ONCOLYTIC HERPES SIMPLEX VIRUSES (HSV) EXPRESSING IMMUNOMODULATORY FUSION PROTEINS
Recombinant oncolytic viruses that produce and secrete novel immunomodulatory fusion proteins are described. The fusion proteins encode a single chain variable fragment antibody (ScFv) that specifically binds PD-1 OR PD-L1 fused via an antibody Fc region to the ectodomain of the TGFβ receptor II (TGFβRII ecto ). The immunomodulatory fusion proteins have dual function: blocking inhibitory pathways mediated by PD-1/PD-L1 and blocking the immune-dampening activity of TGFβ. In addition, dual gene oncolytic herpes simplex viruses (HSVs) are provided that include, in addition to a gene encoding an ScFv-Fc-TGFβRII ecto fusion protein, a gene encoding IL12, a T cell stimulatory factor.
1 . A recombinant oncolytic herpes simplex virus (HSV) comprising a nucleic acid construct encoding a fusion protein comprising an ScFv that specifically binds an immune checkpoint protein, wherein the ScFv is fused to a TGFβRII ectodomain (TGFβRII ecto ).
2 . A recombinant oncolytic HSV according to claim 1 , wherein the ScFv is derived from an anti-PD-1 monoclonal antibody or anti-PD-L1 monoclonal antibody.
3 . A recombinant oncolytic HSV according to claim 2 , wherein the ScFv is derived from an anti-PD-1 monoclonal antibody.
4 . A recombinant oncolytic HSV according to claim 3 , wherein the anti-PD-1 ScFv comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:8 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:9.
5 . A recombinant oncolytic HSV according to claim 4 , wherein the anti-PD-1 ScFv has at least 95% identity to SEQ ID NO:11.
6 . A recombinant oncolytic HSV according to claim 3 , wherein the anti-PD-1 ScFv comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:12 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:13.
7 . A recombinant oncolytic HSV according to claim 6 , wherein the anti-PD-1 ScFv has at least 95% identity to SEQ ID NO:15.
8 . A recombinant oncolytic HSV according to claim 3 , wherein the anti-PD-1 ScFv comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:16 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:17.
9 . A recombinant oncolytic HSV according to claim 8 , wherein the anti-PD-1 ScFv has at least 95% identity to SEQ ID NO:19.
10 . A recombinant oncolytic HSV according to claim 2 , wherein the ScFv is derived from an anti-PD-L1 monoclonal antibody.
11 . A recombinant oncolytic HSV according to claim 10 , wherein the anti-PD-L1 ScFv comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:20 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:21.
12 . A recombinant oncolytic HSV according to claim 11 , wherein the anti-PD-L1 ScFv has at least 95% identity to SEQ ID NO:23.
13 . A recombinant oncolytic HSV according to claim 10 , wherein the anti-PD-L1 ScFv comprises a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:24 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:25.
14 . A recombinant oncolytic HSV according to claim 13 , wherein the anti-PD-L1 ScFv has at least 95% identity to SEQ ID NO:27.
15 . A recombinant oncolytic HSV according to claim 10 , wherein the anti-PD-1 ScFv comprising a heavy chain variable region sequence having at least 95% identity to SEQ ID NO:28 and a light chain variable region sequence having at least 95% identity to SEQ ID NO:29.
16 . A recombinant oncolytic HSV according to claim 15 , wherein the anti-PD-L1 ScFv has at least 95% identity to SEQ ID NO:31.
17 . A recombinant oncolytic HSV according to claim 1 , wherein the ScFv is fused to TGFβRII ecto via an Fc region.
18 . A recombinant oncolytic HSV according to claim 1 , wherein the nucleic acid construct comprises a promoter operable in a mammalian cell operably linked to the fusion protein-encoding sequence.
19 . A recombinant oncolytic HSV according to claim 18 , wherein the promoter is selected from the group consisting of EF1α/HTLV, CMV, and Jet.
20 . A recombinant oncolytic HSV according to any of the previous claims, wherein the nucleic acid construct comprises a sequence encoding a signal peptide 5′ of the sequence encoding the ScFv.
21 . A recombinant oncolytic HSV according to any of the previous claims, wherein TGFβRII ectodomain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:7.
22 . A recombinant oncolytic HSV according to claim 21 , wherein TGFβRII ectodomain comprises SEQ ID NO:7.
23 . A recombinant oncolytic HSV according to claim 17 , wherein the Fc region is an IgG1 Fc region or an IgG4 Fc region.
24 . A recombinant oncolytic HSV according to claim 23 , wherein the Fc region has at least 95% identity to SEQ ID NO:5.
25 . A recombinant oncolytic HSV according to claim 24 , wherein the Fc region comprises SEQ ID NO:5.
26 . A recombinant oncolytic HSV according to claim 23 , wherein the Fc region has at least 95% identity to SEQ ID NO:2.
27 . A recombinant oncolytic HSV according to claim 26 , wherein the Fc region comprises SEQ ID NO:2.
28 . A recombinant oncolytic HSV according to any of claims 1 - 27 , further comprising a gene encoding IL12.
29 . A recombinant oncolytic HSV according to claim 28 , wherein the gene encoding IL12 encodes a polypeptide having at least 90% identity to human IL12 (SEQ ID NO:52).
30 . A recombinant oncolytic HSV according to claim 28 , wherein the gene encoding IL12 encodes a polypeptide having at least 90% identity to murine IL12 (SEQ ID NO:54).
31 . A recombinant oncolytic HSV according to any of claims 28 - 30 , wherein the IL12 gene is operably linked to a second promoter operable in a mammalian cell.
32 . A recombinant oncolytic HSV according to claim 31 , wherein the promoter is selected from the group consisting of EF1α/HTLV, CMV, and Jet.
33 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:11) linked to TGFβRII ecto (SEQ ID NO:7) via an Fc4 region (SEQ ID NO:2).
34 . A recombinant oncolytic HSV according to claim 33 , wherein the fusion protein comprises SEQ ID NO:40.
35 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:15) linked to TGFβRII ecto (SEQ ID NO:7) via an Fc4 region (SEQ ID NO:2).
36 . A recombinant oncolytic HSV according to claim 37 , wherein the fusion protein comprises SEQ ID NO:42.
37 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:19) linked to TGFβRII ecto (SEQ ID NO:7) via the Fc4 region (SEQ ID NO:2).
38 . A recombinant oncolytic HSV according to claim 41 , wherein the fusion protein comprises (SEQ ID NO:44).
39 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:23) linked to TGFβRII ecto (SEQ ID NO:7) via an Fc4 region (SEQ ID NO:2).
40 . A recombinant oncolytic HSV according to claim 33 , wherein the fusion protein comprises SEQ ID NO:46.
41 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:27) linked to TGFβRII ecto (SEQ ID NO:7) via an Fc4 region (SEQ ID NO:2).
42 . A recombinant oncolytic HSV according to claim 37 , wherein the fusion protein comprises SEQ ID NO:48.
43 . A recombinant oncolytic HSV comprising a nucleic acid construct encoding a fusion protein comprising an anti-PD-1 ScFv (SEQ ID NO:31) linked to TGFβRII ecto (SEQ ID NO:7) via the Fc4 region (SEQ ID NO:2).
44 . A recombinant oncolytic HSV according to claim 41 , wherein the fusion protein comprises (SEQ ID NO:50).
45 . A recombinant oncolytic HSV according to any of claims 33 - 44 , further comprising a gene encoding human IL12.
46 . A recombinant oncolytic HSV according to claim 45 , wherein the gene encoding human IL12 encodes the polypeptide of SEQ ID NO:52 or a polypeptide having at least 95% identity thereto.
47 . A recombinant oncolytic HSV according to any of the previous claims, wherein the oncolytic HSV is an HSV-1.
48 . A recombinant oncolytic HSV according to claim 33 , wherein the oncolytic HSV is derived from HSV-1 strain 17, HSV-1 strain F, HSV-1 strain KOS, or HSV-1 strain JS1.
49 . A recombinant oncolytic HSV according to claim 34 , wherein the oncolytic HSV is derived from HSV strain 17.
50 . A recombinant oncolytic HSV according to any of the previous claims, wherein the oncolytic HSV does not encode a functional ICP34.5-encoding gene.
51 . A recombinant oncolytic HSV according to claim 36 , wherein all or a portion of the ICP34.5-encoding gene is deleted.
52 . A recombinant oncolytic HSV according to claim 36 or 37 , wherein the nucleic acid construct encoding the fusion protein and/or the gene encoding IL12 are inserted into the ICP34.5-encoding gene locus.
53 . A recombinant oncolytic HSV for use in a method of treating cancer, wherein the method comprises administering an oncolytic HSV according to any of claims 1 - 38 to a subject having cancer.
54 . A recombinant oncolytic HSV according to claim 53 , for use in a method comprising administering the oncolytic HSV by intravenous, intracavitary, intraperitoneal, intratumoral, or peritumoral delivery.
55 . A recombinant oncolytic HSV according to claim 53 or 54 , wherein the method comprises administering more than one dose of the oncolytic HSV to the patient.
56 . A recombinant oncolytic HSV according to any of claims 53 - 55 , wherein the cancer is a solid tumor.
57 . A recombinant oncolytic HSV according to any of claims 53 - 56 , wherein the subject is a human.
58 . A recombinant oncolytic HSV according to any of claims 53 - 56 , wherein the subject is a dog.
59 . A pharmaceutical composition comprising a recombinant oncolytic HSV according to any of claims 1 - 58 .
60 . A pharmaceutical composition according to claim 59 , wherein the oncolytic HSV is at a concentration of at least 10 6 per ml.
61 . A pharmaceutical composition according to claim 60 , wherein the oncolytic HSV is at a concentration of at least 10 7 per ml.
62 . A method of treating cancer in a subject, comprising administering an oncolytic HSV or pharmaceutical composition according to any of claims 1 - 61 to a subject having cancer.
63 . A method according to claim 62 , wherein the subject is a human.
64 . A method according to claim 62 , wherein the subject is a dog.
65 . A method according to claim 63 or 64 , comprising administering the oncolytic HSV by intravenous, intra-arterial, intracavitary, intratumoral, or peritumoral delivery.
66 . A method according to claim 65 , comprising administering more than one dose of the oncolytic HSV to the subject.
67 . A method according to any of claims 60 - 66 , wherein the cancer is a solid tumor.
68 . A fusion protein comprising a single chain variable fragment (ScFv) that binds an immune checkpoint protein, a TGFβRII ecto ectodomain (TGFβRII ecto ), and an Fc antibody region linking the ScFv to the TGFβRII ecto .
69 . A fusion protein according to claim 68 , wherein the immune checkpoint protein is PD-1 or PD-L1.
70 . A fusion protein according to claim 69 , wherein the immune checkpoint protein is PD-1.
71 . A fusion protein according to claim 70 , wherein the ScFv is derived from a BB9 anti-PD-1 monoclonal antibody, an RG1H10 anti-PD-1 monoclonal antibody, or pembrolizumab.
72 . A fusion protein according to claim 71 , wherein the ScFv comprises a sequence having at least 95% identity to SEQ ID NO: 11, SEQ ID NO:15, or SEQ ID NO:19.
73 . A fusion protein according to claim 69 , wherein the immune checkpoint protein is PD-L1.
74 . A fusion protein according to claim 73 , wherein the ScFv is derived from a Combi5 anti-PD-L1 monoclonal antibody, an H6B1LEM anti-PD-L1 monoclonal antibody, or avelumab.
75 . A fusion protein according to claim 74 , wherein the ScFv comprises a sequence having at least 95% identity to SEQ ID NO:23, SEQ ID NO:27, or SEQ ID NO:31.
76 . A fusion protein according to any of claims 68 - 75 , wherein the TGFβRII ecto comprises an amino acid sequence having at least 95% identity to SEQ ID NO:7.
77 . A fusion protein according to claim 76 , wherein the TGFβRII ecto comprises SEQ ID NO:7.
78 . A fusion protein according to any of claims 68 - 77 , wherein the Fc is an IgG1 Fc or an IgG4 Fc.
79 . A fusion protein according to claim 68 , wherein the Fc is a human Fc.
80 . A fusion protein according to claim 79 , wherein the Fc comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2 or SEQ ID NO:5.
81 . A conditioned media composition comprising a fusion protein according to any of claims 68 - 80 .
82 . A conditioned media composition according to claim 81 , wherein the cell supernatant is virus-free.
83 . A pharmaceutical composition comprising a fusion protein according to any of claims 68 - 80 .
84 . A method of treating cancer, comprising administering a pharmaceutical composition according to claim 83 to a subject having cancer.
85 . A method according to claim 84 , wherein the subject is a human.
86 . A method according to claim 84 , wherein the subject is a dog.
87 . A nucleic acid construct comprising a nucleic acid sequence encoding a fusion protein according to any of claims 68 - 80 .
88 . A nucleic acid construct according to claim 87 , wherein the nucleic acid sequence encoding a fusion protein is operably linked to a promoter.
89 . A nucleic acid construct according to claim 88 , wherein the promoter is a eukaryotic promoter.
90 . A nucleic acid construct according to claim 89 , wherein the promoter is operable in a mammalian cell.