antibody according to claim 1.

29. A method of inhibiting Fas ligand in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody according to claim 1, thereby inhibiting Fas ligand in the subject.

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

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

32. A method of treating cancer, sepsis, myocardial infarction, stroke, hepatic ischemia-reperfusion injury, renal ischemia-reperfusion injury, interstitial lung disease, an autoimmune disease, myelodysplastic syndrome (MDS) or coronavirus disease 2019 (COVID-19) in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody according to claim 1.

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

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