TARGETED COSTIMULATORY POLYPEPTIDES AND METHODS OF USE TO TREAT CANCER
Compositions are provided that are targeted to tumors or tumor-associated neovasculature and enhance the function of tumor-infiltrating T cells. The compositions include fusion proteins that contain a T cell binding domain and a tumor/tumor-associated neovasculature targeting domain. The fusion proteins optionally contain a peptide/polypeptide linker domain and a domain that mediates dimerization or multimerization. The T cell binding domain can be a costimulatory molecule. Methods for using the fusion proteins to enhance an immune response are provided. Therapeutic uses for the disclosed compositions include the induction of tumor immunity.
1 . A fusion protein comprising a first fusion partner comprising a T cell costimulatory polypeptide, or a fragment and/or variant thereof, fused (i) directly to a second fusion partner and, (ii) optionally fused to a linker peptide or polypeptide sequence that is fused to the second fusion partner,
wherein the costimulatory molecule or costimulatory fragment and/or variant thereof increases antigen-specific proliferation of T cells, enhances production of cytokines by T cells, stimulated differentiation or effector function of T cells, or promotes the survival of T cells, and
wherein the second fusion partner comprises a polypeptide that targets the fusion protein to cells of a tumor, tumor vasculature, or tissue involved in activation of an immune response.
2 . The fusion protein of claim 1 , wherein the costimulatory polypeptide comprises a B7 family costimulatory molecule or a fragment and/or variant thereof.
3 . The fusion protein of claim 2 , wherein the costimulatory molecule comprises a soluble fragment of a B7 family costimulatory molecule.
4 . The fusion protein of claim 3 , wherein the costimulatory molecule comprises the extracellular domain of a B7 family costimulatory molecule.
5 . The fusion protein of claim 2 , wherein the costimulatory molecule is selected from the group consisting of B7-DC, B7-1, B7-2, B7-H5, and fragments and/or variants thereof.
6 . The fusion protein of claim 5 , wherein the costimulatory molecule is a variant costimulatory molecule or fragment thereof,
wherein the costimulatory molecule or fragment thereof is a variant of a wild-type costimulatory molecule,
wherein the variant costimulatory molecule or fragment thereof comprises a substitution, deletion or insertion of one or more amino acids.
7 . The fusion protein of claim 5 , wherein the B7-DC polypeptide is murine B7-DC.
8 . The fusion protein of claim 5 , wherein the B7-DC polypeptide is human B7-DC.
9 . The fusion protein of claim 5 , wherein the B7-DC polypeptide is non-human primate B7-DC.
10 . The fusion protein of claim 6 , wherein the substitution, deletion or insertion of one or more amino acids is in the A′, B, C, C′, C″, D, E, F, or G strand of B7-DC, or any combination thereof.
11 . The fusion protein of claim 1 , wherein the second fusion partner comprises a polypeptide that binds to an antigen on a tumor or on tumor-associated neovasculature.
12 . The fusion protein of claim 11 , wherein the second fusion partner comprises a polypeptide that binds to a tumor-specific or a tumor-associated antigen.
13 . The fusion protein of claim 12 , wherein the tumor-specific or tumor-associated antigen is selected from the group consisting of alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.
14 . The fusion protein of claim 11 , wherein the second fusion partner comprises a polypeptide that binds to an antigen that is specific to tumor-associated neovasculature or is more highly expressed in tumor neovasculature relative to normal vasculature.
15 . The fusion protein of claim 14 , wherein the antigen is selected from the group consisting of VEGF/KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin and α 5 β 3 integrin/vitronectin.
16 . The fusion protein of claim 1 , wherein the second fusion partner comprises a chemokine or a chemokine receptor or a soluble fragment thereof.
17 . The fusion protein of claim 16 , wherein the second fusion partner comprises a soluble fragment of a chemokine receptor selected from the group consisting of CXCR2, CXCR4, CCR2 and CCR7, wherein the soluble fragment binds to a chemokine.
18 . The fusion protein of claim 17 , wherein the second fusion partner comprises a chemokine selected from the group consisting of CXC, CC, CX3C and C chemokines or a fragment thereof.
19 . The fusion protein of claim 1 , wherein the linker peptide or polypeptide comprises a flexible peptide or polypeptide, wherein the peptide or polypeptide comprises 2 or more amino acids, and
wherein the peptide or polypeptide comprises an amino acid sequence selected from the group consisting of Gly-Ser, Gly-Ser-Gly-Ser, Ala-Ser, Gly-Gly-Gly-Ser, (Gly 4 -Ser) 3 , (Gly 4 -Ser) 4 , and (Gly 4 -Ser) 4 .
20 . The fusion protein of claim 1 , wherein the linker peptide or polypeptide comprises the hinge region of a human immunoglobulin, and optionally, further comprises an additional region of an immunoglobulin selected from the group consisting of the Fc domain, the C H 1 region or the C L region.
21 . The fusion protein of claim 1 , further comprising a domain that mediates dimerization or multimerization of the fusion protein to form homodimers, heterodimers, homomultimers, or heteromultimers.
22 . The fusion protein of claim 21 , wherein the domain that mediates dimerization or multimerization is selected from the group consisting of one or more cysteines that are capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein, a coiled-coil domain, an acid patch, a zinc finger domain, a calcium hand domain, a C H 1 region, a C L region, a leucine zipper domain, an SH2 (src homology 2) domain, an SH3 (src Homology 3) domain, a PTB (phosphotyrosine binding) domain, a WW domain, a PDZ domain, a 14-3-3 domain, a WD40 domain, an EH domain, a Lim domain, an isoleucine zipper domain, and a dimerization domain of a receptor dimer pair.
23 . The fusion protein of claim 22 , wherein the dimerization or multimerization domain is contained within the first fusion partner, the second fusion partner, or the linker peptide or polypeptide.
24 . The fusion protein of claim 22 , wherein the dimerization or multimerization domain is separate from and not contained within the first fusion partner, the second fusion partner, or the linker peptide or polypeptide.
25 . A dimeric protein comprising a first and a second fusion protein, wherein the first and the second fusion proteins comprise the fusion protein of claim 1 , wherein the first and the second fusion proteins are bound to one another by covalent or noncovalent bonds to form a dimer.
26 . The dimeric protein of claim 25 , wherein the dimer is a homodimer.
27 . The dimeric protein of claim 25 , wherein the dimer is a heterodimer.
28 . A multimeric protein comprising more than two fusion proteins, wherein each of the fusion proteins comprise the fusion protein of claim 1 , wherein the fusion proteins are bound to one another by covalent or noncovalent bonds to form a multimer.
29 . The multimeric protein of claim 28 , wherein the multimer is a homomultimer.
30 . The multimeric protein of claim 29 , wherein the multimer is a heteromultimer.
31 . The dimeric or multimeric protein of claim 25 , wherein the fusion proteins are bound together by disulfide bonds.
32 . The dimeric or multimeric protein of claim 31 wherein the disulfide bonds are formed between cysteines in the linker peptide sequence.
33 . An isolated nucleic acid molecule comprising a nucleic acid sequence that encodes the fusion protein of claim 1 .
34 . A vector comprising the nucleic acid of claim 33 .
35 . The vector of claim 34 , wherein said nucleic acid sequence is operably linked to an expression control sequence.
36 . A host cell comprising the vector of claim 35 .
37 . A pharmaceutical composition for use with an antigen or a vaccine to increase the immunogenicity of the antigen or vaccine comprising:
a) the isolated fusion protein, dimeric protein, or multimeric protein of claim 1 , and
b) a pharmaceutically and immunologically acceptable excipient or carrier.
38 . An immunogenic composition useful for inducing a T cell immune response against a tumor, comprising
(a) a source of antigen to which an immune response is desired;
(b) a fusion protein, dimeric protein, or multimeric protein of claim 1 ,
(c) optionally, a general immunostimulatory agent or adjuvant; and
(d) a pharmaceutically and immunologically acceptable excipient or carrier for (a), (b) and, optionally, (c).
39 . A method for costimulating T cells comprising contacting a T cell with the fusion protein, dimeric protein, or multimeric polypeptide of claim 1 .
40 . The method of claim 39 , wherein the method comprises administering the fusion protein to a mammal.
41 . A method for increasing the activation of tumor-infiltrating T cells in a subject by administering to a mammal in need thereof an effective amount of a fusion protein, dimeric protein, or multimeric protein of claim 1 , or a nucleic acid encoding the same, to activate the mammal's T cells.
42 . A method for increasing the population of tumor-infiltrating T cells in a subject by administering to a mammal in need thereof an effective amount of a fusion protein, dimeric protein, or multimeric protein of claim 1 , or a nucleic acid encoding the same, to activate the mammal's T cells.
43 . A method for stimulating or augmenting an effective anti-tumor T cell response by administering to a mammal in need thereof an effective amount of a fusion protein, dimeric protein, or multimeric protein of claim 1 , or a nucleic acid encoding the same, to activate the mammal's T cells.
44 . A method for potentiating an immune response to an antigen or a vaccine in a mammalian subject, comprising administering to the mammal, in combination with the antigen or vaccine, the fusion protein, dimeric protein, or multimeric protein of claim 1 , or a nucleic acid encoding the same, in an effective amount to activate the subject's T cells.