NKG2d multimers for cancer immunotherapy

Abstract

Methods and compositions for cancer immunotherapy are provided. The methods involve the use of a multimeric molecule (e.g., a fusion protein) comprising three (or more) NKG2D-ligand binding homodimers. In some embodiments, the molecule further comprises a drug moiety (e.g., a cytotoxic agent or a radioligand). The methods disclosed herein are useful for the treatment of cancer that is associated with abnormal expression of one or more NKG2D ligands.

Description

NKG2D MULTIMERS FOR CANCER IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 USC §119(e) of US Provisional Application No. 63 / 730,627, filed December 11, 2024, which is incorporated herein by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING

[0002] This application incorporates by reference a computer readable Sequence Listing in ST.26 XML format, titled 294880_Sequence, created on December 10, 2025 and containing 6,696 bytes.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to the field of medicine, and to NKG2D multimeric molecules and their use in the treatment of NKG2D-ligand expressing cancers.BACKGROUND

[0004] NKG2D is a type II transmembrane glycoprotein having an extracellular lectin-like domain. This domain lacks the recognizable calcium-binding sites found in true C-type lectins and binds protein rather than carbohydrate ligands. NKG2D is an activating receptor that is expressed in a variety of immune cells. Human NKG2D is expressed on CD8+ αβ T cells, γδ T cells, NK cells and NKT cells. Human ligands for NKG2D include MHC class I chain-related molecules (MICA and MICB), UL16-binding proteins (ULBP1, ULBP2, ULBP 3 and ULBP4) and RAET-1G. Expression of NKG2D ligands also occurs in intestinal epithelial cells, tumor cells and under conditions of stress or infection.

[0005] NKG2D exists as a disulfide-linked homodimer that delivers an activating signal upon ligand binding. Signaling requires association with an adapter protein. Alternative splicing of the NKG2D mRNA results in isoforms with different cytoplasmic domains that can associate either with DAP12 to deliver a true activating signal or with DAP10 resulting in a costimulatory signal. NKG2D has been implicated in immune surveillance and immune response against viral infection. In addition, elevated levels of NKG2D ligands have been detected in proliferating cells and many types of cancer.BRIEF SUMMARY OF THE DISCLOSURE

[0006] In one aspect, the present disclosure provides a multimeric NKG2D-ligand binding molecule, comprising: (a) first and second polypeptides comprising, from N-terminus to C-terminus, a first NKG2D monomer or fragment thereof, a polypeptide linker, a second NKG2D monomer orfragment thereof, and a first multimerization domain, wherein the first NKG2D monomer or fragment thereof and the second NKG2D monomer or fragment thereof form an NKG2D-ligand binding dimer (e.g., a homodimer); and (b) third and fourth polypeptides comprising, from N-terminus to C-terminus, a third NKG2D monomer or fragment thereof, and a second multimerization domain, wherein the third NKG2D monomer or fragment thereof of each of the third and fourth polypeptides form an NKG2D-ligand binding dimer (e.g., a homodimer), wherein the first and second polypeptides and the third and fourth polypeptides of associate with one another via disulfide bonding between the first multimerization domains and the second multimerization domains.

[0007] In some embodiments, the first multimerization domain is an immunoglobulin (Ig) light chain constant domain, and the second multimerization domain is an Ig CH1 heavy chain constant domain.

[0008] In some embodiments, the third and fourth polypeptides further comprise an Ig Fc domain including an Ig CH2 heavy chain constant domain and an Ig CH3 heavy chain constant domain at the C-terminus. In some embodiments, the Ig CH1 heavy chain constant domain, the Ig CH2 heavy chain constant domain, and the Ig CH3 heavy chain constant domain are of human IgG 1 isotype.

[0009] In some embodiments, the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof each comprise an extracellular fragment of NKG2D.

[0010] In some embodiments, the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof are identical. In some embodiments, the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof are different. In some embodiments, the first NKG2D monomer or fragment thereof and the second NKG2D monomer or fragment thereof form an NKG2D-ligand binding homodimer, and the third NKG2D monomer or fragment thereof of each of the third and fourth polypeptides form an NKG2D-ligand binding homodimer.

[0011] In some embodiments, the molecules further comprise a peptide linker between the second NKG2D monomer or fragment thereof and the first multimerization domain, and / or a peptide linker between the third NKG2D monomer or fragment thereof and the second multimerization domain. In some embodiments, the polypeptide linker and / or the peptide linker comprises (a) a flexible peptide linker, (b) two or more amino acids selected from glycine, serine, alanine and threonine, or (c) a (G4S)nlinker, wherein n is 1 to 30.

[0012] In some embodiments, the first multimerization domain of the first multimerization domain of the first polypeptide associates with the second multimerization domain of the third polypeptidevia disulfide bonding, and the first multimerization domain of the second polypeptide associates with the second multimerization domain of the fourth polypeptide to form the molecule via disulfide bonding. In some embodiments, the third and fourth polypeptides associate with one another via disulfide bonding between the respective Ig Fc domains.

[0013] In some embodiments, each of the first and second polypeptides comprises the first NKG2DL-binding domain of the first NKG2D monomer, a first polypeptide linker, the second NKG2DL-binding domain of the second NKG2D monomer, and a CL domain, arranged from the N-terminus to the C-terminus, and wherein each of the third and fourth polypeptides comprises the third NKG2DL-binding domain of the third NKG2D monomer, a second polypeptide linker, a CH1 domain, and a Fc domain, arranged from the N-terminus to the C-terminus.

[0014] In some cases, the first polypeptide linker is G4SG4S and the second polypeptide linker is G4S. In some cases, the CH1, Fc, and CL domains are derived from a human IgG antibody. In some cases, each of the third and fourth polypeptides comprises a hinge region located between the CH1 and Fc domains, and wherein the hinge region forms interchain disulfide bonds, thereby crosslinking the third and fourth polypeptides.

[0015] In some cases, each of the first and second polypeptides comprises the sequence of SEQ ID NO: 4. In some cases, each of the third and fourth polypeptides comprises the sequence of SEQ ID NO: 5. In some cases, each of the first and second polypeptides comprises the sequence of SEQ ID NO: 4, and each of the third and fourth polypeptides comprises the sequence of SEQ ID NO: 5.

[0016] In one aspect, the present disclosure provides a recombinant polynucleotide molecule encoding the first polypeptide or the second polypeptide of any one of the molecules discussed above or herein.

[0017] In one aspect, the present disclosure provides a recombinant polynucleotide molecule encoding the third polypeptide or the fourth polypeptide of any one of the molecules discussed above or herein.

[0018] In one aspect, the present disclosure provides an isolated host cell comprising either of the recombinant polynucleotide molecules discussed above.

[0019] In one aspect, the present disclosure provides an isolated host cell comprising both of the recombinant polynucleotide molecules discussed above.

[0020] In some embodiments, the isolated host cell is a bacterium, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

[0021] In one aspect, the present disclosure provides a method for producing any one of the molecules discussed above or herein, the method comprising culturing the host cell discussedabove under conditions to produce the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide, and isolating the molecule so produced.

[0022] In one aspect, the present disclosure provides a composition comprising any one of the molecules discussed above or herein, and a pharmaceutically acceptable carrier.

[0023] In one aspect, the present disclosure provides a method of treating an NKG2D-ligand expressing cancer, comprising administering to a subject in need thereof any one of the molecules discussed above or herein.

[0024] In some embodiments, the NKG2D-ligand expressing cancer is selected from the group consisting of melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic cancer, and prostate cancer.

[0025] In one aspect, the present disclosure provides a conjugate comprising any one of the multimeric NKG2D-ligand binding molecules discussed above or herein, and a drug moiety.

[0026] In some embodiments, the drug moiety is linked to the C-terminus of the third or fourth polypeptide via a polypeptide conjugate linker. In some embodiments, the drug moiety is selected from the group consisting of a cytokine, a chemokine, a cytotoxic agent, and a radionuclide. In some cases, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and IFN-a. In some embodiments, the drug moiety is a cytotoxic agent. In some embodiments, the drug moiety is a radionuclide. In some embodiments, the polypeptide conjugate linker is a protease-cleavable linker. In some cases, the protease-cleavable linker is a linker comprising from 5 to 100 amino acids and containing a substrate for a protease. In some cases, the protease-cleavable linker comprises from 2 to 50 amino acids, or from 5 to 50 amino acids, or from 2 to 25 amino acids, or from 5 to 25 amino acids, or from 2 to 20 amino acids, or from 5 to 20 amino acids, or from 2 to 15 amino acids, or from 5 to 15 amino acids, or from 2 to 10 amino acids, or from 5 to 10 amino acids.

[0027] In one aspect, the present disclosure provides a method of treating an NKG2D-ligand expressing cancer, comprising administering to a subject in need thereof any one of the conjugates discussed above or herein.

[0028] In some embodiments, the NKG2D-ligand expressing cancer is selected from the group consisting of melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic cancer, and prostate cancer.

[0029] In some embodiments, the method further comprises administering an additional cancer therapy.

[0030] In one aspect, the present disclosure provides a composition comprising any one of the conjugates discussed above or herein, and a pharmaceutically acceptable carrier.

[0031] In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.

[0032] Other embodiments will become apparent from a review of the ensuing detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is an illustration of a multimeric NKG2D-ligand binding molecule according to an embodiment of the present disclosure comprising three homodimeric NKG2D-ligand binding moieties formed by the association of four polypeptides comprising multimerizing domains (e.g., immunoglobulin CL and CH1 domains).

[0034] FIG. 2 is an illustration of a multimeric NKG2D-ligand binding molecule according to an embodiment of the present disclosure comprising three homodimeric NKG2D-ligand binding moieties formed by the association of four polypeptides comprising multimerizing domains (e.g., immunoglobulin CL and CH1, CH2 and CH3 domains).

[0035] FIG. 3 is an illustration of a multimeric NKG2D-ligand binding molecule according to an embodiment of the present disclosure comprising three homodimeric NKG2D-ligand binding moieties formed by the association of four polypeptides comprising multimerizing domains (e.g., immunoglobulin CL and CH1, CH2 and CH3 domains) conjugated to a drug moiety (e.g., a cytotoxic agent or a radionuclide shown as a ✱).

[0036] FIG. 4A shows an image of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a purified NKG2D trident, in accordance with an embodiment. “M” indicates the molecular marker lane, “R” indicates the reduced SDS-PAGE lane, and “NR” indicates the nonreduced SDS-PAGE lane.

[0037] FIG. 4B shows a size-exclusion chromatography (SEC) chromatogram of a purified NKG2D trident (top), along with the chromatogram of standard molecular markers (bottom), in accordance with an embodiment.

[0038] FIG. 5 shows a surface plasmon resonance sensorgram (relative response vs. time) of a purified NKG2D trident in accordance with an embodiment.

[0039] FIG. 6 shows an internalization for a NKG2D trident as compared to the controls at 6 hours and 24 hours, in accordance with an embodiment. The controls include anti-MICA mAb1, anti-MICA mAb2, an irrelevant IgG1, and a cells-only control.

[0040] FIG. 7 is an illustration of a structure of a NKG2D trident conjugated with a cytotoxic agent, in accordance with an embodiment. The NKG2D trident has four chains. Two of the chains comprise a NKG2D domain, a G4S linker, a CH1 domain, a CH2 domain, and a CH3 domain from N-terminus to C-terminus (“NKG2D-G4S linker-CH1-CH2-CH3”). The other two chains comprise a first NKG2D domain, a G4SG4S linker, a second NKG2D domain, and a CL domain from N-terminus to C-terminus (“NKG2D-G4SG4S-CL”).DETAILED DESCRIPTION

[0041] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0043] As used herein, the terms “include,” “includes,” and “including,” are meant to be nonlimiting and are understood to mean “comprise,” “comprises,” and “comprising,” respectively.

[0044] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entireties.Definitions

[0045] The term “associated” in the context of the molecules of the present disclosure refers to a functional relationship between two or more polypeptide chains. In particular, the term “associated” means that two or more polypeptides are associated with one another, e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinkages, so as to produce a functional multimeric NKG2D-ligand binding molecule. Examples of associations that might be present in a molecule of the disclosure include (but are not limited to) associations between Fc domains in an Fc region, and associations between CH1 and CL.

[0046] The term “cancer” refers to a disease characterized by the uncontrolled (and often rapid) growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, adrenal gland cancer, autonomic ganglial cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital tract cancers, large intestinal cancer, cancer of the meninges, oesophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleura cancer, salivary gland cancer, small intestinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, vaginal cancer, vulva cancer, lymphoma, leukemia, lung cancer and the like, e.g., any NKG2D-ligand expressing cancers of any of the foregoing types.

[0047] The term “Fc domain” refers to a portion of an immunoglobulin heavy chain that pairs with the corresponding portion of another immunoglobulin heavy chain. The term “Fc region” refers to the region of immunoglobulin molecules formed by association of two heavy chain Fc domains. The two Fc domains within the Fc region may be the same or different from one another. In a native immunoglobulin molecule, the Fc domains are typically identical, but one or both Fc domains might advantageously be modified to allow for heterodimerization, e.g., via a knob-in-hole interaction.

[0048] The terms “host cell” or “recombinant host cell” refer to a cell that has been genetically-engineered, e.g., through introduction of a heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host cell may carry the heterologous nucleic acid transiently, e.g., on an extrachromosomal heterologous expression vector, or stably, e.g., through integration of the heterologous nucleic acid into the host cell genome. For purposes of expressing a multimeric NKG2D-ligand binding molecule or one or more polypeptides of the disclosure, a host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof.The engineered variants include, e.g., derivatives that grow at higher density than the original cell lines and / or glycan profile modified derivatives and and / or site-specific integration site derivatives.

[0049] The term “linker” as used herein, including polypeptide linkers and peptide linkers, refers to a non-cleavable linker, or in the case of a polypeptide conjugate linker to either a non-cleavable linker or a protease-cleavable linker, unless otherwise specifically defined.

[0050] A "multimerization domain" or “multimerizing domain” is any macromolecule that has the ability to associate (covalently or non-covalently) with a second macromolecule of the same or similar structure or constitution. For example, a multimerization domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerization domain is an Fc portion of an immunoglobulin, e.g., an Fc domain of an IgG selected from the isotypes lgG1, lgG2, lgG3, and lgG4, as well as any allotype within each isotype group. In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. In some embodiments, the multimerizing domain is an immunoglobulin CL domain and / or an immunoglobulin CH1 domain and the multimeric NKG2D-ligand binding molecules of the present disclosure are formed by association of two such domains e.g., CL and CH1, or CH1 and CH1) via interchain disulfide bonding. In some embodiments, the multimerizing domain is an immunoglobulin Fc domain and the multimeric NKG2D-ligand binding molecules of the present disclosure are formed by association of two such Fc domains via interchain disulfide bonding as in a conventional immunoglobulin.

[0051] A “non-cleavable linker” refers to a peptide whose amino acid sequence lacks a substrate sequence for a protease.

[0052] The terms "nucleic acid" or "polynucleotide" refer to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with stericallyand electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single stranded or double stranded.

[0053] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.

[0054] The term “protease” as used herein refers to any enzyme that catalyzes hydrolysis of a peptide bond. Generally, the proteases useful in the present disclosure, e.g., the proteases described herein, recognize and cleave a specific sequence motif, e.g., a substrate as described herein. Preferably, the proteases are expressed at higher levels in cancer tissues as compared to normal tissues.

[0055] As used herein, the term “protease cleavable linker” refers to a peptide whose amino acid sequence contains one or more (e.g., two or three) substrate sequences for one or more proteases.

[0056] The term "recombinant," as used herein, is intended to include all molecules that are prepared, expressed, created or isolated by recombinant means, such as multimeric NKG2D-ligand binding molecules expressed using a recombinant expression vector or vectors transfected into a host cell, multimeric NKG2D-ligand binding molecules isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or multimeric NKG2D-ligand binding molecules prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin and / or MHO gene sequences to other DNA sequences.

[0057] As used herein, the term “spacer” refers to a peptide, the amino acid sequence of which is not a substrate for a protease, incorporated into a linker containing a substrate. A spacer can be used to separate the substrate from other domains in a molecule, e.g., NKG2D-ligand binding domains. In some aspects, residues in the spacer minimize aminopeptidase and / or exopeptidase action to prevent cleavage of N-terminal amino acids.

[0058] The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. In preferred embodiments, the subject is human.

[0059] The term “tumor” is used interchangeably with the term “cancer” herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0060] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more molecules or polypeptides of the disclosure. In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0061] The terms “vector” and “expression vector” include, but are not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. In some cases, the vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, and lentivirus.General Description

[0062] The present disclosure relates to multimeric NKG2D-ligand binding molecules (e.g., trimeric NKG2D-ligand binding molecules) and the use of such molecules or compositions containing such molecules for the treatment of NKG2D-ligand expressing cancers. Compositions and methods of the present disclosure are based, at least in part, on the surprising discovery that molecule comprising three NKG2D-ligand binding homodimers induces tumor cell death with improved efficacy compared to molecules comprising a single NKG2D-ligand binding domain or a dimeric molecule comprising two NKG2D-ligand binding domains.

[0063] The multimeric (e.g., trimeric) NKG2D-ligand binding molecules of the present disclosure provide increased binding avidity (e.g., an improved avidity index of at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compared to the prior art monomeric and dimeric constructs by providing three or more NKG2D receptors on the same molecule. Without wishing to be bound by any particular theory, the presence of three or more NKG2D receptors on a single molecule is thought to increase the number and duration of NKG2D-NKG2D-ligand binding interactions, leading to increased anti-tumor activity.

[0064] NKG2D ligand(s) are known to be expressed on cancer cells. Therefore, in some embodiments, the disclosure provides methods for cancer therapy in a subject (e.g., a human subject), the method comprising administering to a subject having an NKG2D-ligand-expressing cancer a multimeric NKG2D-ligand binding molecule as described herein. Unlike an immunotherapy that employs a monoclonal antibody against an NKG2D ligand, such as MICA, the methods provided herein are believed to have broad effects against cancer, on the basis that NKG2D binds to multiple ligands.

[0065] The multimeric NKG2D-ligand binding molecules can target any or all NKG2D ligands that are expressed on human tumor cells, and thus are capable of mediating tumor cell destruction through complement lysis and ADCC. The multimeric NKG2D-ligand binding molecules are also capable of opsonizing any tumor cells that express at least one NKG2D ligand. The multimeric NKG2D-ligand binding molecules can promote efficient cross-presentation (e.g., priming) by dendritic cells, leading to the induction of potent T cell responses against the tumor. Moreover, these multimeric NKG2D-ligand binding molecules are capable of binding and sequestering any “shed” (e.g., soluble or released) NKG2D ligand(s) produced by tumor cells, thereby alleviating immune suppression due to down-regulation of NKG2D expression in response to tumor-derived soluble ligands.Multimeric NKG2D-Ligand Binding Molecules

[0066] In some aspects the disclosure provides a multimeric NKG2D-ligand binding molecule, comprising: (a) first and second polypeptides comprising, from N-terminus to C-terminus, a first NKG2D monomer or fragment thereof, a polypeptide linker, a second NKG2D monomer or fragment thereof, and a first multimerization domain, wherein the first NKG2D monomer or fragment thereof and the second NKG2D monomer or fragment thereof form an NKG2D-ligand binding homodimer; and (b) third and fourth polypeptides comprising, from N-terminus to C-terminus, a third NKG2D monomer or fragment thereof, and a second multimerization domain, wherein the third NKG2D monomer or fragment thereof of each of the third and fourth polypeptides form an NKG2D-ligand binding homodimer, wherein the first and second polypeptides and the third and fourth polypeptidesof associate with one another via disulfide bonding between the first multimerization domains and the second multimerization domains.

[0067] The first multimerization domain may be an immunoglobulin (Ig) light chain constant domain, and the second multimerization domain may be an Ig CH1 heavy chain constant domain, as illustrated in Fig. 1. In some embodiments, the third and fourth polypeptides further comprise an Ig Fc domain including an Ig CH2 heavy chain constant domain and an Ig CH3 heavy chain constant domain at the C-terminus, as illustrated in Fig. 2.(A) NKG2D Monomers and NKG2D-Ligand Binding Dimers

[0068] The NKG2D monomers, e.g., the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof may be identical, or they may be different, but upon dimerization (e.g., to form a homodimer or a heterodimer) they are capable of binding to an NKG2D ligand.

[0069] In some embodiments, the multimeric NKG2D-ligand binding molecules discussed herein bind with increased avidity to an NKG2D ligand as compared to a monomeric or dimeric NKG2D binding molecule. As used herein, “avidity” refers to overall strength across multiple affinities of individual non-covalent binding interactions between a ligand and a receptor. Methods of measuring binding avidity are known in the art and include, for example, ELISA, surface plasmon resonance analysis, CD analysis, fluorescence quenching, size-exclusion binding assay and isothermal titration calorimetry. For brief descriptions of these assays, see, for example, Lengyel et al., 2007, J. Biol. Chem., 282: 30658-30666. In some embodiments, binding avidity is determined by measuring avidity index. In some embodiments, the binding avidity of the multimeric NKG2D-ligand binding molecules to a NKG2D ligand is increased between about 2-fold and about 2000-fold as compared to the monomeric or dimeric NKG2D binding molecule. In some embodiments, the binding avidity is increased between about 2-fold and 1000-fold. In some embodiments, the binding avidity is increased between about 2-fold and 100-fold. In some embodiments, the binding avidity is increased between about 5-fold and 1000-fold. In some embodiments, the binding avidity is increased between about 5-fold and 200-fold. In some embodiments, the binding avidity is increased between about 2-fold and about 20-fold. In some embodiments, the binding avidity is increased 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold. In some embodiments, the binding avidity is increased at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold. In some embodiments, the multimeric NKG2D-ligand binding molecules have an increased binding avidity index as compared to the monomeric or dimeric NKG2D binding molecules, e.g., an improved avidity index of at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0070] In some aspects the disclosure provides a multimeric NKG2D-ligand binding molecule comprising: six NKG2D monomers or fragments thereof, which form three dimers (homodimers or heterodimers) that bind to an NKG2D ligand. NKG2D, also referred to as KLRK1; killer cell lectin-like receptor subfamily K, member 1; CD314; KLR; NKG2-D; F1117759; F1175772 or D12S2489E, is one of the major triggering receptors of NK cells and is well known in the art. In some embodiments, the portion of the NKG2D receptor used for multimeric NKG2D-ligand binding molecules is based on the known sequences of NKG2D (e.g., Accession: NP 031386) or derivatives thereof that bind at least one NKG2D ligand when in dimeric form. Derivatives of NKG2D that can be used in the compositions and methods of the disclosure include, but are not limited to, NKG2D sequences containing one or more mutations, such as a point mutation, a substitution, a deletion mutation and / or an insertion mutation. One of ordinary skill in the art can readily determine suitable derivatives of NKG2D according to the teaching of the present disclosure and knowledge available in the art. At the cDNA level, such a mutation may be a silent mutation. Alternatively, the mutation may result in a change in the corresponding amino acid residue. Where the latter is the case, the change may constitute a conservative change, such that an amino acid residue is replaced with another amino acid residue of similar characteristics. In some cases, however, a mutation may result in a substitution that is non-conservative. Such mutations are acceptable to the extent that the multimeric NKG2D-ligand binding molecules is capable of binding to an NKG2D ligand.

[0071] The NKG2D monomers, e.g., the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof may each comprise an extracellular fragment of NKG2D. In some cases, the extracellular fragment is or comprises or the amino acid sequence of SEQ ID NO: 1. In some embodiments, the extracellular domain of NKG2D may be shortened at the N-terminus, at the C-terminus, or both. In some embodiments, the N-terminus of the extracellular domain used to generate an NKG2D monomer as discussed herein may be shortened by one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 30, about 40, about 50, about 60, and so forth, relative to the full extracellular portion of the polypeptide. In some embodiments, the C-terminus of the extracellular domain used to generate an NKG2D monomer as discussed herein may be shortened by one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 30, about 40, about 50, about 60, and so forth, relative to the full extracellular portion of the polypeptide. In some embodiments, the NKG2D monomer or fragment thereof comprises, consists essentially of, or consists of, an amino acid sequence having at least85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to SEQ ID NO: 1.

[0072] In some embodiments, each NKG2D monomer is a full length NKG2D polypeptide. The full length sequence of NKG2D has been described in the literature. See, for example, RefSeq Accession: NP 031386. Additionally, alternative splice variants of NKG2D have been described. For purposes of the instant disclosure, any one of such alternatively spliced variants may be used, provided that the resulting polypeptide, when constructed as a multimeric NKG2D-ligand binding molecules, is capable of binding its ligand(s).

[0073] It has been shown that the extracellular portion of NKG2D contributes to the formation of homodimers and forms a ligand-binding site(s). Thus, it is possible to delete part or all of the intracellular portion of NKG2D and still maintain the ability to bind its ligand(s). For example, the multimeric NKG2D-ligand binding molecules discussed herein may contain predominantly an extracellular fragment of the NKG2D receptor. Structural analyses have revealed that amino acid residues 78 to 216 of the human NKG2D sequence correspond to the extracellular portion of the NKG2D, containing ligand-binding sites. Accordingly, in some embodiments, each NKG2D monomer of the multimeric NKG2D-ligand binding molecules comprises the extracellular portion of the NKG2D sequence, e.g., amino acid residues 78-216 of human NKG2D. In some embodiments, a multimeric NKG2D-ligand binding molecule comprises a portion of the extracellular domain, shortened as discussed above. Using a human NKG2D as an example, the multimeric NKG2D-ligand binding molecules may contain a fragment of the extracellular domain, wherein the N-terminus of the domain begins at amino acid residue 79, 80, 81, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140 or about 150. Similarly, the multimeric NKG2D-ligand binding molecules may contain a fragment of the extracellular domain, wherein the C-terminus of the domain ends at amino acid residue 231, 230, 229, 228, 227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 217, 216, 215, 214, 213, 212, 211, 210, 209, 208, 207, 206, 205, and so forth. Such deletions at each end of the extracellular domain of the NKG2D sequence may be combined.

[0074] In any of the embodiments of the multimeric NKG2D-ligand binding molecules of the present disclosure, the resulting NKG2D-ligand binding dimers formed from the association of the various polypeptides are each capable of binding ligand(s). For a comprehensive review of the amino acid residues that are involved in receptor-ligand contact, see, for example, Strong and McFarland, 2004, Advances in Protein Chemistry, 68: 281-213. According to published studies, key residues that are thought to be important in the interaction with the ligand have been mapped to amino acid residues approximately from 150 to 207 in human NKG2D. Therefore, each NKG2Dmonomer or fragment thereof of the multimeric NKG2D-ligand binding molecules of the disclosure may comprise a fragment of NKG2D spanning at least most of these residues (e.g., residues 150 to 207 in human NKG2D). Likewise, it will be understood that conservative substitutions, deletions or mutations outside these regions can potentially be tolerated with ease in many instances.

[0075] The multimeric NKG2D-ligand binding molecules of the present disclosure may also include variants of NKG2D that contain one or more amino acid changes as described above, to the extent that the multimeric NKG2D-ligand binding molecules bind to the native ligand or ligands. To determine whether a multimeric NKG2D-ligand binding molecule containing a variant of NKG2D with a particular mutation retains ligand binding activity, binding assays can be carried out, in which binding affinity and / or binding capacity of the particular multimeric NKG2D-ligand binding molecule to its ligand(s) may be evaluated. A number of methods are known in the art by which receptorligand interactions may be measured. These methods for assaying ligand binding include, without limitation, ELISA, surface plasmon resonance analysis, CD analysis, fluorescence quenching, sizeexclusion binding assay and isothermal titration calorimetry.(B) Multimerization Domains

[0076] In some embodiments, a multimeric NKG2D-ligand binding molecule comprises a fragment crystallizable region (Fc) of an immunoglobulin. The Fc region of immunoglobulins plays a significant role in mediating immune defense. FcyRs are widely expressed as transmembrane glycoproteins on a number of cell types, including macrophages, NK cells, dendritic cells, B cells, neutrophils and mast cells. Fc-mediated activities include recruitment of effector cells via Fc-FcyR interactions. There are two classes of Fc receptors that can be distinguished functionally: the activating Fc receptor class and the inhibitory Fc receptor class. Activating Fc receptors include human FcyRIA, FcyRIIA and FcyRIIIA. Activating FcyRs mediate ADCC and ADCP, induce endocytosis of immune complexes leading to antigen presentation, and contribute to the production and release of cytokines and proinflammatory factors. The Fc portion of a multimeric NKG2D-ligand binding molecule is a pair of Fc domains that bind an activating Fc receptor, and preferably an activating Fc Ig domain and includes the hinge region that allows for dimerization.

[0077] For use in a human subject, e.g., for cancer treatment, the Fc domains of the polypeptides used to generate multimeric NKG2D-ligand binding molecules are preferably that of a human origin, preferably IgG Fc domains. Among the four human IgG isotypes, an activating Fc domain of IgG 1 is preferred for the preparation of multimeric NKG2D-ligand binding molecules. The art appreciates that different antibody isotypes have a varying degree of cytotoxic potential in vivo. With respect to human IgG isotypes, IgG 1 and lgG3 have a stronger interaction with FcRs than lgG2 or lgG4. Moreover, certain polymorphic allotypes of a given isotype may influence affinity for an Fc receptor.Indeed, there are allelic variants of activating FcRs that will significantly affect the affinity for certain antibody isotypes. For example, the FcyRllla receptor 158V allotype displays a higher affinity for human lgG1 and increased antibody-dependent cellular cytotoxicity (Cartron G. et al., Blood, 99: 754-758, 2002).

[0078] Without wishing to be bound by any particular theory, it is possible to improve the interaction between the Fc portion of the multimeric NKG2D-ligand binding molecules to its corresponding Fc receptor by strategically selecting or modifying the Fc allele used for preparing the multimeric NKG2D-ligand binding molecule. Accordingly, the disclosure contemplates using a mutant or an allotype of an Fc fragment. A number of useful mutations within an Fc domain have been described, which can affect the interaction of an Fc and its receptor, the effector function of the Fc, as well as the half-life of the Fc-containing molecule. These include specific amino acid substitutions and / or modifications to carbohydrate moieties in the Fc. For review, see, for example, Liu et al., Immunological Reviews, 222:9-27, 2008; Nimmerjahn & Ravetch, Curr. Opin. Immunol., 19(2): 239-45, 2007.

[0079] The structure of Fc domains generally is known in the art. Briefly, the Fc region of a typical IgG molecule is a symmetric homodimer of the carboxy-terminal portion of heavy chains and is composed of the CH2 and CH3 domains, which are generally separated from the Fab by a flexible hinge region. The Fc region is stabilized by non-covalent interactions between domains. The Fc region interacts with FcRs to exert effector functions or to regulate the catabolism of IgG.

[0080] In general, the interaction of an antibody with complement initiates complement-dependent cytotoxicity (GDC), and FcyR interactions mediate antibody-dependent cell toxicity (ADGC) and antibody-dependent cell phagocytosis (ADCP). The classical activation pathway of GDC is triggered when C1, the first component of the pathway, binds to the hinge-Fc portion of the IgG in an antigen-antibody complex. Subsequent activation of the complement cascades eventually induces the formation of a C5-C9 membrane attack complex that leads to the death of the target cell. ADGC, on the other hand, is dependent upon the ability of the FcyR-bearing cells of the innate immune system (e.g., NK cells, monocytes, macrophages and granulocytes) to recognize the Fc domain of antibody bound to target cells. This recognition triggers effector cells to release cytoplasmic perforin, granulysin, and granzymes that induce apoptosis and lysis of target cells. The major effector cells in ADGC are NK cells, which express the type of FcyRs that recognize the IgG 1 and lgG3 subclasses and trigger cytotoxic effects in vivo.

[0081] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containingpeptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. In some embodiments, the multimerizing domain is an immunoglobulin CL domain and / or an immunoglobulin CH1 domain and the multimeric NKG2D-ligand binding molecules of the present disclosure are formed by association of two such domains (e.g., CL and CH1, or CH1 and CH1) via interchain disulfide bonding.(C) Linkers

[0082] The multimeric NKG2D-ligand binding molecules of the present disclosure may further comprise at least one linking moiety that connects one portion of the molecule to another portion of the molecule. In some embodiments, the multimeric NKG2D-ligand binding molecules of the present disclosure may further comprise at least one linking moiety that connects a first NKG2D monomer or fragment thereof with a second NKG2D monomer or fragment thereof, or that connects a second NKG2D monomer or fragment thereof with a first multimerization domain, or that connects a third NKG2D monomer or fragment thereof with a second multimerization domain, and / or that connects a multimerization domain (e.g., an Fc domain) to a drug moiety.

[0083] In some embodiments, the at least one linking moiety (e.g., a polypeptide linker, a peptide linker, or a polypeptide conjugate linker) is not a contiguous portion of an NKG2D monomer or fragment thereof, a multimerization domain, or drug moiety and covalently joins: an amino acid of a first NKG2D monomer or fragment thereof to an amino acid of a second NKG2D monomer or fragment thereof, an amino acid of a second NKG2D monomer or fragment thereof to an amino acid of a first multimerization domain, an amino acid of a third NKG2D monomer or fragment thereof to a second multimerization domain, or a multimerization domain to a drug moiety. Nonlimiting examples of a linking molecule that is not a contiguous portion of either an NKG2D monomer or fragment thereof, a multimerization domain, or a drug moiety are discussed below.

[0084] The linking molecule may be a peptide linker or a polypeptide linker. In some embodiments, the peptide linker or polypeptide linker ranges from about 2 to about 25 amino acids in length. In some embodiments, the peptide linker or the polypeptide linker is 20 amino acids in length. In some embodiments, the peptide linker or the polypeptide linker ranges from about 4 to about 16 amino acids in length. In some embodiments, the peptide linker or the polypeptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, the peptide linker or polypeptide linker is longer than 25 amino acids in length.

[0085] In some embodiments, a flexible peptide linker or polypeptide linker is used. A flexible peptide linker or polypeptide linker is preferably about 25 or fewer amino acids in length. In some embodiments, a peptide linker or polypeptide linker contains about 20 or fewer amino acid residues,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, a peptide linker or polypeptide linker contains about 12 or fewer amino acid residues, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some cases, a peptide linker or polypeptide linker comprises two or more of the following amino acids: glycine, serine, alanine, and threonine. In some embodiments, the flexible peptide linker or polypeptide linker is a glycine-serine linker. In some embodiments, the glycine-serine linker is represented by the formula (GS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the glycine-serine linker is represented by the formula (GGGGS)n (SEQ ID NO: 2), wherein n is 1, 2, 3, 4, or 5. In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 2), wherein n is 1. In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 2), wherein n is 2.

[0086] In some embodiments, a polypeptide conjugate linker provides a protease-dependent cleavable site. A protease-cleavable linker can range from 2amino acids to 80 or more amino acids, and in certain aspects a non-cleavable peptide linker ranges from 2 amino acids to 60 amino acids, 2 amino acids to 40 amino acids, from 2 amino acids to 50 amino acids, from 4 amino acids to 80 amino acids, or from 4 amino acids to 70 amino acids in length. In various embodiments, the protease-cleavable linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 amino acids.

[0087] The protease cleavable linkers comprise one or more substrate sequences for one or more proteases. The one or more substrate sequences are typically flanked by one or more spacer sequences. Each protease-cleavable linker can include one, two, three or more substrate sequences. The spacer sequences can be adjoining, overlapping, or separated by spacer sequences. Preferably, the C- and N-termini of the protease-cleavable linkers contain spacer sequences.

[0088] In particular embodiments, the protease is matrix metalloprotease (MMP)-2, MMP-9, legumain asparaginyl endopeptidase, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloprotease (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3 / 4), cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urokinase / urokinase-type plasminogen activator (uPA), adisintegrin and metalloprotease (ADAM)10, ADAM12, ADAM17, ADAM with thrombospondin motifs(ADAMTS), ADAMTS5, beta secretase (BACE), granzyme A, granzyme B, guanidinobenzoatase, hepsin, matriptase, matriptase 2, meprin, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor-converting enzyme (TACE), kallikrein-related peptidase (KLK)3, KLK5, KLK7, KLK11, NS3 / 4 protease of hepatitis C virus (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, y-secretase component, antiplasmin cleaving enzyme (APCE), decysin 1, apoptosis-related cysteine peptidase, or N-acetylated alpha-linked acidic dipeptidase-like 1.(D) Drug Moieties

[0089] In some embodiments, the multimeric NKG2D-ligand binding molecules of the present disclosure are used to produce conjugates further comprising a drug moiety. As used herein, “drug moiety” refers to a therapeutic agent that is intended for delivery to a targeted cell (e.g., a cancer cell). Generally, a drug moiety is conjugated (e.g., directly or indirectly covalently bound) to the carboxy terminus of a multimeric NKG2D-ligand binding molecule, as illustrated in Fig. 3. However, the skilled artisan recognizes that in some embodiments, a drug moiety is conjugated to the amino terminus of a multimeric NKG2D-ligand binding molecule. Examples of “drug moieties” include drugs (e.g., small molecules), agonists, antagonists, inhibitors, toxins (e.g., molecules of the lymphotoxin family or cytotoxic agents), radionuclides, enzymes, cytokines, chemokines, antibody single chain variable fragments directed against activating compounds or blocking angiogenesis, or essentially any anti-tumor compound.

[0090] In some embodiments, the drug moiety comprises, consist essentially of, or consists of a cytotoxic agent. In some embodiments, the cytotoxic agent may include any compound or biologic capable of inhibiting cell proliferation or inducing cell death. Exemplary cytotoxic agents include topoisomerase inhibitors, such as topoisomerase I inhibitors and topoisomerase II inhibitors (e.g., etoposide, doxorubicin); microtubule-targeting agents, including taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vincristine, vinblastine), and epothilones; alkylating agents, such as nitrogen mustards (e.g., cyclophosphamide, melphalan), nitrosoureas (e.g., carmustine, lomustine), and other DNA crosslinkers; anti metabolites, including folate antagonists (e.g., methotrexate), purine analogs (e.g., 6-mercaptopurine), and pyrimidine analogs (e.g., 5-fluorouracil, cytarabine); DNA-damaging agents or intercalators, such as anthracyclines (e.g., doxorubicin, daunorubicin), actinomycin D, and bleomycin; proteasome inhibitors, including bortezomib and carfilzomib; other small-molecule cytotoxics, such as mitomycin C, hydroxyurea, cisplatin, and carboplatin; and cytotoxic biologies, including protein toxins (e.g., ricin, saporin) or enzyme-toxin conjugates. In some embodiments, the cytotoxic agent is conjugated to the multimeric NKG2D-ligand binding molecules, including the trident molecules, through a cleavable linker. In certainembodiments, the cleavable linker comprises a peptide bond or protease-cleavable moiety that is susceptible to cleavage by one or more proteases present in the human body.

[0091] In some embodiments, the drug moiety comprises, consist essentially of, or consists of a topoisomerase 1 inhibitor. Topoisomerase 1 inhibitors interfere with the action of topoisomerase 1, an enzyme crucial for DNA replication. By inhibiting topoisomerase 1, these drugs can disrupt DNA replication, leading to DNA damage and cell death, particularly in rapidly dividing cancer cells. Topoisomerase 1 inhibitors can be used in the treatment of various cancers, including colorectal cancer, small-cell lung cancer, ovarian cancer, cervical cancer, leukemia, and other solid tumors. In some embodiments, these drugs can be administered in combination with other chemotherapeutic agents to enhance efficacy and reduce the risk of resistance. In some embodiments, the topoisomerase I inhibitor is conjugated to the multimeric NKG2D-ligand binding molecules, including the trident molecules, through a cleavable linker. In certain embodiments, the cleavable linker comprises a peptide bond or protease-cleavable moiety that is susceptible to cleavage by one or more proteases present in the human body.

[0092] Topoisomerase 1 inhibitors include, but are not limited to, camptothecin-based inhibitors and non-camptothecin-based inhibitors. Non-limiting examples of camptothecin-based inhibitors include irinotecan, topotecan, Belotecan, Gimatecan, Exatecan, Lurtotecan, deruxtecan. These inhibitors can stabilize the topoisomerase 1 -DNA complex after the DNA strand is cut, preventing topoisomerase 1 from resealing the break. This action ultimately leads to DNA damage and apoptosis (cell death).

[0093] Non-limiting examples of non-camptothecin-based inhibitors include indenoisoquinolines (e.g., LMP744, LMP400), indolocarbazoles, dibenzonaphthyridones (e.g., Genz-644282), natural products like doxorubicin (which primarily targets Topoisomerase II), nitidine, fagaronine, and novel small molecules such as B-2 / B-3 or riccardin D. These compounds also target topoisomerase 1 but differ from camptothecins in their chemical structures and mechanisms of action.

[0094] In some embodiments, the drug moiety comprises, consists essentially of, or consists of a Toll-like receptor (TLR) agonist. TLRs are members of the interleukin-1 receptor family and function to initiate signaling pathways that promote the expression of genes involved in host defense. These gene products regulate innate immune responses and support the development of antigen-specific adaptive immunity by recognizing molecular patterns associated with foreign pathogens. Ten functional TLRs have been identified in humans. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are primarily expressed on the cell surface and translocate to phagosomes upon activation, whereas TLR3, TLR7, TLR8, and TLR9 are located intracellularly, predominantly within endosomes and the endoplasmic reticulum. These receptors differ in their ligand specificities,expression patterns, and downstream target genes. Upon binding to their cognate ligands, TLRs induce the expression of a variety of host defense genes, including inflammatory cytokines, chemokines, antimicrobial peptides, co-stimulatory molecules, and major histocompatibility complex (MHC) molecules. TLR agonists are molecules that activate TLRs and thereby stimulate potent immune responses. They mimic natural immune stimuli and may be used to enhance immunity in the context of infections, cancer, vaccination, and other therapeutic applications.

[0095] TLR agonists include agents that activate specific TLR receptors. Exemplary TLR1 / TLR2 agonists include Pam3CSK4, FSL-1, and various synthetic or natural lipopeptides. Exemplary TLR3 agonists include poly(I:C) and Ampligen. Exemplary TLR4 agonists include lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), and synthetic lipid A analogs such as E5564. TLR5 agonists may include flagellin and recombinant variants such as FlaB. Exemplary TLR7 agonists include imiquimod and resiquimod. Exemplary TLR8 agonists include CL075 and VTX-2337. TLR9 agonists include CpG oligodeoxynucleotides (CpG ODNs) and CpG-containing sequences such as ISIS 2302. In some embodiments, the TCR agonist is conjugated to the multimeric NKG2D-ligand binding molecules, including the trident molecules, through a cleavable linker. In certain embodiments, the cleavable linker comprises a peptide bond or protease-cleavable moiety that is susceptible to cleavage by one or more proteases present in the human body.

[0096] In some embodiments, the drug moiety comprises a cytokine or functional portion thereof. Cytokines are proteins and peptides that are capable of modulating immune cell function. A “functional portion” of a cytokine is a cytokine fragment that retains the ability to modulate immune cell function (e.g., bind to one or more cytokine receptors). Examples of cytokines include, but are not limited to interferon-alpha (IFN-a), interferon-beta (IFN-p), and interferon-gamma ( I FN-y), interleukins (e.g., IL-1 to IL-29, in particular, IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15 and IL-18), tumor necrosis factors (e.g., TNF-alpha and TNF-beta), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and granulocytemacrophage colony stimulating factor (GM-CSF). In some embodiments, the drug moiety comprises a cytokine selected from the group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and IFN-a. In some embodiments, a drug moiety comprises a cytokine / cytokine receptor heterocomplex.Cytokine / cytokine receptor heterocomplexes are known in the art.

[0097] In some embodiments, the drug moiety is an antibody single chain variable fragment (ScFv). As used herein, an “antibody single chain variable fragment” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. ScFv proteins retain the specificity of the original immunoglobulin, despiteremoval of the constant regions and the introduction of the linker. In some embodiments, a ScFv binds to an immune checkpoint protein (e.g., PD1 or CTLA4). In some embodiments, an ScFv blocks angiogenesis (e.g., binds to a regulator of angiogenesis, such as VEGF).

[0098] In some embodiments, the drug moiety is a chemokine. As used herein, “chemokines” refers to low-molecular-weight proteins that stimulate recruitment of leukocytes. Generally, chemokines are secondary pro-inflammatory mediators that are induced by primary pro-inflammatory mediators such as interleukin-1 (IL-1) or tumor necrosis factor (TNF). Chemokines can be classified into four families: CC chemokines (e.g., CCL1 to CCL-28), CXC (e.g., CXCL1 to CXCL17), C (e.g., XCL1, XCL2), and CX3C (CX3CL1).

[0099] In some embodiments, the drug moiety is a small molecule. As used herein, “small molecule” refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Non-limiting examples of small molecule drugs include small molecule kinase inhibitors (e.g., everolimus, gefitinib, imatinib, etc.), bromodomain inhibitors (e.g., JQ1, l-BET 151, RVX-208, etc.), antibiotics (e.g., kanamycin, neomycin, ciprofloxacin, etc.), and antivirals (e.g., ribavirin, rimantadine, zidovudine, etc.). In some embodiments, the small molecule is an anti-tumor compound. Anti-tumor compounds are discussed in further detail elsewhere in this disclosure.

[0100] In some embodiments, the drug moiety is a radionuclide. As used herein, “radionuclide” refers to medically useful radionuclides. Examples of radionuclides include99mTc,188Re,186Re,153Sm,166Ho,90Y,89Sr,67Ga,68Ga,111In,183Gd,59Fe,225Ac,212Bi,211At,45Ti,60Cu,61Cu, and67Cu.(E) Other Moieties

[0101] In some embodiments, multimeric NKG2D-ligand binding molecules useful for the methods described herein may further comprise one or more accessory moieties, such as a tag sequence and a signal sequence. For example, a tag sequence can be used for detecting and / or isolating the polypeptide. Examples of tags include, without limitation: HA, Flag, Myc, Glu, His and Maltose basic protein. The tag sequence may be located at the amino terminus, carboxyl terminus, or located somewhere in the middle of the multimeric NKG2D-ligand binding molecule (e.g., between modular peptide fragments), provided that the presence of such a tag does not interfere with the function of the multimeric NKG2D-ligand binding molecule. In some cases, a tag sequence is cleavable.

[0102] In some embodiments, multimeric NKG2D-ligand binding molecules or the polypeptides forming such molecules may optionally comprise a signal sequence. A signal sequence is a short (typically about 3-60 amino acids long) peptide chain that directs the post-translational transport of a polypeptide, thereby allowing a greater yield of the polypeptide. The amino acid sequences of asignal sequence direct polypeptides (which are synthesized in the cytosol) to certain subcellular compartments, e.g., organelles. A signal sequence is also referred to as a targeting signal, a signal peptide, a transit peptide, or a localization signal. In some embodiments, a signal sequence is cleaved from the polypeptide by signal peptidase after the polypeptide is transported.Preparation of Multimeric NKG2D-Ligand Binding Molecules

[0103] The skilled artisan would be familiar with molecular biological and biochemical techniques for preparing a multimeric NKG2D-ligand binding molecule discussed herein. Preferably, multimeric NKG2D-ligand binding molecules are produced by conventional recombinant DNA methods. In some embodiments, two or more portions of a multimeric NKG2D-ligand binding molecule are produced as separate fragments and are subsequently linked together to yield a multimeric NKG2D-ligand binding molecule. For example, each respective polypeptide (e.g., first and second polypeptides, or third and fourth polypeptides) of the multimeric NKG2D-ligand binding molecules is produced as a separate recombinant polypeptide, which then associate with one another via disulfide bonding as occurs with a conventional immunoglobulin molecule. Alternatively, the polypeptides can be linked together via a peptide linker.

[0104] For the production of recombinant polypeptides, a variety of host organisms may be used. Suitable hosts include, but are not limited to: bacteria such as E. coli, yeast cells, insect cells, plant cells, and mammalian cells. The skilled artisan will understand how to take into consideration certain criteria in selecting a suitable host for producing the recombinant polypeptides. Factors affecting selection of a suitable host include, for example, post-translational modifications, such as phosphorylation and glycosylation patterns, as well as technical factors, such as the general expected yield and the ease of purification. Host-specific post-translational modifications of a multimeric NKG2D-ligand binding molecule, which is to be used in vivo, should be carefully considered because certain post-specific modifications are known to be highly immunogenic (antigenic).

[0105] Once produced, multimeric NKG2D-ligand binding molecules can be purified by any suitable means, such as chromatographic methods known to those of skill in the art. In some embodiments, multimeric NKG2D-ligand binding molecules are purified by Protein A immunoaffinity chromatography. As will be recognized by one of ordinary skill in the art, the various polypeptides of the multimeric NKG2D-ligand binding molecules can be prepared and isolated separately, and joined by chemical synthesis.NKG2D Receptor Ligands

[0106] In any of the embodiments discussed in this disclosure, multimeric NKG2D-ligand binding molecules are capable of binding the endogenous ligands of the NKG2D receptor. Known NKG2D-ligands in humans include MICA, MICB, RAET-1G, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. Preferably, the multimeric NKG2D-ligand binding molecules discussed in the present disclosure are capable of binding more than one type of NKG2D receptor ligand.

[0107] In some embodiments, the multimeric NKG2D-ligand binding molecules bind ligands with high affinity of 10-4M or less, 10-7M or less, or with subnanomolar affinity, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even less. In some embodiments, the binding affinity of the multimeric NKG2D-ligand binding molecule for its ligands is at least 5x106Ka, at least 1x107Ka, at least 2x107Ka, at least 1 x108Ka, or greater.

[0108] A ligand for NKG2D may be expressed on a cell surface. Alternatively, a ligand for NKG2D may be “shed” from the cell surface and is present as a soluble ligand. It has been known in certain cancers that NKG2D ligands such as MICA are over-expressed and in some cases released (e.g., shed) into the bloodstream or surrounding tissues in a soluble form, e.g., in sera. It is believed that this contributes, at least in part, to the pathogenesis and / or progression of cancer. Thus, the multimeric NKG2D-ligand binding molecules are useful for binding such ligand, either present on cell surface or as a released form, in counterbalancing the expression of the ligands that are present at an abnormally elevated level by functioning as a neutralizing agent.

[0109] Where an NKG2D ligand is expressed on the surface of cancer cells of a subject, multimeric NKG2D-ligand binding molecules as discussed in the present disclosure bind to the cell surface ligand when administered to the subject. The binding of the multimeric NKG2D-ligand binding molecule to its ligand may prevent activation of endogenous NKG2D receptors present on NK cells. Where an NKG2D ligand is “shed” from cancer cells, e.g., released into the bloodstream of a subject, multimeric NKG2D-ligand binding molecules as discussed herein bind to the soluble ligand, sequestering it from further action.Methods of Treating Cancer with Multimeric NKG2D-Ligand Binding Molecules

[0110] Normally, expression of the NKG2D ligands appears to be confined to the gastrointestinal epithelium. Little expression is observed in quiescent epithelial cells, but higher levels of expression occur in rapidly proliferating cells. Expression of the NKG2D ligands is also up-regulated in various transformed cells, particularly those of epithelial origin. Accordingly, provided herein are methods for treating cancer or symptoms of cancer in a subject. The methods comprise administering to the subject a therapeutically effective amount of a multimeric NKG2D-ligand binding molecule that binds NKG2D ligands in vivo.

[0111] As used herein, a therapeutically effective amount refers to an amount of the therapeutic that is believed to effectuate a beneficial effect with statistical significance on the subject having the disease or disorder, such as certain types of cancer. Generally, a therapeutically effective amount is determined by administering the composition to a population of subjects with specified conditions (such as progression or stage of a disease) and evaluating the outcome in response. As used herein, therapeutic treatment shall include, for example, complete prevention or abolishment of the symptoms of a disease, a delay in onset of the symptoms of a disease, or lessening in the severity of a disease.

[0112] Multimeric NKG2D-ligand binding molecules, as discussed herein, are believed to be broadly useful for immunotherapy for a wide variety of cancers, where the expression of one or more NKG2D ligands is elevated in a subject. Cancer broadly refers to a proliferative disease involving transformed cells, including both pre-malignant and malignant disorders. The multimeric NKG2D-ligand binding molecules of the present disclosure are useful for treating a subject having cancer that is characterized by over-expression of one or more NKG2D ligands. In some embodiments, the cancer is characterized by over-expression of one (or predominantly one) ligand of the NKG2D receptor. In other embodiments, the cancer is characterized by over-expression of two or more NKG2D ligands.

[0113] The methods disclosed herein are also useful for the treatment of pre-malignant disorders that carry with them a risk of progressing to malignancy. Examples of such disorders include, without limitation, dysplasia, hyperplasia, and plasma cell disorders such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM).

[0114] In some embodiments, the cancer is melanoma, lung, breast, kidney, ovarian, prostate, pancreatic, gastric, and colon carcinoma, lymphoma or leukemia. In some embodiments, the subject has been diagnosed as having a cancer or as being predisposed to cancer. Thus, methods disclosed herein are also useful to treat a subject who has had a metastasis and is therefore susceptible to a relapse or recurrence. The methods are also useful in high-risk individuals who, for example, have a family history of cancer or metastasizing tumors, or show a genetic predisposition for a cancer metastasis. Specifically, the methods are directed to treating cancer that is associated with NKG2D ligand expression.

[0115] Whether a particular subject (e.g., patient) should receive a cancer therapy comprising a multimeric NKG2D-ligand binding molecule can be determined by testing for aberrant expression of one or more NKG2D ligands in the subject. “Aberrant expression of one or more NKG2D ligands” in the subject means over-expression of the ligand(s) in a biological sample obtained from the subject. In some embodiments, a biological sample may include a biopsy sample taken from atissue of the subject suspected to be cancerous. For example, in some cases, a biological sample is collected from a solid tumor to test for malignancy. In other cases, a biological sample may constitute a blood sample, e.g., serum, a stool sample, urine sample, etc. A biological sample may be any cell or tissue sample that is collected from a subject for the purpose of testing for the diagnosis or progression of a disease, such as cancer.

[0116] One of ordinary skill in the art is familiar with a variety of laboratory techniques and protocols used to assay for the presence of and the levels of one or more markers present in a biological sample. To determine whether a subject has cancer that is associated with overexpression of NKG2D ligand(s), typically immunoaffinity assays are performed. In certain situations, depending on the type of biological samples that are available, immunohistological or immunocytochemical analyses may be carried out. A number of antibodies are commercially available for performing these analyses. Methods commonly employed for this purpose include, but are not limited to, ELISA, immunoblotting, and immunohistochemistry.(A) Subjects

[0117] The methods disclosed herein can be applied to a wide range of species, e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice, which are known to develop cancer. Thus, a “subject” as used herein is a mammalian subject having a disease, or at risk of developing a disease, associated with an abnormal expression of at least one NKG2D ligand, such as cancer. In preferred embodiments, the subject is a human subject having a cancer presenting elevated levels of one or more NKG2D ligands.

[0118] If a subject has been shown to express an elevated level of one or more NKG2D ligands, the subject may be treated with the methods described herein. In some circumstances, a subject has received or is receiving another cancer therapy. In some embodiments, the cancer may be in remission. In some cases, the subject is at risk of having recurrence, or of developing metastatic disease. In some embodiments, the over-expression of one or more NKG2D ligands is limited to cancerous cells, e.g., tumors. In some embodiments, at least one of the NKG2D ligands expressed by cancer cells is shed into the blood stream, and thus detectable in the serum of the subject.Depending on the phenotype of a particular cancer, it may be possible to target one or more ligands which are over-expressed (expressed by tumor cells) over the other ligands, whose expression is not significantly affected.(B) Modes of Action

[0119] Without being limited by any particular theory, the multimeric NKG2D-ligand binding molecules of the present disclosure can function through the two major components of the immune system: innate immunity and adaptive immunity. As used herein, innate immunity or the innate immune system refers to non-specific host defense mechanisms against foreign pathogens. Innate immunity includes both physical barriers (e.g., skin, gastric acid, mucus or tears, as well as cells) and active mechanisms such as NK cells, phagocytes and the complement system. NK cells represent a major component of the innate immune system. NK cells are cytotoxic, e.g., are able to attack cells that have been infected by microbes, as well as some kinds of tumor cells. The cytotoxic activity of NK cells is mediated through cell-surface receptors that recognize MHO class I alleles. A number of receptor types are known in the art, including NKG2D, which is one receptor subtype. Phagocytic cells include neutrophils, monocytes, macrophages, basophils and eosinophils. The complement system is a biochemical cascade of the immune system that helps clear pathogens from a host organism.

[0120] In general, adaptive immunity or the adaptive immune system refers to an antigen-specific antibody-mediated immune response. Adaptive immunity is generally mediated via specific antibody production by B lymphocytes and antigen-specific activity of T lymphocytes. The humoral response mediated by B lymphocytes defends primarily against extracellular pathogens through the production of circulating antibodies that mark foreign cells and molecules for destruction by other specialized cells and proteins. The cellular response mediated by T lymphocytes defends predominantly against intracellular pathogens and cancer cells by directly binding to and destroying the affected cells. According to the present disclosure, multimeric NKG2D-ligand binding molecules, which are non-antibody molecules, are believed to functionally mimic what is ordinarily the function of specific antibodies.

[0121] The present disclosure thus contemplates methods for cancer treatment, wherein multimeric NKG2D-ligand binding molecules bind directly to tumor cells that are expressing NKG2D ligands on the cell surface. In this mode of action, multimeric NKG2D-ligand binding molecules can specifically identify for destruction tumor cells that over-express NKG2D ligands, but not healthy cells that do not.

[0122] Multimeric NKG2D-ligand binding molecules can target any or all NKG2D ligands that are expressed on human tumor cells in at least two ways. One mechanism of mediating tumor cell destruction is through the process of complement lysis (also referred to as complement dependent lysis, complement-dependent cytotoxicity or CDC). A second way of mediating tumor cell destruction is by triggering antibody dependent cellular cytotoxicity (ADCC).

[0123] In some embodiments, multimeric NKG2D-ligand binding molecules act as an opsonizing agent. Opsonization is the process where cells or particles become coated with molecules which allow them to bind to receptors on other cells, such as dendritic cells or phagocytes, to promote the uptake. For antigen-presenting cells such as dendritic cells and macrophages, opsonization promotes efficient processing and presentation of antigens. Opsonizing agents that are capable of specifically binding to both the target (e.g., ligands) and particular receptors on antigen-presenting cells (e.g., FcRs) that can mediate internalization and subsequent antigen processing are useful.

[0124] Tumor cells that express one or more ligands of the NKG2D receptor on the cell surface can become opsonized, e.g., coated, with dimeric NKG2D-Fc molecules. For example, the NKG2D portion of the multimeric NKG2D-ligand binding molecules can bind to the ligands on the tumor cell surface, while leaving the Fc portion (if present, as in Fig. 2) exposed. Dendritic cells have FcyRs and therefore can bind to and internalize the tumor antigen (e.g., NKG2D ligands), which then results in antigen presentation to cytotoxic T cells, also known as CD8+ T cells. This is referred to as cross-priming. Similarly, opsonization results in the generation of MHC class Il-restricted CD4+ T cell responses. Through opsonization, therefore, the multimeric NKG2D-ligand binding molecules can promote efficient cross-presentation (e.g., priming) by dendritic cells, leading to the induction of potent T cell responses against the tumor.

[0125] Cancer patients often suffer from immune suppression. In some cases, it is believed that the immune suppression, at least in part, may be caused by impaired NKG2D receptor signaling. Based on a prevailing model, for example, shed NKG2D ligand impairs host defense by inducing the internalization of NKG2D receptor molecules on lymphocytes. Thus, according to this model, tumor cell shedding of NKG2D ligand results in immune suppression through down-regulation of NKG2D surface expression. Therefore, the methods provided herein are useful for counteracting or relieving immune suppression by administering a multimeric NKG2D-ligand binding molecule or a composition comprising multimeric NKG2D-ligand binding molecule, particularly in situations where a patient exhibits elevated levels of soluble (i.e., shed) NKG2D ligand or ligands that are detectable in sera. The mode of action is that multimeric NKG2D-ligand binding molecules administered to the patient bind to (thus sequestering) excess soluble ligands of NKG2D that were shed from tumors, thereby reversing the down-expression of NKG2D receptors on cell surface that led to immune suppression.

[0126] Thus, the multimeric NKG2D-ligand binding molecules can have multiple therapeutic functions, including neutralizing soluble ligands that are shed by tumor cells, promoting ADCC and / or GDC in tumor cells expressing the cell surface ligands and mediating cross presentation and priming of the adaptive immune system, including CD8 cytotoxic T-lymphocytes (CTLs) and tumor-specific antibody producing B-cells. In addition, conjugates of the multimeric NKG2D-ligand binding molecules and a drug moiety (e.g., a cytotoxic agent or a radionuclide) can facilitate tumor cell death via targeting of NKG2D ligands expressed on the cell surface and either internalization of the conjugate or release of the drug moiety in the presence of the tumor cells (e.g., via a protease-cleavable linkage between the multimeric NKG2D-ligand binding molecule and the drug moiety).Compositions

[0127] In some aspects, the present disclosure provides a composition comprising a multimeric NKG2D-ligand binding molecule as discussed herein, and a pharmaceutically acceptable carrier. Generally, the composition comprising the multimeric NKG2D-ligand binding molecule can be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline). Such carriers can include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include mineral oil, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, for example. Aqueous solutions include, without limitation, water, alcohol, saline, and buffered solutions. Preservatives, flavorings, and other additives such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like also may be present. It will be appreciated that any material described herein that is to be administered to a mammal can contain one or more pharmaceutically acceptable carriers.Administration

[0128] The multimeric NKG2D-ligand binding molecule or composition can be administered directly to a subject. The subject is preferably a mammal. The terms “administration” and “administer” refer to a means of providing a pharmaceutical agent to a subject such that the pharmaceutical agent is to contact its target cells, e.g., cancer cells, in vivo, i.e., in the body of the subject. In some embodiments, the composition comprising the multimeric NKG2D-ligand binding molecule is systematically administered to a subject. In preferred embodiments, a systematic administration is delivered via an intravenous injection. In some embodiments, the multimeric NKG2D-ligand binding molecule or composition comprising the multimeric NKG2D-ligand binding molecule is administered locally. For example, in some cases, the multimeric NKG2D-ligand binding molecule or composition may be delivered directly to or within close proximity of a solid tumor.

[0129] Any multimeric NKG2D-ligand binding molecule or composition described herein can be administered to any part of the subject's body via various administration routes. The multimeric NKG2D-ligand binding molecule or composition can be administered by intravenous, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal,intratracheal, intradermal, or transdermal injection, by oral or nasal administration, by inhalation, or by gradual perfusion over time. The multimeric NKG2D-ligand binding molecule or composition can be delivered to specific tissue. For example, the composition can be delivered to, without limitation, the joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or peritoneal cavity of a mammal. In a further example, an aerosol preparation of a multimeric NKG2D-ligand binding molecule or composition can be given to a subject by inhalation.Dosage

[0130] The dosage required depends on the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages are typically in the range of 0.01 to 1,000 pg / kg. Wide variations in the needed dosage are to be expected in view of the variety of multimeric NKG2D-ligand binding molecules available and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100, 150-, or more fold).

[0131] An effective amount of any multimeric NKG2D-ligand binding molecule or composition described herein can be administered to a subject. The term “effective” as used herein refers to any amount that induces a desired therapeutic effect, such as an immune response or tumor suppression while not inducing significant toxicity in the subject. Such an amount can be determined by assessing a subject's biological reaction, e.g., immune response and improvement in a symptom, after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing a subject's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular multimeric NKG2D-ligand binding molecule or composition administered to a subject can be adjusted according to a desired outcome as well as the subject's response and level of toxicity. Significant toxicity can vary for each particular subject and depends on multiple factors including, without limitation, the subject's disease state, age, and tolerance to pain.Treatment Regimens

[0132] The duration of treatment with any multimeric NKG2D-ligand binding molecule or composition provided herein can be any length of time from as short as one day to as long as the life span of the subject (e.g., many years). For example, a multimeric NKG2D-ligand binding molecule can be administered weekly, once every other week, once every three weeks, once a month, once every other month, once every three months, once every six months, or once a yearfor a period ranging from one week to ten years, or more. It is also noted that the frequency of treatment can be variable. For example, multimeric NKG2D-ligand binding molecules can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly. Multimeric NKG2D-ligand binding molecules can be administered together, e.g., at the same point in time or sequentially, with one or more other cancer therapies, as discussed below.Combination Therapies

[0133] In some embodiments, the subject in need of cancer treatment is treated with the multimeric NKG2D-ligand binding molecules of the present disclosure in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy includes a cytotoxic agent and / or non-cytotoxic agent. A “cytotoxic agent” refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g.,131I,125I,90Y and186Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and / or does not cause destruction of cells. A “non-cytotoxic agent” may include an agent that can be activated to be cytotoxic. A non-cytotoxic agent may include a bead, liposome, matrix or particle. Such agents may be conjugated, coupled, linked or associated with a multimeric NKG2D-ligand binding molecule described herein.

[0134] In some embodiments, conventional cancer medicaments are administered with the multimeric NKG2D-ligand binding molecules discussed herein. I n some cases, the subject in need of cancer treatment is treated with the multimeric NKG2D-ligand binding molecules discussed herein in conjunction with one or more additional agents directed to target cancer cells. Highly suitable agents include those agents that promote DNA-damage, e.g., double stranded breaks in cellular DNA, in cancer cells. Any form of DNA-damaging agent know to those of skill in the art can be used. DNA damage can typically be produced by radiation therapy and / or chemotherapy. DNA-damaging agents are also referred to as genotoxic agents. As used herein, “in conjunction with” or “in combination with” shall mean that the multimeric NKG2D-ligand binding molecule is administered to a subject concurrently with one or more additional therapies (either simultaneously or separately but in close proximity), prior to, or after administration of one or more additional therapies.

[0135] Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy). Energy sources for external radiation therapy include x-rays, gamma rays and particle beams, energy sources used in internal radiation include radioactive iodine (iodine125or iodine131), strontium89, or radioisotopes of phosphorous, palladium,cesium, indium, phosphate, or cobalt. Methods of administering radiation therapy are well known to those of skill in the art.

[0136] Examples of DNA-damaging chemotherapeutic agents that may be particularly useful include, without limitation: Busulfan (Myleran), Carboplatin (Paraplatin), Carmustme (BCNU), Chlorambucil (Leukeran), Cisplatin (Platmol), Cyclophosphamide (Cytoxan, Neosar), Dacarbazme (DTIC-Dome), Ifosfamide (Ifex), Lomustme (CCNU), Mechlorethamme (nitrogen mustard, Mustargen), Melphalan (Alkeran), and Procarbazine (Matulane).

[0137] A number of other chemotherapeutic agents may be also used in combination with the multimeric NKG2D-ligand binding molecules of the present disclosure. These include: methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA / LY231514, LY264618 / Lometexol, Glamolec, CI-994, TNP-470, Hycamtin / Topotecan, PKC412, Valspodar / PSC833, Novantrone / Mitroxantrone, Metaret / Su ramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, lncel / VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / Marmistat, BB2516 / Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / Valrubicin,(125) Metastron / strontium derivative, Temodal / Temozolomide, Evacet / liposomal doxorubicin, Yewtaxan / Paclitaxel, Taxol / Paclitaxel, Xeload / Capecitabine, Furtulon / Doxifluridine, Cyclopax / oral paclitaxel, Oral Taxoid, SPU-077 / Cisplatin, HMR 1275 / Flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT(Tegafur / Uracil), Ergamisol / Levamisole, Eniluracil / 776C85 / 5FU enhancer, Campto / Levamisole, Camptosar / lrinotecan, Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex / Paclitaxel, Doxil / liposomal doxorubicin, Caelyx / liposomal doxorubicin, Fludara / Fludarabine, Pharmarubicin / Epirubicin, DepoCyt, ZD1839, LU 79553 / Bis-Naphtalimide, LU 103793 / Dolastain, Caetyx / liposomal doxorubicin, Gemzar / Gemcitabine, ZD 0473 / Anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / Dexifosamide, Ifes / Mesnex / lfosamide, Vumon / Teniposide, Paraplatin / Carboplatin, Plantinol / cisplatin, Vepeside / Etoposide, ZD 9331, Taxotere / Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as melphelan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorambucil, cisplatin, Cytarabine HCI, Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine (CCNU), Mechlorethamine HCI (nitrogen mustard), Mercaptopurine,Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26), and Vindesine sulfate, but it is not so limited.

[0138] In addition, the following agents may be also useful for combination therapy with the multimeric NKG2D-ligand binding molecules of the present disclosure: alkylating agents, such as carboplatin and cisplatin, nitrogen mustard alkylating agents, nitrosourea alkylating agents, such as carmustine (BCNU), antimetabolites, such as methotrexate, folinic acid, purine analog antimetabolites, mercaptopurine, pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®), hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen, natural antineoplastics, such as aldesleukin, mterleukin-2, docetaxel, etoposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin (ATRA), antibiotic natural antineoplastics, such as bleomycin, dactmomycin, daunorubicin, doxorubicin, daunomycin and mitomycins including mitomycin C, and vinca alkaloid natural antineoplastics, such as vinblastine, vincristine, vindesine, hydroxyurea, acetone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxan®), Schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thiomosine, thiotepa, tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11 (irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, carbomycin, esperamicins, neocarzinostatin, OK 432, bleomycin, furtulon, broxundine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon-O, mepitiostane, mitobromtol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur / uracil, estramustine (estrogen / mechlorethamine), thalidomide, and lenalidomide (Revlmid®).

[0139] Other suitable chemotherapeutics include proteasome inhibiting agents. Proteasome inhibitors block the action of proteasomes, cellular complexes that degrade proteins, particularly those short-lived proteins that are involved in cell maintenance, growth, division, and cell death. Examples of proteasome inhibitors include bortezomib (Velcade®), lactacystin, eponemycin, epoxomycin, aclacinomycin A, the dipeptide benzamide, and vinyl sulfone tripeptide proteasome inhibitors.

[0140] In some embodiments, the compositions and methods described herein are used in conjunction with one or more other cancer treatments, including cancer immunotherapy. Cancer immunotherapy is the use of the immune system to reject cancer. The main premise is stimulating the subject's immune system to attack the tumor cells that are responsible for the disease. This canbe either through immunization of the subject, in which case the subject's own immune system is rendered to recognize tumor cells as targets to be destroyed, or through the administration of therapeutics, such as antibodies, as drugs, in which case the subject's immune system is recruited to destroy tumor cells by the therapeutic agents. Cancer immunotherapy includes an antibodybased therapy and cytokine-based therapy.

[0141] A number of therapeutic monoclonal antibodies have been approved by the FDA for use in humans. These and other antibodies targeting one or more cancer-associated antigens are thus suitable for use in a combination therapy to be administered in conjunction with the multimeric NKG2D-ligand binding molecules of the present disclosure. Examples of monoclonal antibodies approved by the FDA or undergoing clinical studies for cancer therapy include, without limitation: Rituximab (available as Rituxan™), Trastuzumab (available as Herceptin™), Alemtuzumab (available as Campath-IH™), Cetuximab (available as Erbitux™), Bevacizumab (available as Avastin™) Panitumumab (available as Vectibix™), Gemtuzumab ozogamicin (available as Mylotarg™) Ibritumomab tiuxetan (available as Zevalin™) Tositumomab (available as Bexxar™), WX-G250 (available as Rencarex™), Ipilimumab (available as MDX-010), Zanolimumab (available as HuMax-CD4), Ofatunumab (available as HuMax-CD20), Zalutumumab (available as HuMax-EGFr), Oregovomab (available as B43.13, OvalRex™), Edrecolomab (available as IGN-101, Panorex™),131l-chTNT-l / B (available as Cotara™), Pemtumomab (available as R-1549, Theragyn™), Lintuzumab (available as SGN-33), Labetuzumab (available as hMN14, CEAcide™) Catumaxomab (available as Removab™), CNTO 328 (available as cCLB8), 3F8, 177Lu-J591, Nimotuzumab, SGN-30, Ticilimumab (available as CP-675206), Daclizumab (available as Zenapax™), Epratuzumab (available as hl_L2, LymphoCide™),90Y-Epratuzumab, Galiximab (available as IDEC-114), MDX-060, CT-011, CS-1008, SGN-40, Mapatumumab (available as TRM-I), Apolizumab (available as HulDIO, Remitogen™) and Volociximab (available as M200).

[0142] Cancer immunotherapy also includes a cytokine-based therapy. The cytokine-based cancer therapy utilizes one or more cytokines that modulate a subject's immune response. Nonlimiting examples of cytokines useful in cancer treatment include interferon-a (IFN-a), interleukin-2 (IL-2), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12).

[0143] The entire content of all references, including literature references, issued patents, and published patent applications, cited throughout this application are hereby expressly incorporated by reference.

[0144] Example 1: Preparation of Multimeric NKG2D-Ligand Binding Molecules

[0145] Vector(s) containing gene(s) encoding a multimeric NKG2D-ligand binding molecule, as disclosed herein, were introduced into CHO cells for transient or stable expression. In anembodiment, the CHO cell is a CHOK1 cell. In an embodiment, the gene(s) also encode a signal peptide at the N-terminus of the NKG2D-ligand binding molecule to facilitate transmembrane secretion. In an embodiment, the signal peptide comprises the sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 3).

[0146] Following cell culture and protein expression, fraction(s) containing the NKG2D-ligand binding molecule were collected. The NKG2D-ligand binding molecule can be purified by coloum chromatographies, such as size exclsuion, affinity, ion-exchange, hydrophobic chromatographies. In an embodiment, the NKG2D-ligand binding molecule is purified by a process including sizeexclusion chromatography. In an embodiment, the NKG2D-ligand binding molecule is purified by a multi-step size-exclusion chromatography process. In a further embodiment, the size-exclusion chromatography is performed using a buffer containing 20 mM histidine (pH 5.5) and 150 mM NaCI. After purfication, the purifity of the NKG2D-ligand binding molecule can be assessed by reducing and / or non-reducing SDS-PAGE. In an embodiment, the purifity can be further assessed by Size-Exclusion Chromatography (SEC) - High-Performance Liquid Chromatography (HPLC).

[0147] In an embodiment, the multimeric NKG2D-ligand binding molecule is a NKG2D trident for binding a NKG2D ligand (NKG2DL). The NKG2D trident is formed by the association of a first, a second, a third, and a fourth polypeptides. Each of the first and second polypeptides comprises, consists essentially of, or consists of a first NKG2DL-binding domain, a first linker, a second NKG2DL-binding domain, and a CL domain (hereinafter “NKG2D-linker-NKG2D-CL”), arranged from the N-terminus to the C-terminus. Each of the third and fourth polypeptides comprises, consists essentially of, or consists of a NKG2DL-binding domain, a second linker, a CH1 domain, and a Fc domain (hereinafter “NKG2D-linker-CH1-Fc”), arranged from the N-terminus to the C-terminus. The NKG2DL-binding domain binds NKG2DL. In an embodiment, the NKG2DL-binding domain is the NKG2DL-binding domain of a NKG2D molecule or any derivative thereof. In an embodiment, the NKG2DL-binding domain is the extracellular domain of human NKG2D or any derivative thereof. It will be appreciated that any suitable linker may be employed herein. In an embodiment, the first and second linkers each have the amino acid sequence (GGGGS)n(SEQ ID NO: 2). In another embodiment, the first linker has the amino acid sequence G4SG4S, corresponding to (GGGGS)2(SEQ ID NO: 2). In a further embodiment, the second linker has the amino acid sequence G4S, corresponding to GGGGS (SEQ ID NO: 2). It will be appreciated that in some embodiments the linker between the NKG2DL-binding domain and the CH1 domain of the third or fourth polypeptides is optional. In certain embodiments, the third and fourth polypeptides each do not have a linker between the NKG2DL-binding domain and the CH1 domain. CH1 refers to the CH1 domain of an antibody, Fc refers to the Fc region of an antibody (including the CH2 andCH3 domains), and CL refers to the constant region of the light chain of an antibody. In an embodiment, the NKG2D trident has an IgG based design in which at least CH1, Fc, and CL domains are derived from a human IgG antibody. In an embodiment, CH1, Fc, and CL domains are derived from a human IgG antibody.

[0148] The two NKG2D-linker-CH1-Fc polypeptides associate through their Fc regions. In an embodiment, each NKG2D-linker-CH1-Fc polypeptide includes a hinge region located between the CH1 and Fc domains. Similar to hinge regions of natural antibodies, these hinge regions can form interchain disulfide bonds, thereby crosslinking the two NKG2D-linker-CH1-Fc polypeptides. In an embodiment, the hinge region comprises the sequence CPPC, enabling formation of the interchain disulfide bonds.

[0149] In an embodiment, each of the first and second polypeptides of the NKG2D trident comprises, consists essentially of, or consists of NKG2D-G4SG4S-NKG2D-CL that has the following sequence:NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYIGMQR TVGGGGSGGGGSNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASL LKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIE NCSTPNTYICMQRTVRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4). In some embodiments, each of the first and second polypeptides of the NKG2D trident comprises, consists essentially of, or consists of, an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to SEQ ID NO: 4.

[0150] In an embodiment, each of the third and fourth polypeptides of the NKG2D trident comprises, consists essentially of, or consists of NKG2D-G4S-CH1-Fc that having the following sequence:NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQR TVGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 5). In some embodiments, each of the third and fourth polypeptidesof the NKG2D trident comprises, consists essentially of, or consists of, an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to SEQ ID NO: 5.

[0151] In an embodiment, two of the four polypeptides of the NKG2D trident have the sequence of SEQ ID NO: 4, and the other two have the sequence of SEQ ID NO: 5.

[0152] In an embodiment, the NKG2D trident is expressed in CHOK1 cell and purified by size exclusion using a buffer containg 20mM Histidine (pH5.5) and 150mM NaCI. The purity of the NKG2D trident is assessed using SDS-PAGE and SEC-HPLC, as shown in Fig. 4A and 4B. The NKG2D trident has a purifity of about 98.6% based on the SEC-HPLC.

[0153] Example 2: Binding Affinities of NKG2D Trident

[0154] The binding affinities of NKG2D-ligand binding molecules, including the NKG2D trident, can be determined using any sutiable methods. In an embodiment, the binding affinities were measured using Biacore. In an embodiment, the binding assay was performed on a Biacore 8K instrument using a CM5 sensor chip and 1 x HBS-EP+ as the running buffer at 25 °C. In an embodiment, the binding assay was further performed with biotinylated human Major Histocompatibility Complex class I chain-related protein A (MICA) captured on the chip at a working concentration of 0.6 pg / mL with a 30-second contact time. MICA is a ligand of NKG2D. In an embodiment, samples were injected as analytes at concentrations of 0, 0.41, 1.23, 3.70, 11.11, 33.33, and 100 nM, followed by an association phase of 120 seconds and a dissociation phase of 3600 seconds. In an embodiment, the surface was regenerated with 3 M MgCI2between cycles. In an embodiment, data were analyzed using a 1:1 binding model with the Biacore Insight evaluation software.

[0155] The binding affinity of the NKG2D trident to human MICA was determined using the biacore method described above. The results are shown in Fig. 5. The NKG2D trident exhibits a Ka(1 / Ms) of 1.46 x 106, a Kd(1 / s) of 1.39 x 10-5and a KD(M) of 9.51 x 10-12. NKG2D trident binds much more avidly to MICA than divalent NKG2D (6.33 x 10-10) and monovalent NKG2D (1.51 x 107). Therefore, trivalent binding of NKG2D to MICA results in significantly enhanced binding affinity.

[0156] Example 3: Internalization of NKG2D Trident

[0157] The internalization of the NKG2D trident was assessed by the following procedures.

[0158] Antibody Labeling

[0159] NKG2D trident, anti-MICA mAb1, anti-MICA mAb2, and irrelevant IgG1 were changed into PBS (pH7.4) with the antibody concentration adjusted to no less than 1 mg / mL, and then 1 / 10 of the final volume of 1 M NaHCO3solution was added to obtain an antibody solution. pHrodoTMiFL RedSTP ester (amine reactive dye) was dissolved in DMSO at a concentration of 2mg / mL to obtain a dye solution. An appropriate volume of the dye solution was added to the antibody solution, mixed thoroughly by pipetting up and down several times, and incubated at room temperature for 60 minutes to allow the labeling reaction, protected from light. Following the labeling reaction, the mixture was subjected to centrifugation using an ultrafiltration tube to remove unreacted dye, and the labeled antibody was resuspended in an appropriate volume of PBS. An appropriate aliquot of the resuspended solution was diluted with 8 M guanidine hydrochloride to a concentration of about 0.1 mg / mL to obtain a solution. The absorbance of the antibody in the solution was measured at 280nm and 560nm (A max), and the concentration and degree of labeling (DOL) of the labeled antibody were calculated using the following formula:~ (pHro o:“ iF A„.w* pHrodcT iFL CFO * dilution factor Proteus concon-ratioo i. l - -;- -■ - proteirs extinction coefficientThe degree of labeling (DOL) calculation formula is as follows:.,x dilution iac-orMoles dye per mole protein « - - - s.-jys x protein concentration

[0160] Endocytosis Assay

[0161] HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (PBS) at 37 °C in a humidified incubator with 5% CO2. HCT 116 cultures were subcultured at a ratio of 1:3 to 1:20 when confluence exceeded 80%.

[0162] HCT116 cell layer was digested using recombinant enzyme solution (T rypLE™ Express Enzyme). After counting, an appropriate volume of the cell suspension was centrifuged and resuspended in complete medium. The cell density was adjusted to 3.5 x 105cells / mL, and 100 pL per well was seeded into 96-well culture plates and incubated at 37 °C in a humidified atmosphere with 5% CO2. Once the cells adhered, pHrodo Red-labeled antibody was diluted to 200 nM in complete medium and added to the cells at 100 pL per well, resulting in a final drug concentration of 100 nM. The cells were incubated with the labeled drugs at 37 °C and 5% CO2for 6 hours and 24 hours, respectively. Following incubation, cells in the 96-well plate were digested with recombinant enzyme solution and washed once with washing buffer (PBS containing 1% FBS). The cells were then resuspended in 100 pL washing buffer, and the mean fluorescence intensity (MFI) of each well was measured by flow cytometry. Based on the degree of labeling (DOL) of the antibody, the MFI value obtained by Fluorescence-Activated Cell Sorting (FACS) was normalized by dividing by the DOL to obtain the normalized MFI when the DOL was 1. GraphPad Prism software (XY plot, points with connecting lines) was used for data visualization. The results are shown in Fig. 6

[0163] As shwon in Fig. 6, the NKG2D trident internalized much more efficiently than the controls, as reflected by normalized mean fluorescence intensity (MFI) values. The normalized MFI for the NKG2D trident increased steadly over the time course, reaching a level of approximately 22500 at 24 hours from approximately 8000 at 6 hours. The normalized MFI for the NKG2D trident is also markedly higher than those observed for the controls including anti-MICA mAb1, anti-MICA mAb2, irrelevant IgG 1 and cells only. The elevated normalized MFI values for the NKG2D trident confirm that a greater proportion of bound NKG2D trident was internalized and retained intracellularly.

[0164] Example 4: Drug Moieties or Conjugates of NKG2D Trident

[0165] A drug moiety can be conjudated to NKG2D trident. In certain embodiments, an NKG2D trident-drug conjugate promotes tissue-specific delivery of otherwise highly cytotoxic agents. In some embodiments, the drug moiety is conjugated to NKG2D trident via an a conjugation linker. The drug moiety can be conjugated to the NKG2D trident in a site-specific manner. In an embodiment, the drug moiety is a cytotoxic agent. In some embodiments, the cytotoxic agent can be conjugated to a residue of the NKG2D trident in a site specific manner. In certain embodiments, the cytotoxic agent moiety may be conjugated to one or more site-specific asparagine residues of NKG2D trident. Fig. 7 illustrates the conjugation of a cytotoxic agent (represented as a star) to the NKG2D trident. As depicted in Fig. 7, in some embodiments, each NGK2D domain of the NKG2D trident can contain 3 site-specific asparagine residues, while each CH2 domain can contain one site-specific asparagine residue, each of which can serve as a potential conjugation site. In some embodiments, each NKG2D domain of the NKG2D trident may be conjugated to more than three cytotoxic agents, for example, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cytotoxic agents, in a site-specific manner. In other embodiments, each NKG2D domain may be conjugated to fewer than three cytotoxic agents, for example, 1 or 2 cytotoxic agents, in a site-specific manner. In certain embodiments, each CH2 domain of the NKG2D trident may be conjugated to more than one cytotoxic agent, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cytotoxic agents, in a site-specific manner.

[0166] Various conjugation linker technologies are available, including those designed to be cleaved by lysosomal enzymes such as cathepsin B, p-glucuronidase, legumain, sulfatase, and phosphatase. In certain embodiments, the conjugation linker facilitates release of the payload — such as the drug moiety or a portion thereof — upon uptake into antigen-expressing cells. In some embodiments, the conjugation linker contributes to in vitro and in vivo stability of the drug moieties. In certain embodiments, the payload comprises an amine group. In other embodiments, the payload comprises a hydroxyl functional group. In some embodiments, the payload undergoes rapid self-immolation following release.

[0167] A variety of cytotoxic agents are suitable for such conjugation, and the invention is not limited to any particular class or subclass of cytotoxic agent.

[0168] In an embodiment, the conjugation may occur through the primary amine group of the asparagine side chain, forming a stable covalent bond between the cytotoxic agent and antibody.

[0169] In some embodiments, the cytotoxic agent moiety may include functional groups such as hydroxyl, ketone, or carboxyl groups that facilitate conjugation via standard coupling chemistries. For example, coupling strategies such as carbodiimide chemistry, maleimide or isothiocyanate-based linkers, or click chemistry can be employed to achieve site-specific attachment of the cytotoxic agent to the NGK2D trident.

[0170] In some embodiments, the site-specific asparagine residues are those bold, underlined in the sequence of the NGK2D trident below.NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQR TVGGGGSGGGGSNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASL LKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIE NCSTPNTYICMQRTVRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQR TVGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 5).

[0171] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:

1. A multimeric NKG2D-ligand binding molecule, comprising:(a) first and second polypeptides comprising, from N-terminus to C-terminus, a first NKG2D monomer or fragment thereof, a polypeptide linker, a second NKG2D monomer or fragment thereof, and a first multimerization domain, wherein the first NKG2D monomer or fragment thereof and the second NKG2D monomer or fragment thereof form an NKG2D-ligand binding dimer;(b) third and fourth polypeptides comprising, from N-terminus to C-terminus, a third NKG2D monomer or fragment thereof, and a second multimerization domain, wherein the third NKG2D monomer or fragment thereof of each of the third and fourth polypeptides form an NKG2D-ligand binding dimer,wherein the first and second polypeptides and the third and fourth polypeptides of associate with one another via disulfide bonding between the first multimerization domains and the second multimerization domains.

2. The molecule of claim 1, wherein the first multimerization domain is an immunoglobulin (Ig) light chain constant domain, and the second multimerization domain is an Ig CH1 heavy chain constant domain.

3. The molecule of claim 2, wherein the third and fourth polypeptides further comprise an Ig Fc domain including an Ig CH2 heavy chain constant domain and an Ig CH3 heavy chain constant domain at the C-terminus.

4. The molecule of any one of claims 1-3, wherein the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof each comprise an extracellular fragment of NKG2D.

5. The molecule of any one of claims 1-4, wherein the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and the third NKG2D monomer or fragment thereof are identical.

6. The molecule of any one of claims 1-4, wherein the first NKG2D monomer or fragment thereof, the second NKG2D monomer or fragment thereof, and / or the third NKG2D monomer or fragment thereof are different.

7. The molecule of any one of claims 1-6, wherein the first NKG2D monomer or fragment thereof and the second NKG2D monomer or fragment thereof form an NKG2D-ligandbinding homodimer, and the third NKG2D monomer or fragment thereof of each of the third and fourth polypeptides form an NKG2D-ligand binding homodimer.

8. The molecule of any one of claims 3-7, wherein the Ig CH1 heavy chain constant domain, the Ig CH2 heavy chain constant domain, and the Ig CH3 heavy chain constant domain are of human lgG1 isotype.

9. The molecule of any one of claims 1-8, wherein, further comprising a peptide linker between the second NKG2D monomer or fragment thereof and the first multimerization domain, and / or a peptide linker between the third NKG2D monomer or fragment thereof and the second multimerization domain.

10. The molecule of any one of claims 1 -9, wherein the polypeptide linker and / or the peptide linker comprises (a) a flexible peptide linker, (b) two or more amino acids selected from glycine, serine, alanine and threonine, or (c) a (G4S)nlinker, wherein n is 1 to 30.

11. The molecule of any one of claims 1-10, wherein the first multimerization domain of the first multimerization domain of the first polypeptide associates with the second multimerization domain of the third polypeptide via disulfide bonding, and the first multimerization domain of the second polypeptide associates with the second multimerization domain of the fourth polypeptide to form the molecule via disulfide bonding.

12. The molecule of any one of claims 3-11, wherein the third and fourth polypeptides associate with one another via disulfide bonding between the respective Ig Fc domains.

13. The molecule of any one of claims 1-12, wherein each of the first and second polypeptides comprises the first NKG2DL-binding domain of the first NKG2D monomer, a first polypeptide linker, the second NKG2DL-binding domain of the second NKG2D monomer, and a CL domain, arranged from the N-terminus to the C-terminus, andwherein each of the third and fourth polypeptides comprises the third NKG2DL-binding domain of the third NKG2D monomer, a second polypeptide linker, a CH1 domain, and a Fc domain, arranged from the N-terminus to the C-terminus.

14. The molecule of claim 13, wherein the first polypeptide linker is G4SG4S and the second polypeptide linker is G4S.

15. The molecule of any one of claims 13-14, wherein the CH1, Fc, and CL domains are derived from a human IgG antibody.

16. The molecule of any one of claims 13-15, wherein each of the third and fourth polypeptides comprises a hinge region located between the CH1 and Fc domains, and wherein the hinge region forms interchain disulfide bonds, thereby crosslinking the third and fourth polypeptides.

17. The molecule of any one of claims 13-16, each of the first and second polypeptides comprises the sequence of SEQ ID NO: 4.

18. The molecule of any one of claims 13-17, wherein each of the third and fourth polypeptides comprises the sequence of SEQ ID NO:

5.

19. A recombinant polynucleotide molecule encoding the first polypeptide or the second polypeptide of any one of claims 1 -18.

20. A recombinant polynucleotide molecule encoding the third polypeptide or the fourth polypeptide of any one of claims 1-19.

21. An isolated host cell comprising the recombinant polynucleotide molecule of claim 19, or the recombinant polynucleotide molecule of claim 20.

22. An isolated host cell comprising the recombinant polynucleotide molecules of claims 19 and 20.

23. The isolated host cell of claim 15 or 16, wherein the host cell is a bacterium, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

24. A method for producing the molecule of any one of claims 1-18, comprising culturing the host cell of claim 21 under conditions to produce the first polypeptide, the second polypeptide, the third polypeptide, and the fourth polypeptide, and isolating the molecule so produced.

25. A composition comprising the molecule of any one of claims 1 -18, and a pharmaceutically acceptable carrier.

26. A method of treating an NKG2D-ligand expressing cancer, comprising administering to a subject in need thereof the molecule of any one of claims 1-18.

27. The method of claim 26, wherein the NKG2D-ligand expressing cancer is selected from the group consisting of melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic cancer, and prostate cancer.

28. A conjugate comprising the multimeric NKG2D-ligand binding molecule of any one of claims 1-18, and a drug moiety.

29. The conjugate of claim 28, wherein the drug moiety is linked to the C-terminus of the third or fourth polypeptide via a polypeptide conjugate linker.

30. The conjugate of claim 28 or 29, wherein the drug moiety is selected from the group consisting of a topoisomerase 1 inhibitor, a Toll-like receptor agonist, a cytokine, a chemokine, a cytotoxic agent, and a radionuclide.

31. The conjugate of claim 30, wherein the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and IFN-a.

32. The conjugate of claim 30, wherein the drug moiety is a cytotoxic agent.

33. The conjugate of claim 30, wherein the drug moiety is a radionuclide.

34. The conjugate of any one of claims 28-33, wherein the polypeptide conjugate linker is a protease-cleavable linker.

35. The conjugate of claim 34, wherein the protease-cleavable linker is a linker comprising from 5 to 100 amino acids and containing a substrate for a protease.

36. The conjugate of claim 34, wherein the protease-cleavable linker comprises from 2 to 50 amino acids, or from 5 to 50 amino acids, or from 2 to 25 amino acids, or from 5 to 25 amino acids, or from 2 to 20 amino acids, or from 5 to 20 amino acids, or from 2 to 15 amino acids, or from 5 to 15 amino acids, or from 2 to 10 amino acids, or from 5 to 10 amino acids.

37. The conjugate of claim 28, wherein the drug moiety is a toxin.

38. The conjugate of claim 32, wherein the cytotoxic agent is conjugated at one or more site-specific residues of the molecule of any one of claims 1-18.

39. The conjugate of any one of claims 37-38, wherein the cytotoxic agent is conjugated via a linker.

40. The conjugate of any one of claims 37-39, wherein the cytotoxic agent is conjugated to one or more asparagine residues in the multimeric NKG2D-ligand binding molecule.

41. The conjugate of any one of claims 37-40, wherein the cytotoxic agent is selected from the group consisting of topoisomerase inhibitors, microtubule-targeting agents, alkylating agents, antimetabolites, DNA-damaging agents / intercalators, proteasome inhibitors, and cytotoxic biologies.

42. The conjugate of claim 41, wherein the cytotoxic agent is a topoisomerase inhibitor.

43. A method of treating an NKG2D-ligand expressing cancer, comprising administering to a subject in need thereof the conjugate of any one of claims 28-42.

44. The method of claim 43, wherein the NKG2D-ligand expressing cancer is selected from the group consisting of melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic cancer, and prostate cancer.

45. The method of any one of claims 26, 27, 43 or 44, further comprising administering an additional cancer therapy.

46. A composition comprising the conjugate of any one of claims 28-42, and a pharmaceutically acceptable carrier.

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