NKG2D-IG fusion proteins for cancer immunotherapy

Abstract

To provide methods and compositions for cancer immunotherapy.SOLUTION: The methods involve the use of a chimeric molecule (e.g., fusion protein) comprising a dimeric NKG2D portion and a Fc portion, which binds one or more NKG2D ligands. In some embodiments, the molecule further comprises a drug moiety (e.g., an IL15 / Ra moiety). The methods disclosed herein are useful for the treatment of cancer that is associated with abnormal expression of one or more NKG2D ligands.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application is a joint venture of U.S. Provisional Application No. 62 / 255,016, filed November 13, 2015. No. 60 / 133,393, filed on Oct. 13, 2003, and claims priority under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety. Be absorbed. [Background technology]

[0002] NKG2D is a type II transmembrane glycoprotein that contains an extracellular lectin-like domain. Main lacks the recognizable calcium-binding site found in authentic C-type lectins. NKG2D binds proteins rather than carbohydrate ligands. It is expressed in various immune cells. Human NKG2D is an activating receptor that expresses CD8+αβT cells, γδT cells, NK cells, and NKT cells. In the mouse system, NKG2D is also expressed in macrophages. Human ligands for NKG2D include MHC class I chain-related molecules (MICA and MICB), UL16 binding domains, and NKG2D-associated molecules. ULBP1, ULBP2, ULBP3, and ULBP4, and RAET-1G; The minor ligands are minor histocompatibility antigen 60 (H60) and retinoic acid-induced early transcription factor Expression of NKG2D ligands is also increased in the intestine under conditions of stress or infection. It occurs in epithelial cells and tumor cells.

[0003] NKG2D is a disulfide-linked homodimer that transmits activation signals upon ligand binding. It exists as a dimer and requires association with adaptor proteins for signal transduction. Alternative splicing of NKG2D mRNA may result in the expression of DAP12, which transmits the true activation signal, or the coactivator These antibodies have distinct cytoplasmic domains that can associate with either DAP10 or DAP20, which mediates stimulation of the cytoplasmic domains. NKG2D isoforms are involved in immune surveillance and immune responses to viral infections. In addition, elevated levels of NKG2D ligands are involved in the regulation of proliferative cells and many types of cancer. It has been detected in.

[0004] Certain NKG2D-Fc chimeras and their uses are described, for example, in published PCT applications WO 2004 / 023363 and WO 2004 / 023664. This invention was previously disclosed in WO / 2010 / 080124, the entire contents of which are incorporated herein by reference. , which is incorporated herein. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2010 / 080124 Brochure [Patent Document 2] U.S. Patent No. 6,992,174B2 [Patent Document 3] International Publication No. WO2006 / 107124 Brochure [Patent Document 4] U.S. Patent Publication No. 2003 / 0028071 [Patent Document 5] U.S. Patent Publication No. 2003 / 0032995 [Patent Document 6] U.S. Patent No. 4,675,187 [Non-patent literature]

[0006] [Non-Patent Document 1] Lengyel et al., 2007, J. Biol. Chem., 282:30658-666. [Non-Patent Document 2] Strong and McFarland, 2004, Advances in Protein Chemistry, 68:281-213. [Non-Patent Document 3] Liu et al., 2008; Immunological Reviews, 222:9-27 [Non-Patent Document 4] Nimmerjahn F. and Ravetch J.V., 2006, Immunity, 24:19-28. [Non-Patent Document 5] Cartron G. et al., 2002, Blood, 99:754-758 [Non-Patent Document 6] Nimmerjahn and Ravetch, 2007, Curr. Opin. Immunol. 19(2):239-45 [Non-Patent Document 7] Chen et al., Adv. Drug Deliv. Rev. (2013), 65(10):1357-69 [Non-Patent Document 8] Rowley et al., Eur J Immunol. 2009 February;39(2):491-506 [Non-Patent Document 9] Zhang et al., 2004, J. Gene Med., 7:354-65. [Non-Patent Document 10] Caine et al., Protein Expr. Purif., 1996, 8:159-66. Summary of the Invention [Means for solving the problem]

[0007] In the present disclosure, novel compositions and methods for the treatment of cancer are provided. Chimeric molecules comprising at least in part two NKG2D fragments and an Fc fragment (e.g., dimeric NKG2D-Fc chimera) has the ability to bind to one or more NKG2D ligands and comprises a single NKG2D fragment and an Fc With improved efficacy compared to chimeric molecules containing fragments (e.g., monomeric NKG2D-Fc chimeras). In some embodiments, the present invention is based on the surprising discovery that: The dimeric NKG2D-Fc chimera described herein, compared to the monomeric NKG2D-Fc chimera, has the following advantages: binds to an NKG2D ligand with increased avidity. Avidity is increased by 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold.

[0008] Thus, in some embodiments, the present disclosure provides a dimeric NKG2 comprising NKG2D1-NKG2D2-Fc. D-Fc chimera, wherein NKG2D1 and NKG2D2 each comprise NKG2D or a fragment thereof, and and Fc comprises a fragment of an immunoglobulin, the crystallizable region (Fc). In some embodiments, the present disclosure provides a dimeric NKG2D-Fc chimera, including Compositions are provided that include the dimeric NKG2D-Fc chimeras described above and a pharma- ceutically acceptable carrier.

[0009] In some embodiments, the dimeric NKG2D-Fc chimera further comprises a drug moiety. In some embodiments, the drug moiety is attached to the amino or carboxy terminus of the chimera. In some embodiments, the drug moiety is attached to the carboxy terminus of the chimera. is.

[0010] In some embodiments, the dimeric NKG2D-Fc chimera comprises at least one joining molecule. and wherein at least one linking molecule is a contiguous portion of an NKG2D1, NKG2D2, Fc, or drug moiety. NKG2D1 amino acids and NKG2D2 amino acids, NKG2D2 amino acids and Fc amino acids, or The Fc amino acid and the drug moiety are covalently linked.

[0011] In some embodiments, at least one linking molecule is a peptide linker. In some embodiments, the peptide linker ranges in length from about 2 to about 25 amino acids. In some embodiments, at least one linking molecule is a glycine-serine lysine. In some embodiments, the glycine-serine linker has the formula (GS): n (formula where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the glycine-serine linker has the formula (GGGGS): n (SEQ ID NO: 2) (wherein n is , 1, 2, 3, 4 or 5).

[0012] In some embodiments, the chimera comprises three linking molecules, X1, X2 and X3, where X1 is N X2 covalently links an amino acid of NKG2D1 to an amino acid of NKG2D2; and X3 covalently links an amino acid of Fc to the drug moiety. In some embodiments, X1 is (GS)3 (SEQ ID NO: 4), and X2, X3 and X4 are are (GGGGS)4 (SEQ ID NO:3), respectively.

[0013] In some embodiments, the NKG2D fragment comprises an extracellular fragment of NKG2D. In an embodiment, the NKG2D extracellular fragment is represented by SEQ ID NO:1.

[0014] In some embodiments, the Fc is a fragment crystallizable region (Fc) of human immunoglobulin (IgG). In some embodiments, the human immunoglobulin is IgG1.

[0015] In some embodiments, the present disclosure provides a method for administering to a subject having a cancer that expresses an NKG2D ligand: administering the dimeric NKG2D-Fc chimera described herein in an effective amount to treat cancer. The present invention provides a method for treating cancer, comprising:

[0016] In some embodiments, the cancer expressing an NKG2D ligand is melanoma, lung cancer. , plasma cell cancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreatic cancer, or prostate cancer. Optionally, one or more of these cancers may be present in a subject.

[0017] In some embodiments, the method further comprises treating the subject with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer treatment includes surgery, radiation therapy, From chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy and immunotherapy is selected from the group consisting of:

[0018] In some embodiments, the additional cancer treatment is a DNA damaging chemotherapy. .

[0019] In some embodiments, the NKG2D ligand is MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6.

[0020] Each limitation of the invention may encompass various embodiments of the invention. Each of the limitations of the invention including one or combination of elements of It is anticipated that the present invention may be included in the following embodiments: The present invention is not limited to the details of construction and the arrangement of components illustrated in the drawings or shown in the drawings. , other embodiments are possible and that the present invention may be practiced or carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description. The terms "including" and "common" in this specification are intended to be illustrative and not limiting. "containing" or "comprising" or "having" or "containing" The use of "including" and variations thereof refers to the items listed below and to It is intended to encompass these equivalents, as well as additional items. [Brief description of the drawings]

[0021] [Figure 1] Schematic representation of a dimeric NKG2D-Fc chimera with (right) and without (left) a drug moiety. [Diagram 2] FIG. 13 shows that hNKG2Dx2-hIgG1-hIL15 / Ra is produced as a single fusion protein and purified by Protein A. [Diagram 3] FIG. 13 shows that hNKG2Dx2-hIgG1-IL15 / Ra promotes the proliferation of human NK cells, similar to IL-15. [Figure 4]FIG. 1 shows that hNKG2Dx2-hIgG1-IL15 / Ra promotes potent killing of multiple cell lines and is superior to hNKG2Dx2-hIgG1 in cell lines with moderate ligand expression. Panel A shows that neither construct promotes killing of the B16 tumor cell line, which does not express NG2D-L. Panel B shows that both constructs equally promote killing of the synthetic B16 tumor cell line, which expresses high levels of NKG2D ligands. Panel C shows that various tumors express different levels of NKG2D ligands on their cell surface, as measured by binding of NKG2D fusion proteins. Panel D shows that hNKG2Dx2-hIgG1-IL15 / Ra kills cells expressing moderate NKG2D ligands more efficiently than hNKG2Dx2-hIgG1. [Diagram 5] 1 shows that resting NK cells are activated by fusion proteins to produce IFN-γ, but maximal production requires all three components: NKG2D, hIgG1, and IL-15. N297Q is a mutation in hIgG1 that prevents CD16 (expressed on NK) from binding to hIgG1. [Figure 6] FIG. 1 shows that preactivated NK cells require CD16 binding, but not IL-15, to kill target cells. [Figure 7] FIG. 1 shows that optimal activation and killing by resting NK cells requires CD16 binding and IL-15 activation. [Figure 8] FIG. 1 shows ELISA analysis demonstrating that NKG2Dx2-hIgG1 binds to MICA*008 with improved avidity compared to the monomeric NKG2D-Fc chimera, hNKG2Dx1-hIgG1. [Figure 9] FIG. 1 shows flow cytometry analysis demonstrating that hNKG2Dx2-hIgG1 binds to cells expressing NKG2D ligands with improved avidity compared to hNKG2Dx1-hIgG1. [Figure 10]NKG2D-Fc induces NK cell killing of ligand-positive targets. Dimeric NKG2D-Fc chimeras are more effective in mediating killing than monomeric NKG2D-Fc chimeras. * indicates p<0.5, ** indicates p<0.01. [Figure 11] 1 shows that dimeric NKG2D-Fc chimeras (e.g., hNKG2Dx2-hIgG1) kill cells expressing NKG2D ligands more efficiently than monomeric NKG2D-Fc chimeras (e.g., hNKG2Dx1-hIgG1). ** indicates p<0.01. [Figure 12] Figure 1 shows that soluble MICA neutralizes NKG2Dx2-hIgG1 better than NKG2Dx1-hIgG1.* represents p<0.05, ** represents p<0.01, *** represents p<0.005, **** represents p<0.001. [Figure 13] FIG. 1 shows a structural model of dimeric hNKG2D-hIgG1 in complex with human MICA (hMICA). (G4S)4 is SEQ ID NO:3; GGSGGGSG is SEQ ID NO:5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Disclosed herein are novel compositions and methods for the immunotherapy of cancer. The products and methods comprise, at least in part, a chimeric molecule comprising two NKG2D fragments and an Fc fragment (e.g. For example, a dimeric NKG2D-Fc chimera has the ability to bind to one or more NKG2D ligands and is a single compared to a chimeric molecule comprising a single NKG2D fragment and an Fc fragment (e.g., a monomeric NKG2D-Fc chimera) , based on the surprising discovery that anti-tumor agents induce tumor cell death with improved efficacy.

[0023] Monomeric NKG2D-Fc chimeras described in the prior art (e.g., published PCT application WO 2013 / 023363) The construct described in the / 2010 / 080124 brochure) is a low-binding NKG2D ligand. The compounds exhibit low binding avidity (e.g., low binding avidity index). The dimeric NKG2D-Fc construct described in is capable of expressing multiple NKG2D receptors (or portions thereof) on the same molecule. minutes), resulting in increased yields compared to the monomeric constructs of the prior art. Avidity (e.g., at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) Without wishing to be bound by any particular theory, the present invention provides an improved avidity index for However, the presence of multiple NKG2D receptors on a single molecule may be a factor in determining whether NKG2D-NKG2D ligand binding occurs. This is believed to increase the number and duration of binding interactions, resulting in increased antitumor activity. As shown in the Examples section, the dimeric NKG2D-Fc chimera is different from the monomeric NKG2D-Fc chimera of the prior art. In comparison, the chimeric conjugates show up to 100-fold improved binding avidity. Increasing the number of receptors (or portions thereof) in the construct increases the binding phase (e.g., via steric hindrance). Whether or not they inhibit the interaction or cause aggregation of the chimeras is prevented by the stability of the molecule. Since it was not known whether or not the drug would be effective, the success of this approach was not predictable.

[0024] NKG2D ligands are known to be expressed on cancer cells. In embodiments, the present disclosure provides a method for treating cancer in a subject (e.g., a human subject). The method includes administering to a subject having a cancer that expresses an NKG2D ligand two or more of the two described herein. The method includes administering a monomeric NKG2D-Fc chimera to an NKG2D ligand, e.g., MICA. Unlike immunotherapy using clonal antibodies, the methods provided herein are directed to the treatment of cancer in which NKG2D is involved It is believed to have a broad range of effects against cancer based on its binding to multiple ligands. do.

[0025] The dimeric NKG2D-Fc chimera is capable of binding to any or all of the NKG2Ds expressed on human tumor cells. The ligand can be targeted, thus inducing tumor cell destruction by complement lysis and ADCC. The NKG2D-Fc chimera also has the ability to express at least one NKG2D ligand. The NKG2D-Fc chimera has the ability to opsonize any tumor cells expressing NKG2D. This can promote efficient cross-presentation (e.g., priming) and potentiate tumor resistance. Furthermore, the chimera can be used to induce T cell responses in a variety of tumor cell-produced tumor cells. Ability to bind and capture any "shed" (e.g., soluble or released) NKG2D ligand. and thereby inhibiting NKG2D expression in response to tumor-derived soluble ligands. Relieves immunosuppression caused by downregulation.

[0026] NKG2D-Fc In some embodiments, the present disclosure provides a dimeric NKG2D-Fc chimera comprising NKG2D1-NKG2D2-Fc. NKG2D1 and NKG2D2 each contain NKG2D or a fragment thereof and bind to an NKG2D ligand. and Fc comprises a fragment of an immunoglobulin crystallizable region (Fc), In some embodiments, the NKG2D-Fc chimera is an extracellular fragment of NKG2D. In some embodiments, the NKG2D extracellular fragment is represented by SEQ ID NO:1. do.

[0027] As used herein, a "dimeric NKG2D-Fc chimera" refers to a chimeric NKG2D-Fc chimera that comprises two NKG2D ligand-binding sites. a chimeric molecule comprising a ligand binding site of the NKG2D receptor, The ligand-binding site comprises part or all of the NKG2D ligand-binding domain and is capable of binding to an NKG2D ligand. In the Examples section and figures, the two NKG2D fragments of the dimeric NKG2D-Fc chimera are fused to an Fc fragment. The ligand-binding sites are also collectively referred to as "NKG2Dx2." Monomeric NKGs described in the prior art The 2D-Fc chimera can be referred to as "NKG2Dx1." Terms such as "chimera" and "chimeric molecule" are used herein. The term generally refers to a molecule that is composed of parts from multiple origins or sources. In one embodiment, the dimeric NKG2D-Fc is produced as a recombinant chimeric fusion protein.

[0028] In some embodiments, the dimeric NKG2D-Fc chimera described herein is a monomeric NKG2 D-Fc chimera binds to NKG2D ligands with increased avidity compared to the D-Fc chimera. As used herein, "avidity" refers to the individual non-covalent interactions between a ligand and a receptor. It refers to the overall strength of a binding interaction across multiple affinities. Methods for measuring are known in the art, for example, ELISA, surface plasmon resonance, etc. These include ELISA, CD analysis, fluorescence quenching, size exclusion binding assays and isothermal titration calorimetry. For a brief description of these assays, see, e.g., Lengyel et al., 2007, J. Biol. Chem. 282:30658-666. In some embodiments, the binding avidity is determined by measuring the avidity index. In this embodiment, the binding avidity of the dimeric NKG2D-Fc chimera for the NKG2D ligand is The increase is between about 2-fold and about 2000-fold compared to the monomeric NKG2D-Fc chimera. 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 embodiments, the binding avidity is increased between about 5-fold and 200-fold. In embodiments, the binding avidity is increased between about 2-fold and about 20-fold. In embodiments, the binding avidity is increased by 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold. In some embodiments, the binding avidity is at least 2-fold, at least 5-fold , at least 10-fold, at least 100-fold, or at least 1000-fold. The dimeric NKG2D-Fc construct exhibited increased morphology compared to the monomeric NKG2D-Fc chimera. A significant binding avidity index, e.g., at least 1.1, 2, 3, 4, 5, 6, 7 , 8, 9, 10 or more improved avidity index.

[0029] NKG2D In some embodiments, the present disclosure provides a dimeric NKG2D-Fc comprising two NKG2Ds or fragments thereof. The chimera is provided. NKG2D (KLRK1; killer cell lectin-like receptor subfamily K, member NKG2-D; FLJ17759; also called FLJ75772 or D12S2489E) is a major NK cell It is one of the trigger receptors and is well known in the art. See, e.g., Garrity et al. (2005) The portion of the NKG2D receptor used for the dimeric NKG2D-Fc comprises at least A known sequence of NKG2D (e.g., Accession: NP_031386) or a derivative thereof that binds to one ligand. Derivatives of NKG2D that can be used in the compositions and methods of the invention Examples of such mutations include, but are not limited to, one or more mutations, such as point mutations, substitutions, deletion mutations, and / or Examples of NKG2D sequences include NKG2D sequences containing insertion mutations. According to the knowledge available in the art, suitable NKG2D derivatives can be easily determined. At the cDNA level, such a mutation may be a silent mutation. The mutation may result in a change in the corresponding amino acid residue. Conservative changes are made in which an amino acid residue is replaced by another amino acid residue of similar characteristics. However, in some cases, the mutation may result in a substitution that is non-conservative. Such mutations may affect the ability of the dimeric NKG2D-Fc chimera to bind to NKG2D ligands. It is acceptable to have

[0030] In some embodiments, each NKG2D portion of the dimeric NKG2D-Fc chimera is a full-length The full length sequence of NKG2D is described in the literature. See eq Accession:NP_031386. In addition, alternative splice variants of NKG2D have been identified. For purposes of the present invention, when constructed as a dimeric NKG2D-Fc chimera: If the resulting polypeptide has the ability to bind to its ligand, such selection is Any one of the alternative splice variants may be used.

[0031] In some embodiments, when constructed as a dimeric NKG2D-Fc chimera, the resulting If the polypeptide maintains the ability to bind its ligand, the dimeric NKG2D-Fc chimera Each NKG2D portion is a subsequence (i.e., a fragment) of an NKG2D receptor polypeptide. For example, each NKG2D moiety of the dimeric NKG2D-Fc construct may comprise one or more amino acids. The NKG2D sequence may be truncated at either end by an amino acid residue. The N-terminus of the NKG2D sequence is 1, 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, 7, 18, 19, 20, about 30, about 40, about 50, about 60, about 70, about 80 or more residues are deleted. Similarly, the C-terminus of the NKG2D sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, 20 or more residues may be deleted. In embodiments, both the N-terminus and C-terminus may be truncated as indicated.

[0032] The extracellular portion of NKG2D contributes to the formation of homodimers and forms the ligand-binding site. Thus, it has been shown that some or all of the intracellular portion of NKG2D can be deleted and still However, it is possible for the antibody to retain its ability to bind to its ligand. The dimeric NKG2D-Fc chimera may mainly contain the extracellular fragment of the NKG2D receptor. Structural analysis revealed that the amino acid residues 78 to 216 of the human NKG2D sequence contain the ligand-binding site. We have shown that NKG2D corresponds to the extracellular portion of NKG2D. The outer domain is the amino acid residues 78 to 232, 94 to 232, or 92 to 232.

[0033] Thus, in some embodiments, each of the dimeric NKG2D-Fc constructs NKG2D refers to the extracellular portion of the NKG2D sequence, e.g., amino acid residues 78 to 216 of human NKG2D; The amino acid sequence includes amino acid residues 78 to 232, 94 to 232, or 92 to 232 of mouse NKG2D. In some embodiments, the dimeric NKG2D-Fc construct comprises a portion of the extracellular domain. Thus, the extracellular domain of the dimeric NKG2D-Fc construct may be N-terminal, C-terminal or For example, the dimeric NKG2D-Fc may be generated by The N-terminus of the extracellular domain may be one or more amino acids with respect to the entire extracellular portion of the polypeptide. amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 It may be truncated by 8, 19, 20, about 30, about 40, about 50, about 60, etc. amino acid residues. The C-terminus of the extracellular domain used to generate c is the extracellular domain of the polypeptide. With respect to the moiety, 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 amino acid residues, etc. Using human NKG2D as an example, the dimeric NKG2D-Fc construct may be The N-terminus of the domain is 79, 80, 81, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , an extracellular domain beginning at about amino acid residue 110, about 120, about 130, about 140, or about 150; Similarly, the dimeric NKG2D-Fc construct may contain a fragment of the domain C Ends: 231, 230, 229, 228, 227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 21 Amino acid residues at positions 7, 216, 215, 214, 213, 212, 211, 210, 209, 208, 207, 206, 205, etc. The extracellular domain of the NKG2D sequence may contain a fragment of the extracellular domain that terminates in the 5'-terminal end of ... Such deletions at each end of the gene may be combined.

[0034] Those skilled in the art will appreciate that the dimeric NKG2D-Fc chimera described by the present disclosure is composed of two identical NKG2D fragments, It will be appreciated that the NKG2D fragment may comprise two different NKG2D fragments. In structure, the dimeric NKG2D-Fc chimera corresponds to amino acid residues 78 to 216 of human NKG2D. In some other embodiments, the dimeric NKG2D-Fc chimera comprises two NKG2D fragments that The antibody comprises two NKG2D fragments, the first of which corresponds to amino acid residues 78 to 216 of human NKG2D. and the second fragment is a different portion of the NKG2D extracellular domain (e.g., residues 140-210 of human NKG2D). The amino acid position corresponds to the first amino acid position.

[0035] One or more mutations in the NKG2D portion of the construct at the NKG2D-ligand binding interface. Dimeric NKG2D-Fc derivatives, including those described herein, are also contemplated. In particular, certain mutations are contemplated for the NKG2D receptor and its It is known that the binding affinity between the ligands (e.g., MICA) of See Lengyel et al., 2007, J. Biol. Chem. 282:30658-666. The three-dimensional structure of the complex between the β-lactam and the ligand has been described. The amino acid residues of NKG2D that contribute to the interaction can be determined and the key residues can be systematically varied. In either embodiment, the effect of the mutation can be tested by The Fc chimera has the ability to bind to a ligand. For a comprehensive review of amino acid residues, see, for example, Strong and McFarland, 2004, Advances in in Protein Chemistry, 68:281-213. The key residues thought to be important for interaction with the NKG2D domain are located at approximately 150–200 residues in human NKG2D. Located at amino acid residue 7, which corresponds to approximately residues 166-223 of mouse NKG2D Thus, the respective NKG2D fragments of the dimeric NKG2D-Fc constructs of the present invention are preferred. or at least most of these residues (e.g., residues 150-207 of human NKG2D). Similarly, conservative substitutions, deletions or mutations outside these regions are possible in many instances. It will be understood that there may be readily acceptable possibilities in the above.

[0036] Several amino acid residues have been identified as being particularly important for mediating ligand binding. Specifically, the residues of human NKG2D that are important for binding to MICA are Y152 and Y162. , Q185, K197, Y199, E201 and N207. Residues in 2D include I182, Y199, and Y152. These residues are important for binding to RAE-1β. Residues in mouse NKG2D include K166, Y168, Y215, K213, E217, and N223. Therefore, in a preferred embodiment, these (of the corresponding dimeric NKG2D constructs) Most or all of the residues are broadly tolerated (e.g., specific) for multiple ligands. The desired position (based on the gene) is maintained without mutation or deletion. Mutations are strategically introduced at one or more of these key residues that confer ligand recognition and binding. By injecting NKG2 into the NKG2 receptor, the NKG2 receptor binds preferentially to one ligand over the other. It is also possible to design 2D-Fc constructs. For example, in the mouse counterpart, Y168 and Y215, respectively, are involved in the binding of various ligands. Y152 and Y199 in human NKG2D, which are equivalent to Y152 and Y199, contribute to the binding of MICA and ULBP3. Thus, in some embodiments, these residues maintain broad ligand specificity. It is not modified to do so.

[0037] In the examples provided below, representative dimeric NKG2D-Fc chimeras are shown, each of which The fragment corresponds to amino acid residues 78-216 of human NKG2D. The same approach was used with NKG2D sequences from any other species in which cancer is known to occur. For example, the NKG2D fragment of the dimeric NKG2D-Fc may be a dimeric N As long as KG2D-Fc maintains its ligand-binding activity, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid alterations, such as deletions, insertions and substitutions.

[0038] The present invention relates to a dimeric NKG2D-Fc chimera that binds to its natural ligand or ligands. A dimeric NKG2D-F that contains one or more of the amino acid alterations described above and that binds to the The dimeric NKG2D-Fc mutants containing specific mutations are To determine whether the specific dimeric NKG2D-Fc chimeras retain their binding activity, Binding assays can be performed to assess the binding affinity and / or binding capacity of the compound to a ligand. Many methods are known in the art that can measure receptor-ligand interactions. These methods for assaying ligand binding include, but are not limited to, ELISA, surface plasmon resonance analysis, CD analysis, fluorescence quenching, size exclusion binding assay and isothermal For a brief description of these assays, see, for example, Lengye et al. See l et al. (2007).

[0039] Fc fragment In some embodiments, the dimeric NKG2D-Fc chimera is a crystallographic fragment of an immunoglobulin. The Fc region of immunoglobulins plays an important role in mediating immune defense. FcγR is expressed by various types of cells including macrophages, NK cells, dendritic cells, B cells, neutrophils, and mast cells. It is widely expressed on many cell types as a transmembrane glycoprotein. Fc-mediated activities include Examples of these include the recruitment of effector cells via Fc-FcγR interactions. There are two classes of Fc receptors that can bind to the activating Fc receptor class and the inhibitory Fc receptor class. Activating Fc receptors include human FcγRIA, FcγRIIA, and FcγRIIIA, as well as their and their mouse orthologues, namely, FcγRI, FcγRIII, and FcγRIV. γR mediates ADCC and ADCP and induces endocytosis of immune complexes leading to antigen presentation. It contributes to the production and release of cytokines and proinflammatory factors. Structure and Function of IgG For a general overview of the mechanisms, see Liu et al. (2008; Immunological Reviews 222:9-27) As described in more detail herein, the Fc portion of the dimeric NKG2D-Fc , a domain that binds to an activating Fc receptor, preferably an activating FcIg domain, and dimerizes The hinge region allows for:

[0040] The Fc portion of the dimeric NKG2D chimeras useful for the present disclosure can be readily modified to render them species specific. For use in mouse systems, e.g., cells derived from mice, For this purpose, the Fc fragment used to generate the dimeric NKG2D-Fc is preferably derived from a mouse-derived Fc fragment. In some embodiments, the Fc fragment of mouse IgG2a is preferred.

[0041] For example, for use in human subjects for the treatment of cancer, dimeric NKG2D-Fc can be generated. The Fc fragment used to induce the expression of the antibody is preferably of human origin. In an embodiment, NKG2D-Fc comprises an activating Fc Ig domain. The four human IgG isotypes Of these, the activating Fc domain of IgG1 is preferred for the preparation of dimeric NKG2D-Fc. In some embodiments, the Fc is a fragment crystallizable region of human immunoglobulin (IgG) (Fc In some embodiments, the human immunoglobulin is IgG1. Human IgG1 Experimental data on chimeric constructs containing the Fc region of do.

[0042] It has been reported in the art that different antibody isotypes exert different degrees of cytotoxicity in vivo. It is understood that the immune system has a certain degree of immunity (see, for example, Nimmerjahn F. and Ravetch JV, 2006, Immunit For example, mouse IgG2a and IgG2b isotypes are are comparable in efficacy to their IgG1 or IgG3 counterparts in clearing infections, including bacterial and viral infections. These drugs are more efficient at killing tumor cells and tumour cells. This is due, at least in part, to in vitro This may be due to the different ratios of activating to inhibitory FcRs present in human IgG. Regarding isotype, IgG1 and IgG3 have stronger interactions with FcR than IgG2 or IgG4. Furthermore, a particular polymorphic allotype of a given isotype has a specific affinity for the Fc receptor. In fact, it can significantly affect affinity for certain antibody isotypes. There are allelic variants of activating FcRs that may exert their effects. For example, the FcγRIIIa receptor 15 The 8V allotype exhibits higher affinity for human IgG1 and antibody-dependent cellular cytotoxicity. (Cartron G. et al., 2002, Blood, 99:754-758).

[0043] Without wishing to be bound by any particular theory, it is believed that the Fc portion of the dimeric NKG2D-Fc chimera and The interaction between the corresponding Fc receptors of NKG2D and NKG2D-Fc was used to prepare dimeric NKG2D-Fc chimeras. This can be optimized by strategically selecting or modifying the Fc alleles that are involved. Thus, the present invention contemplates the use of mutations or allotypes of the Fc fragment. Many useful mutations within the Fc domain have been described, which may affect the interaction of Fc with its receptors, These may affect effector functions, as well as the half-life of Fc-containing molecules. The sugar moiety in the saccharide may contain specific amino acid substitutions and / or modifications. Iu et al., 2008, Immunological Reviews, 222:9-27; Nimmerjahn and Ravetch, 2007 , Curr. Opin. Immunol. 19(2):239-45.

[0044] Generally, the structure of an Fc fragment is known in the art. Briefly, a typical IgG molecule The Fc region of is a symmetric homodimer of the carboxy-terminal portions of the heavy chains, H 2 and C H 3. Domain The Fc region is composed of the Fab domain and is separated from the Fab domain by a flexible hinge region. The Fc region is stabilized by non-covalent interactions between the Fc domain and the FcR. , affecting effector functions or controlling the catabolism of IgG. The constant region of the heavy chain (Cγ2 and Cγ3), as well as the hinge region located between the variable domain and the constant region, act as C1q and Fc receptors. Therefore, the constant regions of the heavy chains of IgG molecules interact with different On effector cells, the effector contains binding sites for complement and for FcR. Thus, effector cell recruitment is mediated by Fc-FcγR interactions. This is mediated through

[0045] In general, the interaction of antibodies with complement induces complement-dependent cytotoxicity (CDC), and FcγR interaction Mediates antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Classical CDC The activation pathway of IgG begins with the first component, C1, binding to the hinge of IgG in the antigen-antibody complex. -Fc portion of the antibody, which activates the complement cascade and ultimately targets the target ADCC induces the formation of the C5-C9 membrane attack complex, which leads to cell death. It depends on the ability of cells bearing FcγR (e.g., NK cells, monocytes, macrophages, and granulocytes) to The Fc domain of the antibody bound to the target cell is recognized by the effector cell. Activation of cytoplasmic perforin, Glycine max, which induces apoptosis and lysis of target cells The main effector cells in ADCC are NK cells express FcγR of the type that recognizes IgG1 and IgG3 subclasses and In vivo, it causes cytotoxic effects.

[0046] In the context of the present invention, as demonstrated in the Examples, the dimeric NKG2D-Fc kinase described herein Mela is a dimer that can specifically but broadly recognize and bind its ligand. By virtue of having a functional Fc portion coupled to an NKG2D portion, equivalent Has the ability to mediate effects.

[0047] As mentioned above, activating (FcγRI, FcγRIIA, and FcγRIII) and inhibitory (FcγRIIB) receptors Generally, the interaction of IgG with activating FcγR induces Fcγ receptors that inhibit cell activation. During interaction with RIIB, it causes activation of the cells. Except for B cells and NK cells, Inhibitory and inhibitory FcγRs are co-expressed on the same effector cells, thereby mediating cell activation. B cells express only the inhibitory FcγRIIB and therefore cannot differentiate between the two. NK cells cannot be activated by endogenous IgG under physiological conditions. It expresses activating FcγRIII so that it can kill target cells independently of the activation of FcγRIII. Manifest.

[0048] FcγRIIA and FcγRIII (CD16) have low affinity for monomeric IgG and act as effector This is believed to be important for inducing the function and providing antitumor activity. This increases affinity for activating FcγRIII and decreases affinity for inhibitory FcγRIIB. It is possible to engineer a dimeric NKG2D-Fc that is genetically engineered to bind to the NKG2D-Fc domain.

[0049] Thus, it is mainly located in the lower hinge region and directly connects the Cγ2 region to the adjacent FcγR. The amino acid residues of the dimeric NKG2D-Fc molecule that contribute to the functional interaction may be modified, Such mutants are encompassed by the present invention. It has been shown that the IgG-FcγR domain is required for binding to FcγR. Another residue that has been shown to be important for protein activity is located in the Cγ2 domain, Asp265. , Asp270, Ala327, Pro329 and Lys338.

[0050] To generate dimeric NKG2D-Fc chimeras with enhanced activity, several strategies have been developed. In order to design a dimeric NKG2D-Fc with enhanced ADCC ability, at least two Two approaches are considered. The first is to develop a method for binding to activating and inhibitory FcγRs, which is crucial. Based on the amino acid residues in IgG1 that have been identified as having flavors, the present invention provides receptors for these The present invention provides variants of the dimeric NKG2D-Fc chimera that enhance or reduce, respectively, the affinity for Thus, in one embodiment, each residue position is a triplet based on IgG1. The amino acid substitutions Ser298Ala / Glu333Ala / Lys334Ala are provided. The dimeric NKG2D-Fc should show high affinity for FcγRIIIA, but not for FcγRII. B, but not B, thereby promoting ADCC. The dimeric NKG2D-Fc mutant, which contains two sets of mutations in the Fc, Ser239Asp / Ile332Glu, These are expected to show improved ADCC. Other mutations to enhance ADCC include limited However, examples thereof include Ser239Asp / Ala330Leu / Ile332Glu and Ser239Asp / Ser298Ala / Ile332Ala. Similarly, in some embodiments, binding to FcγRIIIA (e.g., an activating receptor) Mutations that combine increased binding to FcγRIIB and decreased binding to FcγRIIB are considered. Examples of the amino acid sequence include, but are not limited to, Phe243Leu / Arg292Pro / Tyr300Leu / Val305Ile / Pro396Leu. (residue positions are based on IgG1).

[0051] The second approach demonstrated that some modifications significantly affect the affinity of Fc for FcγRs. Based on the observation that the Fc domain has two have been shown to contain asparagine N-linked oligosaccharide moieties (reviewed in Liu et al., 2008). ADCC requires the presence of specific oligosaccharides and depends on changes in the structure of the oligosaccharides. In particular, Previous studies have demonstrated that the fucose moiety attached to the innermost GlcNAc of biantennary complex-type oligosaccharides Removal of the CDC domain improves Fc binding to FcγRIIIA without impairing CDC activity. Based on this observation, in one embodiment, In some embodiments, the present invention provides a fucose-depleted dimeric NKG2D-Fc. Alternatively, the chimera is completely devoid of fucose moieties (i.e., non-fucosylated). In some embodiments, the chimera is hypofucosylated.

[0052] To generate dimeric NKG2D-Fc containing modified sugars, host cells are transfected with the desired modifications. For example, the host cell may be engineered to express an enzyme that catalyzes CHO cells were cultured using the enzyme β-(1,4)-N-acetylglucosaminyltransferase (AGGT) Alternatively, the gene may be transfected with GnT-III, which is a nucleotide sequence encoding bisecting, nonfucosylated oligosaccharides. The NKG2D-Fc products generated from these host cells significantly enhanced AD. In addition, in some embodiments, the fucoprotein in NKG2D-Fc can have CC activity. The content of fucosyltransferase was determined by the expression of α-1,6-fucosyltransferase (FUT8) that lacks core-fucosyltransferase activity. Alternatively, small interfering RNA can be used to knock out the FUT8 enzyme. Expression may be constitutively inhibited to achieve the same effect. Host cells lacking guanosine diphosphate (GDP)-mannose 4,6-dehydratase (GMD) were used to Cosylated NKG2D-Fc may be obtained.

[0053] Next, to engineer a dimeric NKG2D-Fc with enhanced complement activity, we investigated the effect of the Fc domain In general, complement is activated by at least three pathways: These membrane-attached complexes can form pores in the plasma membrane of target cells, causing their lysis. Binding of C1q to the Fc domain results in the formation of the membrane attach complex C5b-9. This is a critical step in the process. Among the human IgG subclasses, IgG1 and IgG3 Only the α-terminal β-terminal region of the complement cascade is capable of initiating the complement cascade. , introduced into the Fc domain of dimeric NKG2D-Fc to promote C1q recruitment and C1q-Fc interaction. Residues of Fc that can be targeted for such mutations include, but are not limited to, Asp270, Lys3 22, Pro329 and Pro331. These mutations are substituted with non-polar neutral amino acids, e.g. In certain embodiments, the dimer is substituted with a corresponding residue having Ala, Met, or Trp. The NKG2D-Fc variant contains the mutations Lys326Trp, Clu333Ser, or both.

[0054] To achieve increased C1q binding and enhanced CDC, some embodiments of the invention include The method includes introducing one or more mutations into specific residues in the hinge region of human IgG1. Non-limiting examples of such mutations include Lys222Trp / Thr223Trp, Cys220Asp / Asp221C, ys, Cys220Asp / Asp221Cys / Lys222Trp / Thr223Trp, Lys222Trp / Thr223Trp / His224Trp and As An example is p221Trp / Lys222Trp.

[0055] In addition, when fusion proteins with artificial sequences and activities are used as therapeutic agents, In some circumstances, patients treated with such fusion proteins may experience an unwanted immune response, e.g. It should be noted that the specific structural properties of the Fc fragment may lead to the development of antibodies against the drug. Modifications have been shown to reduce the immunogenicity of therapeutic fusion proteins. No. 6,992,174 B2 to Gillies et al., which are incorporated herein by reference; See Immunological Reviews, vol. 222:9-27, 2008. Such modifications are within the scope of the present disclosure. These findings may be useful for the efficient design of dimeric NKG2D-Fc.

[0056] Linker The dimeric NKG2D-Fc construct used in the methods of the present disclosure comprises a first NKG2D portion ( NKG2D1) with a second NKG2D moiety (e.g., NKG2D2) and an NKG2D moiety (e.g., NKG2D1 or N KG2D2) to the Fc fragment and / or the Fc fragment to the drug moiety. In some embodiments, the linking moiety (e.g., linking molecule) may further comprise: In some cases, the hinge region of the Fc fusion protein molecule is designated X1, X2, or X3. and the fusion peptide (e.g., a soluble receptor), which This allows the two parts of the molecule to function separately (e.g., Ashkenazi et al., 1997 (see

[0057] In some embodiments, at least one linking moiety (e.g., linking molecule) is NKG2D1 NKG2D2, Fc or the adjacent portion of the drug moiety, but not the amino acid of NKG2D1 with the amino acid of NKG2D2 covalently linking an amino acid of NKG2D2 to an amino acid of the Fc, or an amino acid of the Fc to a drug moiety As used herein, a "non-contiguous" joining molecule refers to a Each of the NKG2D portions (e.g., NKG2D1 and NKG2D2), the NKG2D portion and the Fc portion, and / or the F The c moiety and the drug moiety are additional elements that are not part of the NKG2D or immunoglobulin or drug moiety. which is in fact adjacent to the portion of the chimera it links and functions as a linker It means that the NKG2D, Fc or drug molecule is linked via an additional element. Non-limiting examples of linking molecules that are not junctions are described below.

[0058] The linking molecule may be a peptide linker. Drinkers range from about 2 to about 25 amino acids in length. In some embodiments, The peptide linker is 20 amino acids in length. In some embodiments, the peptide The linker ranges in length from about 4 to about 16 amino acids. Petit drinkers are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19. In some embodiments, the peptide is 20, 21, 22, 23, 24, or 25 amino acids in length. A peptide linker is longer than 25 amino acids. The dimeric NKG2D-Fc chimera was synthesized using conventional molecular biology / recombinant DNA methods as a single recombinant polypeptide. It may also be produced as a polypeptide.

[0059] In some embodiments, the peptide linker comprises a protease-dependent cleavable moiety. Examples of protease cleavable peptide linkers include, but are not limited to, The MMP-sensitive linker GGPLGLWAGG (SEQ ID NO: 6) and the factor Xa-sensitive linker IEGR ( SEQ ID NO: 7). Various cleavable sequences, e.g., Chen et al., Adv. Drug Deliv. Rev. (2013), 65(10), ): The one described on pages 1357-69 is known.

[0060] In some embodiments of the invention, flexible peptide linkers are used. The peptide linker is preferably about 25 or less amino acids in length. In some embodiments, the flexible peptide linker is 20 amino acids in length. In the above, the peptide linker is about 20 or less amino acid residues, e.g., 2, 3, 4, 5, 6, 7, Contains 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 amino acid residues. In some embodiments, the peptide linker is about 12 or less amino acid residues, e.g., 3, 4 , 5, 6, 7, 8, 9, 10, 11 and 12 amino acid residues. The linker contains two or more of the following amino acids: glycine, serine, alanine and threonine. In some embodiments, the flexible peptide linker is a glycine-serine linker. -It is.

[0061] In some embodiments, the glycine-serine linker has the formula (GS): n (In the formula, n is 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. wherein the glycine-serine linker has the formula (GGGGS): n (SEQ ID NO:2) (wherein n is 1, 2, 3, 4 or is 5).

[0062] In some embodiments, the dimeric NKG2D-Fc chimera comprises three linking molecules, X1, X2 and X3. X1 covalently links an amino acid of NKG2D1 to an amino acid of NKGD2; X2 covalently links an amino acid of Fc to an amino acid of Fc; and X3 covalently links an amino acid of Fc to a drug moiety. In some embodiments, X1 is (GS)3 (SEQ ID NO: 4). , X2, X3 and X4 are each (GGGGS)4 (sequence number 3).

[0063] In some embodiments, the dimeric NKG2D-Fc chimera comprises the IEGR (SEQ ID NO: 7) peptide linker. Contains anchor.

[0064] Alternatively, the linking molecule may be a non-peptide linker. A "non-peptide linker" is a biocompatible compound that contains two or more repeating units linked together. Examples of non-peptide polymers include, but are not limited to, polyethylene glycol. Poly(ethylene / propylene) glycol (PEG), poly(propylene glycol) Polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharide, dextran , polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl ethyl ether, polyacrylonitrile Arylamide, polyacrylate, polycyanoacrylate, lipid polymer, chitin, hyaline Useful non-peptide linkers for Fc fusion molecules include uronic acid and heparin. For a more detailed description, see, for example, International Publication No. WO 2005 / 023363, which is incorporated herein by reference. See, for example, US Pat. No. 5,399,341, issued Dec. 20, 2006, and US Pat. No. 5,399,341. Typically, such linkers are Depending on the linker, the molecular weight ranges from about 1 kDa to 50 kDa. For example, a typical PEG is The molecular weight is about 1 to 5 kDa, and the polyethylene glycol is about 5 kDa to 50 kDa, more preferably , and has a molecular weight of approximately 10 kDa to 40 kDa.

[0065] Drug portion In some embodiments, the dimeric NKG2D-Fc chimera further comprises a drug moiety. As used herein, a "drug moiety" refers to a compound that is intended for delivery to a target cell (e.g., a cancer cell). Generally, the drug moiety is linked to the carboxy terminus of the dimeric NKG2D-Fc chimera. However, those skilled in the art will appreciate that some In some embodiments, the drug moiety is attached to the amino terminus of the dimeric NKG2D-Fc chimera. Examples of "drug moieties" include drugs (e.g., small molecules), toxins (e.g., phosphorylation Photoxin family of molecules), radionuclides, enzymes, cytokines, chemokines, activation or angiogenesis-inhibiting compounds or virtually any antitumor compound. Examples of single chain antibody variable fragments include single chain antibody variable fragments.

[0066] In some embodiments, the drug moiety comprises a cytokine or a functional portion thereof. Inhibitors are proteins and peptides that have the ability to regulate immune cell function. A "functional portion" of a cytokine is a portion of an immune cell that is involved in the function of an immune cell (e.g., one or more cytokine receptors). Cytokine fragments that retain their ability to regulate the function of cytokines. Examples include, but are not limited to, interferon alpha (IFN-α), interferon beta Interferon-β (IFN-β) and interferon gamma (IFN-γ), interleukins (e.g., IL-1 to IL-2), L-29, especially IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15 and IL-18), tumor necrosis inflammatory cytokines (e.g., TNF-alpha and TNF-beta), erythropoietin (EPO), MIP3a, monocyte chemotaxis Protein (MCP)-1, intracellular adhesion molecule (ICAM), macrophage colony-stimulating factor (M-CSF), Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) In some embodiments, the drug moiety includes IL-2, IL-12, IL-15, IL-18, , IL-21 and IFN-α.

[0067] In some embodiments, the drug moiety is a cytokine / cytokine receptor heterodimer. Cytokine / cytokine receptor heterocomplexes are known in the art. It is known in the art and is described, for example, in Rowley et al., Eur J Immunol. February 2009; Vol. 39 (No. 2): pp. 491-506. In some embodiments, the dimeric NKG2D-Fc chimera is capable of inhibiting IL-15 (e.g., A drug moiety containing IL-15Ra (e.g., GenBank AAX37025) / IL-15Ra (e.g., GenBank AAP69528.1) heteroconjugate In some embodiments, the drug moiety comprises a sequence selected from the group consisting of positions 22-135 of IL-15 (GenBank AAX37025). 31 of human IL-15 receptor alpha (hIL15Ra, GenBank AAP69528.1) fused to the 32 amino acid residues In some embodiments, IL-15 and IL-15Ra comprise amino acids 101 to 107 of the The IL-1 and IL-2 are separated by, for example, a 20 amino acid (G4S)4 (SEQ ID NO: 3) linker. Dimeric NKG2D-Fc chimeras, including IL-15 / IL-15Ra heterocomplexes, are further described in the Examples section. In some embodiments, the dimeric NKG2D-Fc chimera is a heterodimer of IL-12p35 and IL-12p40. In some embodiments, IL-12p35 and IL-12p40 are , separated by a linker, for example a 20 amino acid (G4S)4 (SEQ ID NO: 3) linker. In some embodiments, the dimeric NKG2D-Fc chimera binds to IL-23p19 and IL-23p40. In some embodiments, the drug moiety comprises a heteroconjugate. are separated by a linker, for example a 20 amino acid (G4S)4 (SEQ ID NO:3) linker. In some embodiments, the dimeric NKG2D-Fc chimera is a heterodimer of IL-27p28 and EB1. In some embodiments, the drug moiety comprises a ligand conjugate. The nucleotides are separated by a linker, for example a 20 amino acid (G4S)4 (SEQ ID NO:3) linker. In some embodiments, each of the cytokine / cytokine receptor heterocomplexes The subunits are on different chains of a dimeric NKG2D-Fc chimera.

[0068] In some embodiments, the drug moiety is a single chain antibody variable fragment (ScFv). As used herein, a "single-chain antibody variable fragment" refers to a single-chain antibody variable fragment that is linked by a short linker peptide. This refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins. ScFv protein The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. In some embodiments, the ScFv maintains its affinity to immune checkpoint proteins. In some embodiments, the ScFv binds to a protein (e.g., PD1 or CTLA4). For example, they inhibit angiogenesis by binding to angiogenesis regulators such as VEGF.

[0069] In some embodiments, the drug moiety is a chemokine. In this context, "chemokine" refers to a protein of low molecular mass that stimulates the recruitment of leukocytes. Generally, chemokines are the primary inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor (TNF). Chemokines are secondary inflammatory mediators induced by mediators. CC chemokines (e.g., CCL1 to CCL-28), CXC (e.g., CXCL1 to CXCL17), C (e.g., XCL1, They can be classified into four families: CX3C (CX3CL1), CXCL2, and CX3C.

[0070] In some embodiments, the drug moiety is a small molecule. A "small molecule" is a non-peptide that is either synthesized in the laboratory or found in nature. A small molecule drug is a drug that is a non-oligomeric organic compound. Non-limiting examples of small molecule drugs include small molecule kinase inhibitors (e.g., everolimus, gefitinib, imatinib, etc.), bromodime inhibitors (e.g., JQ1, I-BET 151, RVX-208, etc.), antibiotics (e.g., kanamycin, nephrectomycin, etc.), omycin, ciprofloxacin, etc.) and antiviral drugs (e.g., ribavirin, rimantadine, In some embodiments, the small molecule is an anti-tumor compound. The anti-tumor compounds are described in more detail elsewhere in this disclosure.

[0071] In some embodiments, the drug moiety is a radionuclide. In this context, "radionuclide" refers to a medically useful radionuclide. Examples of radionuclides include: 99m Tc, 188 Re, 186 Re, 153 Sm, 166 Ho, 90 Y, 89 Sr, 67 Ga, 68 Ga, 111 In, 183 Gd, 59 Fe, 22 5 Ac, 212 Bi, 211 At, 45 Ti, 60 Cu, 61 Cu and 67 Cu is an example.

[0072] Other parts In some embodiments, dimeric NKG2D-Fc kinase useful for the methods described herein. The melae may further comprise one or more accessory moieties, such as a tag sequence and a signal sequence. For example, the tag sequence can be used to detect and / or isolate the polypeptide. Examples of tags include, but are not limited to, HA, Flag, Myc, Glu, His, and Malt. The tag sequence may be a sequence that indicates that the presence of such a tag is a dimeric NKG2 As long as it does not interfere with the function of the D-Fc molecule, it may be located at the amino terminus or the carboxyl terminus. or anywhere in the middle of the dimeric NKG2D-Fc chimeric molecule. In this case, the tag sequence is cleavable.

[0073] In some embodiments, the dimeric NKG2D-Fc chimera optionally comprises a signal sequence. A signal sequence is a short (typically 10-fold) sequence that directs the transport of a polypeptide after translation. Typically, it is a peptide chain (of about 3 to 60 amino acids in length) that allows the polypeptide to be more The amino acid sequence of the signal sequence allows for the targeting of the polypeptide to a specific intracellular compartment, e.g. Signal sequences direct polypeptides (synthesized in the cytosol) to organelles. They are also called targeting signals, signal peptides, transport peptides or localization signals. In some embodiments, the signal sequence is a sequence that is generated by the signal peptide after the polypeptide is transported. It is cleaved from the polypeptide by an endonuclease.

[0074] In some embodiments, the dimeric NKG2D chimera comprises an NKG2D chimera modified with an IL-2 signal sequence. The NKG2D-Fc construct contains a nucleotide sequence that allows for optimal expression and secretion of the NKG2D-Fc construct. See, for example, Zhang et al., 2004, J. Gene Med. 7:354-65. In this embodiment, the dimeric NKG2D chimera comprises a signal peptide derived from the polypeptide sequence of CD33. For example, the CD33 signal peptide contains the 1st to 16th residues of the CD33 polypeptide sequence. It is possible for a person skilled in the art to carry out the methods provided in this disclosure. It will be appreciated that there are many other suitable signal peptide sequences that can be used to In addition, if a signal peptide is present in the NKG2D chimera, additional amino acids Residues, e.g., spacers, are optionally present between the N-terminal signal peptide and the Fc portion of the chimera. In some embodiments, for example, a signal sequence may be inserted. This is followed by a Met-Asp dipeptide spacer.

[0075] Preparation of dimeric NKG2D-Fc The art provides molecular biology techniques for preparing dimeric NKG2D-Fc chimeras with desired characteristics. The biological and biochemical techniques are well known in the art. Preferably, the dimeric NKG2D-Fc chimeric construct is The tract is produced by conventional recombinant DNA techniques. The NKG2D-Fc chimera is produced as a single (e.g., unbroken) recombinant polypeptide. In other embodiments, two or more portions of the dimeric NKG2D-Fc are produced as separate fragments. and subsequently linked together to obtain a dimeric NKG2D-Fc molecule. Each NKG2D portion (e.g., NKG2D1, NKG2D2) and the Fc portion of the dimeric NKG2D-Fc are expressed as separate recombinant proteins. Each is produced as a separate polypeptide and then fused together by chemical ligation means. The dimeric NKG2D-Fc is obtained by combining the two peptides. This production methodology allows the use of non-peptide linking molecules. This production methodology may be particularly preferred when a single cut is used. When produced as an unlinked polypeptide, the dimeric NKG2D-Fc chimera did not fold correctly. It may also be preferable if the target molecule is not properly bound to the ligand (e.g., does not bind appropriately to the ligand).

[0076] A variety of host organisms may be used for the production of recombinant polypeptides. Examples include, but are not limited to, bacteria such as E. coli, yeast cells, insect cells, plant cells, and The selection of an appropriate host organism depends on the particular application of the dimeric NKG2D-Fc chimera. Those skilled in the art will be familiar with the selection of suitable hosts for producing recombinant polypeptides. In selecting an appropriate host, the reader will understand how to take into account certain criteria. Influencing factors include, for example, post-translational modifications, such as phosphorylation and glycosylation. The pattern of synthesis and technical factors, such as generally expected yield and ease of purification, Host-specific post-translational modifications of dimeric NKG2D-Fc for use in vivo include those Note that host (post-) specific modifications are known to be highly immunogenic (antigenic). must be carefully considered.

[0077] Once produced, the dimeric NKG2D-Fc can be synthesized by any suitable means, for example, by chromosomal synthesis known to those skilled in the art. The purification can be performed by chromatographic methods. Examples of chromatographic methods include gel filtration chromatography. For example, see Caine et al., Protein Expr. Purif., 1996, Vol. 8: See pages 159-66. In some embodiments, the dimeric NKG2D-Fc is Purified by immunoaffinity chromatography.

[0078] As will be appreciated by those skilled in the art, the dimeric NKG2D chimeric moieties can be prepared and isolated separately. , and can also be linked by chemical synthesis.

[0079] NKG2D receptor ligands In any of the embodiments described herein, the dimeric NKG2D-Fc is Known human NKG2D ligands include MICA, MIC B, RAET-1G, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. The dimeric NKG2D-Fc chimeras described in this disclosure have the ability to bind to two or more NKG2D receptor ligands. Has power.

[0080] In some embodiments, the dimeric NKG2D-Fc chimeric molecule comprises 10 -4 M or less, 10 -7 M or less With high affinity or sub-nM, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1n In some embodiments, the dimer N The binding affinity of the KG2D-Fc molecule for its ligand is at least 5×10 6 Ka, at least 1 ×10 7 Ka, at least 2 × 10 7 Ka, at least 1 × 10 8 Ka or higher.

[0081] In some embodiments, NKG2D-Fc binds to a subset of NKG2D receptor ligands (e.g., Three-dimensional structural data combined with mutational analysis are In addition, NKG2D is permissive in recognizing and binding a diverse array of its endogenous ligands. He revealed that:

[0082] The ligand for NKG2D may be expressed on the cell surface. The ligand may be "shed" from the cell surface and exists as a soluble ligand. NKG2D ligands are overexpressed and, in some cases, are present in the blood, e.g., in a serum-soluble form. It is known that certain cancers release (e.g., shed) leukocytes into surrounding blood vessels or tissues. This is believed to contribute, at least in part, to the initiation and / or progression of cancer. Thus, dimeric NKG2D-Fc may function as a neutralizing agent to suppress abnormally elevated levels of NKG2D. In balance with the expression of ligands present in the nucleus, they are expressed either on the cell surface or as released forms. The present invention is useful for binding to such ligands present anywhere in the body.

[0083] If the NKG2D ligand is expressed on the surface of the cancer cells of the subject, the bilayer described in this disclosure can be used. The dimeric NKG2D-Fc binds to the ligand on the cell surface when administered to a subject. Binding of the Fc chimera to its ligand activates the endogenous NKG2D receptor present on NK cells. NKG2D ligands may be "shed" from cancer cells, e.g., released into the bloodstream of a subject. When the dimeric NKG2D-Fc described herein is used, it binds to a soluble ligand and further Isolate it from action.

[0084] therapeutic application Generally, expression of NKG2D ligands appears to be restricted to the gastrointestinal epithelium. , is observed in quiescent epithelial cells, whereas higher levels of expression are observed in rapidly proliferating cells. Expression of NKG2D ligands is also increased in various transformed cells, particularly those of epithelial origin. Thus, the present invention provides a method for treating cancer or symptoms of cancer in a subject. A method is provided herein for detecting a dimeric NKG2D that binds to an NKG2D ligand in vivo. The method comprises administering to the subject a therapeutically effective amount of -Fc.

[0085] The terms "treating," "treatment," and "treat" in the context of cancer therapy are The term "composition" refers to the administration of a composition comprising a dimeric NKG2D-Fc as described herein to a subject with cancer. The agent is administered to the subject in a therapeutically effective amount. The amount may be statistically significant for a subject with a disease or disorder, e.g., a particular type of cancer. Generally, a therapeutically effective amount refers to an amount that is believed to produce a beneficial effect with The composition is administered to a population of subjects with a particular condition (e.g., disease progression or stage) to assess response. As used herein, therapeutic treatment is determined by evaluating the results of For example, complete prevention, or elimination of symptoms of a disease, delay in the onset of symptoms of a disease, or This may include reducing the severity of the disease.

[0086] cancer The dimeric NKG2D-Fc chimera is a chimeric NKG2D-Fc chimera that is capable of inhibiting the expression of one or more NKG2D ligands in a subject. It is believed to be broadly useful for immunotherapy of a wide range of cancers when Cancer broadly refers to proliferative disorders involving transformed cells, including precancerous and malignant The present invention relates to a method for treating disorders characterized by overexpression of one or more NKG2D ligands. In some embodiments, the method is useful for treating a subject having a cancer that is Cancers are characterized by overexpression of one (or primarily one) ligand for the NKG2D receptor In other embodiments, the cancer is characterized by overexpression of two or more NKG2D ligands. can be done.

[0087] The methods disclosed herein include the treatment of precancerous disorders that confer a risk of progression to malignancy. It is a useful treatment for

[0088] Examples of such disorders include, but are not limited to, dysplasia, hyperplasia, and simple plasia of unknown significance. Plasma cell disorders such as clonal gammopathy (MGUS) and smoldering multiple myeloma (SMM) In some embodiments, the cancer is a melanoma, lung, breast, renal, or ovarian cancer. carcinoma, lymphoma or leukemia of the liver, ovary, prostate, pancreas, stomach and colon. In embodiments, the cancer is melanoma. In some embodiments, the cancer is Malignant tumors of plasma cells, such as multiple myeloma (MM) or precancerous conditions of plasma cells. In some embodiments, the cancer is melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma. In some embodiments, the cancer is ovarian cancer, colon cancer, pancreatic cancer, or prostate cancer. The subject has been diagnosed with cancer or is predisposed to cancer. The methods disclosed herein are directed to treating patients with metastases and therefore susceptible to relapse or recurrence. The method is also useful for treating a subject. For example, the method can be used to treat a cancer or metastatic tumor. In high-risk individuals who have a family history or exhibit a genetic predisposition for metastasis of cancer Specifically, the method is useful for treating cancers in which expression of NKG2D ligands is involved. In some embodiments, the NKG2D ligand is MICA. Thus, in some embodiments, the cancer causes a MICA-associated tumor.

[0089] Whether or not a particular subject (e.g., patient) should receive a cancer treatment that includes NKG2D-Fc depends on the subject. by testing for abnormal expression of one or more NKG2D ligands in the "Abnormal expression of one or more NKG2D ligands" in a subject can be determined by analyzing a biological sample taken from the subject. In some embodiments, the biological sample is Examples of such samples may include biopsy samples taken from tissue of a subject suspected of having cancer. For example, in some cases, biological samples are taken from solid tumors to test for malignancies. In other cases, the biological sample is a blood sample, e.g., serum, a fecal sample, a urine sample, etc. The biological sample may be comprised of a sample for the purpose of testing for the diagnosis or progression of a disease, e.g., cancer. The sample may be any cell or tissue sample obtained from a subject for the purpose of

[0090] One of skill in the art would be able to easily determine the presence and level of one or more markers present in a biological sample. Be familiar with the various laboratory techniques and procedures used to assay To determine whether or not a patient has a cancer associated with overexpression of an NKG2D ligand, typically In certain situations, the type of biological sample available Depending on the assay, immunohistochemical or immunocytochemical analysis can be performed. There are many commercially available methods available for performing these analyses. Examples of assays include, but are not limited to, ELISA, immunoblotting, and immunohistochemistry.

[0091] subject The methods disclosed herein are applicable to a wide range of species in which cancer is known to occur, including, for example, humans, Non-human primates (e.g., monkeys), horses, cows, pigs, sheep, deer, elk, goats, dogs, It can be applied to cats, rabbits, guinea pigs, hamsters, rats and mice. Thus, as used herein, a "subject" refers to a subject having a differential diagnosis of at least one NKG2D ligand. have or are at risk of developing a disease associated with abnormal expression of In a preferred embodiment, the subject is a mammalian subject. In some embodiments, the subject is a human subject having cancer that exhibits elevated levels of NK G2D ligands include MICA.

[0092] If the subject is shown to express elevated levels of one or more NKG2D ligands, In that case, the subject can be treated with the methods described herein. Optionally, the subject may be treated with another cancer therapy. In some embodiments, the cancer is in remission or has been treated with In some cases, the subject is at risk of having a recurrence, e.g., metastasis. In embodiments, overexpression of one or more NKG2D ligands is indicative of a cancer cell, e.g., a tumor In some embodiments, at least one of the following is expressed by the cancer cell: The NKG2D ligands are shed into the bloodstream and are therefore detectable in the serum of a subject.

[0093] Depending on the phenotype of a particular cancer, it may be overexpressed (by tumor cells) relative to other ligands. It may be possible to target one or more ligands (which are expressed), the expression of which is significant is not affected by.

[0094] Mechanism of action The present invention relates, in part, to chimeric molecules comprising two NKG2D fragments and an Fc fragment (e.g., a dimeric NKG 2D-Fc chimera) has the ability to bind to one or more NKG2D ligands, and and improved efficacy compared to chimeric molecules containing an Fc fragment (e.g., monomeric NKG2D-Fc chimera). The discovery is based on the surprising finding that it effectively induces tumor cell death.

[0095] Without being limited by any particular theory, it is believed that dimeric NKG2D-Fc chimeras are involved in the regulation of two types of immune system function. It appears that the immune system can function through two major components: innate and adaptive immunity. As used herein, innate immunity or the innate immune system refers to a non-specific defense against foreign pathogens. Innate immunity refers to the host defense mechanism that protects against physical barriers (e.g., skin, stomach acid, mucus, etc.). These include both innate immune mechanisms (e.g., immune cells, inflammatory bowel disease, and inflammation) and active mechanisms such as NK cells, phagocytes, and the complement system. NK cells are a major component of the innate immune system. NK cells are cytotoxic, e.g. For example, they can attack cells infected with microorganisms and some types of tumor cells. The cytotoxic activity of NK cells is mediated through cell surface receptors that recognize MHC class I alleles. Many receptor types are known in the art, and one receptor subtype Phagocytes include neutrophils, monocytes, macrophages, basophils, and eosinophils. The complement system is a vital component of the immune system that helps eliminate pathogens from the host organism. It is a scientific cascade.

[0096] Generally, adaptive immunity or the adaptive immune system refers to an antigen-specific, antibody-mediated immune response. Adaptive immunity is usually characterized by specific antibodies produced by B lymphocytes and antigen-specific T lymphocytes. Humoral responses mediated by B lymphocytes are mediated by the activity of other specialized cells. Through the production of circulating antibodies that mark foreign cells and molecules for destruction by cells and proteins. It mainly defends against extracellular pathogens. The response is primarily directed against intracellular pathogens and pathogens by directly binding to and destroying affected cells. According to the present disclosure, the dimeric NKG2D-Fc, a non-antibody molecule, specifically binds to NKG2D-Fc and protects against cancer cells. These antibodies are thought to functionally mimic what are normally functions of target antibodies.

[0097] Thus, the present invention provides a method for the preparation of a dimeric NKG2D-Fc antibody that binds to a tumor that expresses an NKG2D ligand on the cell surface. We are investigating a method for the treatment of cancer that directly binds to tumor cells. Monomeric NKG2D-Fc destroys tumor cells that overexpress NKG2D ligands, but not those that do not express NKG2D ligands. Healthy cells that do not overexpress the gene are not destroyed, and therefore can be specifically identified.

[0098] Dimeric NKG2D-Fc is expressed on human tumor cells in at least two ways: can target all NKG2D ligands, a single mechanism mediating the destruction of tumor cells. The mechanism is the process of complement lysis (also called complement-dependent lysis, complement-dependent cytotoxicity, or CDC). The second method that mediates tumor cell destruction is through antibody-dependent cellular cytotoxicity (ADCC). By doing so.

[0099] In some embodiments, the dimeric NKG2D-Fc functions as an opsonizing agent. Opsonization is the process by which a cell or particle is targeted to a receptor on another cell, e.g., a dendritic cell or a phagocyte. This is the process by which the cells are coated with molecules that bind to the receptor and facilitate uptake. For antigen-presenting cells such as macrophages, opsonization facilitates efficient processing and presentation of antigens. It promotes targeting (e.g., ligands) and mediates internalization and subsequent antigen processing. The ability to specifically bind to both specific receptors (e.g., FcR) on antigen-presenting cells that can Potent opsonizing agents are particularly useful.

[0100] Tumor cells that express one or more ligands for the NKG2D receptor on their cell surface are opsonized. For example, the NKG2D portion of the chimera may be coated with a dimeric NKG2D-Fc molecule. The Fc portion of the chimera remains exposed and can bind to ligands on the surface of tumor cells. Dendritic cells have FcγR and therefore can bind tumor antigens (e.g., NKG2D ligands). and internalize, which then induces cytotoxic T cells, also known as CD8+ T cells. This results in antigen presentation to the cells, known as cross-priming. Antibody immunization results in the generation of MHC class II-restricted CD4+ T cell responses. Through immunofluorescence, NKG2D-Fc chimeras are able to efficiently cross-present (e.g., prime) by dendritic cells. These results suggest that immunization with T cells may be beneficial in tumorigenesis, as they can promote T cell proliferation and activation, leading to the induction of strong T cell responses against tumors.

[0101] Cancer patients often suffer from immunosuppression. In some cases, immunosuppression can be at least This may be partly caused by impaired NKG2D receptor signaling. According to the prevailing model, for example, shed MICA binds to the NKG2D receptor on lymphocytes. This model impairs host defense by inducing internalization of the nuclei. Thus, loss of MICA in tumor cells leads to immunosuppression through downregulation of NKG2D surface expression. vinegar.

[0102] Thus, the methods provided herein are particularly useful in treating patients who are receiving soluble (i.e., shed) In the event that the level of NKG2D ligand or detectable ligand in serum is elevated, Useful for combating or reducing immunosuppression by administering a composition comprising NKG2D-Fc The mechanism of action is that NKG2D-Fc administered to patients binds to excess NKG2D shed from tumors. Cells that bind (and thus are sequestered) to soluble ligands, thereby resulting in immune suppression Reverts down-expression of NKG2D receptors on the surface.

[0103] Thus, dimeric NKG2D-Fc chimeras mediate soluble ligands shed by tumor cells. and promoting ADCC and / or CDC in tumor cells expressing the cell surface ligand. and the appropriate expression vectors including CD8 cytotoxic T lymphocytes (CTLs) and B cells producing tumor-specific antibodies. It has multiple therapeutic functions, including mediating cross-presentation and priming of the immune system. This can be done.

[0104] Administration The dimeric NKG2D-Fc composition can be administered directly to a subject. The subject is preferably a mammalian The terms "administration" and "administering" refer to the process by which a pharmaceutical agent is delivered to its target cells, e.g. For example, a pharmaceutical agent may be administered to a subject so as to contact the cancer cells in vivo, i.e., within the subject's body. In some embodiments, a composition comprising NKG2D-Fc is administered to a subject. In a preferred embodiment, the systemic administration is via intravenous injection. In some embodiments, the composition comprising the dimeric NKG2D-Fc is administered locally. For example, in some cases, the composition is administered directly to a solid tumor or in close proximity to a solid tumor. may be delivered to.

[0105] Pharmaceutically acceptable carriers In some embodiments, the present disclosure relates to a dimeric NKG2D-Fc chimera as described herein. and a pharma- ceutically acceptable carrier. Generally, the present invention provides a composition comprising a dimeric NKG2D-Fc. The composition can be suspended in a pharma- ceutically acceptable carrier (e.g., saline). Such carriers include, but are not limited to, sterile, aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include mineral oil, propylene glycol, and the like. These include glycerol, polyethylene glycol, vegetable oils and injectable organic esters. Aqueous carriers include, but are not limited to, water, alcohol, saline and buffered solutions. Preservatives, flavoring agents, and additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Other additives etc. may also be present. Any of the compositions described herein to be administered to a mammal It is understood that the material may contain one or more pharma- ceutically acceptable carriers. It will be possible.

[0106] Route of administration Any of the compositions described herein can be administered to any part of a subject's body via a variety of administration routes. The compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intramuscularly, rectally, vaginally, or intraperitoneally. by intravenous, intrathecal, intratracheal, intradermal or transdermal injection, by oral or nasal administration, It can be administered by inhalation or by gradual perfusion over time. The composition can be delivered to a specific tissue. For example, but not limited to, the composition can be delivered to a mammalian The substance can be delivered to the joints, nasal mucosa, blood, lungs, intestines, muscle tissue, skin or abdominal cavity. In a further example, an aerosol formulation of the composition is administered to a subject by inhalation. can.

[0107] dose The required dose depends on the route of administration, the nature of the formulation, the nature of the patient's illness, the size, weight, and surface area of ​​the subject. Proper use depends on the patient's dosage, age and sex, other medications being taken, and the judgment of the treating physician. Amounts typically range from 0.01 to 1,000 μg / kg. There is a wide variation in the dose required. In view of the different efficiencies of the various available dimeric NKG2D-Fc compositions and the various routes of administration, Variations in these dosage levels are expected, as is well understood in the art. can be adjusted using standard, empirical, routine methods for optimization. Administration may be in a single dose or multiple doses (e.g., 2-fold or 3-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, etc. The composition may be concentrated in a suitable delivery vehicle (e.g., 50-fold, 100-fold, 150-fold or more). Encapsulation (e.g., polymeric microparticles or implantable devices) can improve the efficiency of delivery. It is possible.

[0108] Treatment regimen The duration of treatment with any of the compositions provided herein may range from as little as one day to the lifespan of the host ( For example, the dimeric NKG2D-Fc composition may be administered for any length of time, e.g., for months. It can be administered once a year for three months or once a year for 10 years. The frequency of treatments is variable. For example, the dimeric NKG2D-Fc composition can be administered daily, weekly, monthly, or The dimeric NKG2D-Fc composition can be administered once (or twice, three times, etc.) each year. or multiple other cancer therapies, administered together, e.g., at the same time or sequentially. For example, a patient may administer autologous tumor tumours separately, at intervals of hours, days, months or years. The patient can be treated with a cellular vaccine, followed by an anti-CTL4 antibody, followed by dimeric NKG2D-Fc. do.

[0109] Effective dose An effective amount of any of the compositions described herein can be administered to a subject. The term "effective" as used herein means a compound that exhibits the desired therapeutic effect without inducing significant toxicity in the subject. Such an amount refers to any amount that has a known effect, for example, inducing an immune response. After administration of the composition, the subject's biological response, e.g., immune response, and improvement in symptoms, are evaluated. In addition, the level of toxicity, if any, can be determined based on known By assessing the subject's clinical symptoms before and after administering a particular amount of the composition. The effective amount of a particular composition administered to a subject will depend on the desired outcome, as well as the host's Note that the dose can be adjusted according to the response and level of toxicity of the subject. The nature of the treatment may vary for each individual host, including but not limited to, the disease state, age and pain of the subject. It depends on several factors, including pain tolerance.

[0110] Combination Therapy In some embodiments, a subject in need of cancer treatment is treated with a combination of an additional cancer therapy. In combination, the patient is treated with a dimeric NKG2D-Fc composition as described herein. In this case, the additional cancer treatment may include a cytotoxic agent and / or a non-cytotoxic agent. A "cytotoxic agent" is an agent that inhibits or prevents the function of cells and / or causes the destruction of cells. The term refers to a substance that causes necrosis. 131 I, 125 I, 90 Y and 186 Re), chemotherapeutic agents, and enzymatically active toxins of bacterial, fungal, plant or animal origin, or is intended to include toxins, such as synthetic toxins or fragments thereof. , substances that do not inhibit or interfere with the function of cells and / or cause the destruction of cells. "Non-cytotoxic agents" refer to agents that can be activated to become cytotoxic. Non-cytotoxic agents may include beads, liposomes, matrices or can include particles (see, e.g., U.S. Patent Publication No. 2003 / 0028071 and U.S. Patent Publication No. 2003 / 0028072). (See US Pat. No. 6,299,545, which are incorporated herein by reference). The agent may be conjugated, coupled, linked, or associated with the dimeric NKG2D-Fc composition described herein. They may also be combined.

[0111] In some embodiments, conventional cancer medicaments are used in combination with the compositions described herein. In some cases, a subject in need of cancer treatment is administered a 1 Treatment with the dimeric NKG2D-Fc composition described herein in combination with one or more additional agents. Highly suitable drugs include those that promote DNA damage, e.g. Any form of DNA damaging agent known to those of skill in the art may be used to break the double strand of cellular DNA. DNA damage is typically induced by radiation and / or chemotherapy. DNA damaging agents are also referred to as genotoxic agents. "In conjunction with" refers to the administration of dimeric NKG2D-Fc simultaneously (or concomitantly) with one or more additional therapies. administration of one or more additional therapies, either separately or in close proximity; It is intended to mean administered to a subject before or after.

[0112] Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (sealed The energy source for external radiation therapy is X-rays. , gamma rays, and particle rays. Energy sources used in internal radiation include , radioactive iodine (iodine 125 or iodine 131 ),strontium 89 or radioisotopes Examples of the metal oxide include phosphorous acid, palladium, cesium, indium, phosphate, and cobalt. Methods of administering radiation therapy are well known to those of skill in the art.

[0113] Examples of DNA damaging chemotherapeutic agents that may be particularly useful include, but are not limited to, busul Fenoxacin (Mileran), carboplatin (Paraplatin), carmustine (Carmustme) (BCNU), Chlorambucil (Leukeran), cisplatin (Platmol), cyclophosphamide (Cytoxan, Neosar), Dacarbazme (DTIC-Dome), Ifosf Amido (Ifex), Lomustine (CCNU), Mechlorethamine (Mechloret hamme) (nitrogen mustard, Mustargen), melphalan (Archera Examples include procarbazine (Alkeran) and procarbazine (Matulane).

[0114] Many other chemotherapeutic agents may also be used, alone or in combination, for the methods described herein. These include methotrexate, vincristine, Adriamycin, cisplatin, sugar-free chloroethyl nitrosourea, 5-fluorouracil Rasil, Mitomycin C, Bleomycin, Doxorubicin, Dacarbazine, Taxol le, flagylin, meglumine GLA, valrubicin, carmustine carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farne Farnesyltransferase inhibitors, farnesyltransferase inhibitors, MMP, MTA / LY231514, LY264618 / Lometexol, Glamolec, CI-994, TNP-470, Hycamtin / Topotecan, PKC412, Valspodar / PSC833, Novantrone / Mitoxantrone (Mitrox antrone, Metaret / Suramin, Batimastat, E7070, BCH-4556, CS-682 , 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN 698 , TA 2516 / Marmistat, BB2516 / Marmistat, CDP 845, D2163, PD183805, DX895 1f, Lemonal DP 2202, FK 317, Picibanil / OK-432, AD 32 / Valrubicin, Metast ron / strontium derivatives, Temodar / temozolomide, Evacet / Liposomal Doxorubicin, Yewtaxan / paclitaxel, Taxol / paclitaxel Cell, Xeloda (Xeload) / Capecitabine, Furtulon / Doxifluridine, Cyclopax / Oral paclitaxel, oral taxoids, SPU-077 / Cisplatin, HMR 1275 / Flavopiride CP-358(774) / EGFR, CP-609(754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT( Tegafur / uracil, Ergamisol / levamisole, eniluracil / 776 C85 / 5FU enhancer, Campto / Levamisole, Camptosar / Irinotecan, Tumode Tumodex / Ralitrexed, Leustatin / Cladribine, Paxex Paxex / paclitaxel, Doxil / liposomal doxorubicin, Caelyx ) / Liposomal doxorubicin, Fludara / Fludarabine, Farmorubicin (Pharmarubici n) / Epirubicin, DepoCyt, ZD1839, LU 79553 / Bis-naphthalimide, LU 103793 / Dras Dolastain, Caetyx / Liposomal Doxorubicin, Gemzar / Ge Mucitabine, ZD 0473 / Anormed, YM 116, lodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / Dexifosamide, Ife s) / Mesnex / Ifosamide, Vumon / Teniposide, Para Platinum / Carboplatinum, Plantinol / Cisplatinum, Vepesid de) / Etoposide, ZD 9331, Taxotere / Docetaxel, Guanine Arabinoside Prod. Rags, taxane analogs, nitrosoureas, melphelan and cyclophos Alkylating agents such as famidis, aminoglutethimide, asparaginase, busulfan, Ruboplatin, Chlorambucil, Cisplatin, Cytarabine HCl, Dactyli nomycin, daunorubicin HCl, estramustine sodium phosphate, etoposide (VP16 -213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hyaluronan) droxycarbamide), ifosfamide, interferon alpha-2a, alpha-2b, Leuprolide acetate (LHRH-releasing factor analogue), lomustine (CCNU), mechlorethamine H Cl (nitrogen mustard), mercaptopurine, mesna, mitotane (o.p'-DDD), Xantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozotoxin Synth, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsa Clin (m-AMSA), Azacitidine, Erythropoietin, Hexamethylmera MIN (HMM), interleukin 2, mitoguanidine (methyl-GAG; methylglyoxal bis- guanylhydrazone; MGBG), pentostatin (2'-deoxycoformycin), semustine ( These include, but are not limited to, vindesine sulfate, teniposide (VM-26), and vindesine sulfate. I can't.

[0115] In addition, the following agents may be useful in the present invention: alkylating agents, such as carboplatin. tin and cisplatin, nitrogen mustard alkylating agents, nitrosourea alkylating agents Anti-inflammatory agents such as methotrexate, folivoviral agents, etc. phosphate, purine analog antimetabolites, mercaptopurine, pyrimidine analog antimetabolites, For example, fluorouracil (5-FU) and gemcitabine (Gemzar®), antitumor Hormonal agents such as goserelin, leuprolide and tamoxifen, natural anti-cancer agents For example, aldesleukin, mterleukin-2, docetaxel, etoposide (VP-16), interferon alpha, paclitaxel (Taxol®) and Retinoic acid (ATRA), antibiotic natural anticancer agents, e.g., bleomycin, ductomomycin Dactmomycin, daunorubicin, doxorubicin, daunomycin and mitomycin mitomycins, including vinca C, and vinca alkaloid natural antineoplastic agents, e.g. vinblastine, vincristine, vindesine, hydroxyurea, acetone, adriamycin Isin, Ifosfamide, Enocitabine, Epitiostanol, Aclarubicin, Ancitabine Tabine, Nimustine, Procarbazine hydrochloride, Carboquone, Carboplatin, Carmofu Chromomycin A3, antitumor polysaccharides, antitumor platelet factor, cyclophosphamide (cyclophosphamide) Toxan (registered trademark), Sizofiran, Cytarabine (cytosine arabinoside), Dacarbazine thiomosine, thiotepa, tegafur, dolastatin, dolastatin ana Logs, e.g., auristatin, CPT-11 (irinotecan), mitoxantrone (m itozantrone), vinorelbine, teniposide, aminopterin, carbomycin, espera Sewing machines (see, for example, U.S. Pat. No. 4,675,187, which is incorporated herein by reference). Incorporated), neocarzinostatin, OK 432, bleomycin, furtulon, broccoli Broxundine, Busulfan, Hongban, Peplomycin, Bestatin (Ube Nimex (registered trademark), interferon-β, mepitiostane, mitobronitol (mitob romtol), melphalan, laminin peptide, lentinan, Coriolus versico lor) extract, tegafur / uracil, estramustine (estrogen / mechlorethamine), Thalidomide and lenalidomide (Revlmid®).

[0116] Other suitable chemotherapeutic agents include agents that inhibit the proteasome. Asome inhibitors inhibit the synthesis of proteins, particularly short-chain amino acids, involved in cell maintenance, growth, division and cell death. It blocks the action of the proteasome, a cellular complex that breaks down vital proteins. Examples of endothelial cell inhibitors include bortezomib (Velcade®), lactacystin (AG Scientific, Inc., San Diego, CA), MGl 32 (Biomol International, Plymouth Meeti) ng, PA), PS-519, eponemycin, epoxomycin, acrasinomycin A, dipeptide benzamide, CVT-63417 and vinyl sulfone tripeptide proteasome Examples of such inhibitors include

[0117] In some embodiments, the methods described herein include one or more of the following: It is used in combination with several other cancer treatments. Cancer immunotherapy is a treatment that uses The main premise is to use the immune system to attack the tumor cells that cause the disease. The goal of the study is to stimulate the immune system of the elephant. This can be done through immunization of the subject or through drug therapy, e.g. For example, this can be achieved either through administration of an antibody as a drug, in the former case through the subject's own In the latter case, the subject's immune system recognizes tumor cells as targets for destruction. They are recruited for drug-mediated destruction of tumor cells. These include immunosuppressive and cytokine-based therapies.

[0118] Many therapeutic monoclonal antibodies have been approved by the FDA for use in humans. FDA-approved monoclonal antibodies for cancer immunotherapy Antibodies include CD52, CD33, CD20, ErbB2, vascular endothelial growth factor and epidermal growth factor receptors. Thus, antibodies that target one or more cancer-associated antigens are These and other antibodies are administered in combination with dimeric NKG2D-Fc. Suitable for use in the treatment of cancer. Examples of monoclonal antibodies include, but are not limited to, rituximab (available as Rituxan™). available as Herceptin®; alemtuzumab (cancer); cetuximab (available as Erbitux®); Bevacizumab (available as Avastin®), panitumumab (available as Vectibix®) ), gemtuzumab ozogamicin (available as Mylotarg™), Ibritumomab tiuxetan (available as Zevalin®) and tositumomab (Vexax®) Currently undergoing human clinical trials in the United States for the treatment of cancer. Examples of monoclonal antibodies in development include, but are not limited to, WX-G250 (Lencalex encarex™), ipilimumab (available as MDX-010), zanolimumab (available as HuMax-CD4), Ofatumumab (available as HuMax-CD20) , ch14.18, zalutumumab (available as HuMax-EGFr), oregovomab (B43.13, oval OvalRex™), edrecolomab (IGN-101, Panorex orex™), 131 I-chTNT-I / B (available as Cotara™); Pemtumomab (R-1549, available as Theragyn™), lintuzumab (SGN-3 3), labetuzumab (hMN14, available as CEAcide™), catumaxoma (available as Removab™), CNTO 328 (available as cCLB8), 3F8, 177Lu-J591 , nimotuzumab, SGN-30, ticilimumab (available as CP-675206), daclizumab (Zena Pax™), epratuzumab (hLL2, LymphoCide (trademark) available as 90 Y-Epratuzumab, Galiximab (available as IDEC-114), MD X-060, CT-011, CS-1008, SGN-40, mapatumumab (available as TRM-I), apolizumab ( HuID10, available as Remitogen™) and volociximab (available as M200) Some examples include:

[0119] Cancer immunotherapy also includes cytokine-based therapies. Anti-cancer therapies utilize one or more cytokines to modulate the subject's immune response. Non-limiting examples of cytokines useful in the treatment of cancer include interferon-α (IFN-α). N-α), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and and interleukin-12 (IL-12).

[0120] All references cited throughout this application (including literature references, issued patents, published The entire contents of all pending patent applications, including any co-pending patent applications therefor, are hereby incorporated by reference. Explicitly included. EXAMPLES

[0121] construct hIgG1 This construct contains the hIgG1 portion from the parent pFUSE-hIgG1 vector (Invivogen). do.

[0122] hNKG2Dx2-hIgG1 Two copies of human NKG2D (F78-V216) with an amino acid spacer between them were inserted into the 5' Cloning was performed via restriction-free cloning in the pFUSE-hIgG1 vector (Invivogen). A schematic of this construct is depicted in the left panel of FIG.

[0123] hNKG2D, RefSeq NP_031386.2, amino acids 78-216: FLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGL VHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 1)

[0124] hIgG1-X or hNKG2Dx2-hIgG1-X Using either the hIgG1 vector or the hNKG2Dx2-hIgG1 parent vector described above Various constructs were then cloned into the 3' hIgG1 segment (denoted by "X" above). These constructs are described below.

[0125] hIL15 / hIL15Ra A codon-optimized version of the human IL-15 receptor alpha (hIL15Ra, GenBank AAP6952 8.1, amino acids 31 to 107) was synthesized from a codon-optimized version of IL-15 (IL15, GenBank AAX37 The hIL15Ra and hIL-15 are fused with a 20 amino acid residue (G4S)4 (SEQ ID NO:3) separated by a linker. The amino acid sequence is shown below.

[0126] hIL15Ra, GenBank AAP69528.1, amino acids 31-107: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP( Column number 8)

[0127] IL15, GenBank AAX37025, amino acids 22-135: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNV TESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 9)

[0128] aPD1 Anti-mouse PD1 ScFv was added to the 3' hIgG1 vector or the hNKG2Dx2-hIgG1 vector. It was cloned into one segment.

[0129] aCTLA4 ScFv for anti-mouse CTLA4 was ligated to the 3' hI in the hIgG1 vector or the hNKG2Dx2-hIgG1 vector. It was cloned into the gG1 segment.

[0130] Heterologous expression and purification The indicated fusion constructs were synthesized by co-transfection with calcium phosphate of plasmids encoding the constructs. The fusion construct was produced in 293FT cells by the ELISA method. The supernatant was collected and the fusion construct was The constructs were purified by protein A chromatography. 1 μg of each construct (2- The mixture was reduced with mercaptoethanol and heated at 70°C for 10 min or not, and then diluted with 8-10% The gel was placed on a 100-μL SDS-PAGE gel and run at 100 V for 60-120 min. The proteins were then purified using Coomassie Blue. Figure 2 shows that hNKG2Dx2-hIgG1-hIL15 / Ra expressed as a single fusion protein. It has been shown that the ribozyme can be easily produced and purified by protein A.

[0131] Proliferation assay Human NK cells were isolated from normal donors using RosetteSep (StemCell Technologies). NK cells were isolated from the NK cells by injecting 5 μM carboxyfluorescein succinimidyl ester (CFSE) (I The cells were then labeled with IL-15 constructs (hIgG1-hIL15 / Ra or hNKG2Dx2-20AA-hIgG1-hIL15 / Ra) or various dilutions of IL-15 were incubated in RPMI + 10% FBS for 4 h. The cells were cultured for 1 day, and CFSE dilution was measured using FACSCanto (BD). Incubation with IgG1-IL15 / Ra is similar to incubation with IL-15 Here we provide results showing that it promotes the proliferation of human NK cells.

[0132] Killing assay Human NK cells were isolated from normal donors using RosetteSep (StemCell Technologies). The NK cells were isolated and frozen in BamBanker (Wako). The NK cells were thawed and incubated with RPMI + 10% FBS + 200 IU / mL hIL. The cells were then washed three times with PBS and transfected with CFSE (Invitrogen). The cells were then added to various pre-labeled tumor targets. The cells were centrifuged at 1000 rpm for 1 min. The cells were then co-cultured for 5 hours at 37°C in a humidified CO2 incubator. After 4 hours, 7-amino Actinomycin D (7-AAD) was added and tumor target cell death was analyzed.

[0133] Figure 4 shows that hNKG2Dx2-hIgG1-IL15 / Ra promoted potent killing of multiple cell lines and moderate ligation. Panel A shows that hNKG2Dx2-hIgG1 is superior to hNKG2Dx2-hIgG1 in cell lines with IgG1 expression. The construct did not promote killing of the B16 tumor cell line, which does not express NG2D-L. Both the hNKG2Dx2-hIgG1 and hNKG2Dx2-hIgG1-IL15 / Ra constructs expressed high levels of IgG1. The results of the study equally promoted the killing of the synthetic B16 tumor cell line expressing the NKG2D ligand of IL-1 (Figure 4, Panel B). Various tumors express different levels of NKG2D, as measured by binding of NKG2D fusion proteins. The ligands are expressed on their cell surface (Figure 4, Panel C). hNKG2Dx2-hIgG1 and hNKG2D The x2-hIgG1-IL15 / Ra construct inhibited the expression of NKG2D ligands in tumors that naturally express high levels of NKG2D ligands (K5 62) also promotes the killing of NKG2Dx2-hIgG1-IL15 / Ra, whereas the hNKG2Dx2-hIgG1-IL15 / Ra construct inhibits the expression of NKG2D ligands. This results in superior killing in a stepwise manner that is inversely correlated with expression (Figure 4, panel D).

[0134] IFNγ ELISA Human NK cells were cultured in a medium with no overnight recovery period for NK cells (e.g., RPMI + 10% FBS + 200 IU / mL hIL- 2) Tumor targets were isolated and frozen as described above, except that they were not labeled with CFSE. After 24 hours of co-culture, the plates were spun down and the supernatants were subjected to ELISA for IFNγ. The samples were aspirated for analysis by SA (Becton Dickinson).

[0135] FIG. 5 shows that resting NK cells are activated by the fusion protein to produce IFN-γ. We show that maximal production requires all three components: NKG2D, hIgG1, and IL-15. 97Q is a mutation in hIgG1 that prevents CD16 (expressed on NK cells) from binding to hIgG1. Please note that.

[0136] CD16 and IL-15 activation FIG. 6 shows that preactivated NK cells (e.g., incubated overnight with hIL-2) showed that CD16 binding, but not IL-15, was required for target cell killing. vinegar.

[0137] Human NK cells were isolated, frozen, and added to tumor targets as described above. The cells were not allowed an overnight recovery period. Tumor targets were labeled with CFSE. Optimal activation of and killing by resting NK cells requires CD16 binding and IL-15 activation. This indicates that.

[0138] Improved characteristics of the dimeric NKG2D-Fc construct A protein model showing NKG2Dx2-hIgG1 in complex with the NKG2D ligand MICA is shown in Figure 1. Binding of the NKG2Dx2-hIgG1 and hNKG2Dx1-hIgG1 constructs to MICA*008 was measured by ELI. The results were assayed by SA and flow cytometry. demonstrates that it binds to MICA*008 with improved avidity compared to x1-hIgG1 ELISA data are shown. FIG. 9 shows that hNKG2Dx2-hIgG1 has improved activity compared to hNKG2Dx1-hIgG1. Flow cytometry analysis demonstrated that the antibody binds to cells expressing NKG2D ligands with similar avidity. Represents the tometry data.

[0139] FIG. 10 shows that NKG2D-Fc inhibits NK cell proliferation in ligand-positive (e.g., NKG2D ligand-expressing) targets. In particular, hNKG2Dx2-20AA-hIgG1 was more effective than hNKG2Dx1-20AA-hIgG1 in killing the also mediated significantly increased killing of B16 cells.

[0140] FIG. 11 shows that hNKG2Dx2-hIgG1 suppresses the expression of NKG2D ligand-expressing cells compared to hNKG2Dx1-hIgG1. The results show that IL-16 improves cell death in B16-ULBP2OE and HeyA8 cells.

[0141] Tumors can shed their NKG2D ligands, further compromising the immune response. One potential solution to this problem is the use of soluble NKG2D ligases to restore immune system function. The ability of the soluble MICA to absorb soluble 1 shows that MICA neutralizes NKG2Dx2-hIgG1 better than NKG2Dx1-hIgG1.

[0142] Equivalent Those of ordinary skill in the art will be able to, using no more than routine experimentation, practice the specific embodiments of the invention described herein. Many equivalents to the embodiments will be recognized or may be identified. are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition comprising a recombinant polypeptide, the recombinant polypeptide comprising (a) a first ligand-binding fragment of NKG2D; (b) a second ligand-binding fragment of NKG2D; and (c) a dimerized Fc domain of an immunoglobulin, (i) a CH2 domain; and (ii) CH3 domain Fc domain containing Including, A pharmaceutical composition for use in combination with one or more additional cancer therapies in the treatment of cancers which express an NKG2D ligand.

2. The pharmaceutical composition described in claim 1, wherein the first and second ligand-binding fragments each comprise an extracellular fragment of an NKG2D receptor.

3. The pharmaceutical composition of claim 2, wherein the first and second ligand-binding fragments are the same.

4. The pharmaceutical composition of claim 2, wherein the first and second ligand-binding fragments are different.

5. The pharmaceutical composition described in claim 1, wherein the NKG2D is human NKG2D.

6. The pharmaceutical composition described in claim 2, wherein the NKG2D is human NKG2D.

7. The pharmaceutical composition described in claim 3, wherein the NKG2D is human NKG2D.

8. The pharmaceutical composition described in claim 4, wherein the NKG2D is human NKG2D.

9. The pharmaceutical composition of claim 1, wherein the immunoglobulin is a human immunoglobulin.

10. The pharmaceutical composition of claim 1, wherein the immunoglobulin is IgG1.

11. The pharmaceutical composition described in claim 9, wherein the immunoglobulin is IgG1.

12. The pharmaceutical composition of claim 1, wherein the recombinant polypeptide comprises a linker between the first and second ligand-binding fragments.

13. The pharmaceutical composition described in claim 12, wherein the linker is a flexible peptide linker.

14. The pharmaceutical composition of claim 13, wherein the linker comprises two or more of the following amino acids: glycine, serine, alanine and threonine.

15. The pharmaceutical composition of claim 13, wherein the linker is 25 amino acids or less in length.

16. The pharmaceutical composition of claim 13, wherein the linker is 20 amino acids or less in length.

17. The pharmaceutical composition of claim 13, wherein the linker is 12 amino acids or less in length.

18. The pharmaceutical composition of claim 1, wherein the Fc domain of each polypeptide further comprises a hinge region.

19. The pharmaceutical composition of claim 1, wherein the polypeptide does not contain a cytokine.

20. The pharmaceutical composition described in any one of claims 1 to 19, wherein the cancer expressing the NKG2D ligand is melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer, colorectal cancer, pancreatic cancer, or prostate cancer.

21. A pharmaceutical composition described in any one of claims 1 to 20, wherein the one or more additional cancer treatments include administration of a cytotoxic agent and / or a non-cytotoxic agent.

22. A pharmaceutical composition described in any one of claims 1 to 20, wherein the one or more additional cancer treatments include administration of a radioisotope, a chemotherapeutic agent or a toxin.

23. A pharmaceutical composition described in any one of claims 1 to 20, wherein the one or more additional cancer treatments comprise radiation therapy and / or chemotherapy.

24. A pharmaceutical composition described in any one of claims 1 to 20, wherein the one or more additional cancer treatments comprises immunotherapy.

25. A pharmaceutical composition described in any one of claims 1 to 20, wherein the one or more additional cancer treatments comprises an antibody-based therapy.

26. The pharmaceutical composition of any one of claims 1 to 20, wherein the one or more additional cancer treatments comprises a cytokine-based therapy.

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