MODIFIED ORTHOPOXVIRUS VECTORS

FIELD

The invention relates to the field of immunotherapy, e.g., for the treatment of cell proliferation disorders, such as cancers. Particularly, the invention relates to genetically modified orthopoxviruses, as well as methods of making and using the same.

BACKGROUND

The immune system may be stimulated to identify tumor cells and target them for destruction. Immunotherapy employing oncolytic orthopoxviruses is a rapidly evolving area in cancer research. New approaches are needed to engineer and/or enhance tumor-selectivity for oncolytic viruses in order to maximize efficiency and safety. This selectivity is especially important when potentially toxic therapeutic agents or genes are added to the viruses.

Although the use of orthopoxviruses as clinical oncolytic vectors is a promising paradigm for cancer treatment, due to toxicity, such as pox lesions in patients, and immunosuppressive side effects, most current clinical candidates have shown only modest clinical success. There exists a need for methods to engineer orthopoxviruses that exhibit more robust virus replication, cancer cell killing, and spreading from the point of infection. The present invention addresses this need and provides a solution to selectivity and safety limitations by employing a modified vaccinia virus.

SUMMARY

The present disclosure describes the use of orthopoxviruses for the treatment of cancer. In particular, the disclosure is based in part on the surprisingly enhanced oncolytic activity, spread of infection, and safety results engendered when a orthopoxvirus is genetically modified to contain deletions in one or more, or all, of the following genes: C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G, B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. Genetically modified orthopoxviruses, such as vaccinia viruses (e.g., Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, LC16m8, CV-1, modified vaccinia Ankara (MVA), Dairen I, GLV-1h68, IHD-J, L-IVP, LC16m8, LC16mO, Tashkent, Tian Tan, and WAU86/88-1 viruses) that exhibit mutations in one or more, or all, of these genes may exhibit an array of beneficial features, such as improved oncolytic ability, replication in tumors, infectivity, immune evasion, tumor persistence, capacity for incorporation of exogenous DNA sequences, and/or amenability for large scale manufacturing. The present disclosure describes orthopox viruses further genetically modified to contain deletions in the B8R gene. In various embodiments disclosed below, the invention may or may not include a deletion of the B8R gene. In various embodiments, the modified orthopoxvirus expresses at least one of three transgenes: IL-12-TM, FLT3-L and anti-CLTA4 antibody.

In a first aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 genes, each independently selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R. In some embodiments, the deletion includes each of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R genes. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a deletion of the B8R gene.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of B14R, B16R, B17L, B18R, B19R, and B20R. In some embodiments, the deletion includes at least 2, 3, 4, or 5 genes, each independently selected from the group consisting of B14R, B16R, B17L, B18R, B19R, and B20R. In some embodiments, the deletion includes each of B14R, B16R, B17L, B18R, B19R, and B20R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3L. In some embodiments, the deletion includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 genes, each independently selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3L. In some embodiments, the deletion includes each of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3L. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a caspase-9 inhibitor. In some embodiments, the gene that encodes a caspase-9 inhibitor is F1L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a BCL-2 inhibitor. In some embodiments, the gene that encodes a BCL-2 inhibitor is N1L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a dUTPase. In some embodiments, the gene that encodes a dUTPase is F2L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a IFN-alpha/beta-receptor-like secreted glycoprotein. In some embodiments, the gene that encodes a IFN-alpha/beta-receptor-like secreted glycoprotein is B19R.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes an IL-1-beta-inhibitor. In some embodiments, the gene that encodes an IL-1-beta-inhibitor is B16R.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a phospholipase-D. In some embodiments, the gene that encodes a phospholipase-D is K4L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a PKR inhibitor. In some embodiments, the gene that encodes a PKR inhibitor is K3L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a serine protease inhibitor. In some embodiments, the gene that encodes a serine protease inhibitor is K2L.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a TLR signaling inhibitor. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a TLR signaling inhibitor. In some embodiments, the gene that encodes a TLR signaling inhibitor is N2L.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a kelch-like protein. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a kelch-like protein. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 2 genes that each encodes a kelch-like protein. In some embodiments, the genes that encode a kelch-like protein are, independently, selected from the group consisting of F3L and C2L.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 or 2 genes that encodes a monoglyceride lipase. In some embodiments, the genes that encode a monoglyceride lipase are, independently, selected from the group consisting of K5L and K6L.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1, 2 or 3 genes that encodes an NF-κB inhibitor. In some embodiments, the genes that encode an NF-κB inhibitor are, independently selected from the group consisting of K7R, K1L, and M2L.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1, 2, or 3 genes that encodes an Ankyrin repeat protein. In some embodiments, the genes that encode an Ankyrin repeat protein are, independently, selected from the group consisting of B18R, B20R, and M1L.

In another aspect, the invention features a nucleic acid that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1, 2, 3, or 4, gene selected from the group consisting of K ORF A, K ORF B, B ORF E, B ORF F, and B ORF G. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome further includes a B8R deletion.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group of inverted terminal repeat (ITR) genes consisting of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In some embodiments, the deletion includes at least 2, 3, 4, 5, 6, 7, or 8 genes, each independently selected from the group of ITR genes consisting of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In some embodiments, the deletion includes each of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the vaccinia virus is a strain selected from the group consisting of Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, LC16m8, CV-1, modified vaccinia Ankara (MVA), Dairen I, GLV-1h68, IHD-J, L-IVP, LC16m8, LC16mO, Tashkent, Tian Tan, and WAU86/88-1. In some embodiments, the vaccinia virus is a strain selected from the group consisting of Copenhagen, Western Reserve, Tian Tan, Wyeth, and Lister. In some embodiments, the vaccinia virus is a Copenhagen strain vaccinia virus.

In some embodiments, one or more, or all, of the deletions is a deletion of the entire polynucleotide encoding the corresponding gene. In some embodiments, one or more, or all, of the deletions is a deletion of a portion of the polynucleotide encoding the corresponding gene, such that the deletion is sufficient to render the gene nonfunctional, e.g., upon introduction into a host cell.

In some embodiments, the nucleic acid further includes a transgene encoding a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a tumor-associated antigen listed in any one of Tables 3-30 herein. In some embodiments, the tumor-associated antigen is a tumor-associated antigen selected from the group consisting of CD19, CD33, EpCAM, CEA, PSMA, EGFRvIII, CD133, EGFR, CDH19, ENPP3, DLL3, MSLN, ROR1, HER2, HLAA2, EpHA2, EpHA3, MCSP, CSPG4, NG2, RON, FLT3, BCMA, CD20, FAPα, FRα, CA-9, PDGFRα, PDGFRβ, FSP1, S100A4, ADAM12m, RET, MET, FGFR, INSR, and NTRK. In some embodiments, the tumor-associated antigen includes MAGE-A3, or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes NY-ESO-1, or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes one or more human papillomavirus (HPV) proteins, or fragments thereof. In some embodiments, the tumor-associated antigen includes (i) E6 and E7 proteins, or fragments thereof, of HPV16 and (ii) E6 and E7 proteins, or fragments thereof, of HPV18. In some embodiments, the tumor-associated antigen includes brachyury or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes prostatic acid phosphatase, or one or more fragments thereof.

In some embodiments, the nucleic acid further includes a transgene encoding an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of OX40 ligand, ICOS ligand, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD40/CD40L antibody or antigen-binding fragment thereof, anti-Lag3 antibody or antigen-binding fragment thereof, anti-CTLA-4 antibody or antigen-binding fragment thereof, anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD1 antibody or antigen-binding fragment thereof, and anti-Tim-3 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof or an anti-CTLA-4 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

Antibodies or antigen-binding fragments thereof described herein may be full-length antibodies or antibody fragments, such as a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem scFv (taFv). In some embodiments, the antibody or antigen-binding fragment thereof contains two or more CDRs covalently bound to one another, e.g., by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge, or by a linker, such as a linker described herein. In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain polypeptide. In some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

In some embodiments, the nucleic acid further includes a transgene encoding an interleukin. In some embodiments, the interleukin (IL) is selected from the group consisting of IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-7, IL-10, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, IL-21, and IL-23. In some embodiments, the interleukin is selected from the group consisting of IL-12 p35, IL-12 p40, and IL-12 p70. In some embodiments, the interleukin is membrane-bound.

In some embodiments, the nucleic acid further includes a transgene encoding an interferon. In some embodiments, the interferon is selected from the group consisting of IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, IFN-zeta, and IFN-gamma.

In some embodiments, the nucleic acid further includes a transgene encoding a TNF superfamily member protein. In some embodiments, the TNF superfamily member protein is selected from the group consisting of TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.

In some embodiments, the nucleic acid further includes a transgene encoding a cytokine. In some embodiments, the cytokine selected from the group consisting of GM-CSF, FMS-like tyrosine kinase 3 ligand (Flt3 ligand), CD40 ligand, anti-TGF-beta, anti-VEGF-R2, and guanyl adenylate cyclase (cGAS). In some embodiments, the cytokine is Flt3 ligand.

In another aspect, the invention features a recombinant orthopoxvirus vector that has a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 genes, each independently selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R. In some embodiments, the deletion includes each of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, and B20R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In another aspect, the invention features a recombinant orthopoxvirus vector that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of B14R, B16R, B17L, B18R, B19R, and B20R. In some embodiments, the deletion includes at least 2, 3, 4, or 5 genes, each independently selected from the group consisting of B14R, B16R, B17L, B18R, B19R, and B20R. In some embodiments, the deletion includes each of B14R, B16R, B17L, B18R, B19R, and B20R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In another aspect, the invention features a recombinant orthopoxvirus vector that includes a recombinant orthopoxvirus genome, wherein the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3L. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the recombinant orthopoxvirus vector has a deletion of at least 1 gene selected from the group consisting of C2L, C1L, N1L, N2L, M1L, K1L, K2L, K3L, K4L, K7R, and F2L. In some embodiments, the deletion includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 genes, each independently selected from the group consisting of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3L. In some embodiments, the deletion includes each of C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, and F3. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In another aspect, the invention features a recombinant orthopoxvirus vector that has a deletion of at least 1 gene that encodes a caspase-9 inhibitor.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a BCL-2 inhibitor.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a BCL-2 inhibitor. In some embodiments, the gene that encodes a BCL-2 inhibitor is N1L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a dUTPase.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a dUTPase. In some embodiments, the gene that encodes a dUTPase is F2L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a IFN-alpha/beta-receptor-like secreted glycoprotein.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes an IL-1-beta-inhibitor.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a phospholipase-D.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a phospholipase-D. In some embodiments, the gene that encodes a phospholipase-D is K4L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a PKR inhibitor.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a PKR inhibitor. In some embodiments, the gene that encodes a PKR inhibitor is K3L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a serine protease inhibitor.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a TLR signaling inhibitor.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a TLR signaling inhibitor. In some embodiments, the gene that encodes a TLR signaling inhibitor is N2L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a kelch-like protein.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 or 2 genes that encodes a kelch-like protein. In some embodiments, the genes that encode a kelch-like protein are, independently, selected from the group consisting of F3L and C2L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes a monoglyceride lipase.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes a monoglyceride lipase. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 2 genes that each encodes a monoglyceride lipase. In some embodiments, the genes that encode a monoglyceride lipase are, independently, selected from the group consisting of K5L and K6L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes an NF-κB inhibitor.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1, 2, or 3 genes that encodes an NF-κB inhibitor. In some embodiments, the genes that encode an NF-κB inhibitor are, independently, selected from the group consisting of K7R, K1L, and M2L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene that encodes an Ankyrin repeat protein.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene that encodes an Ankyrin repeat protein. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 2 genes that each encodes an Ankyrin repeat protein. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 3 genes that each encodes an Ankyrin repeat protein. In some embodiments, the genes that encode an Ankyrin repeat protein are, independently, selected from the group consisting of B18R, B20R, and M1L.

In another aspect, the invention features a recombinant orthopoxvirus vector has a deletion of at least 1 gene selected from the group consisting of B15R, B17R, and B14R.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of B15R, B17R, and B14R. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 2 genes selected from the group consisting of B15R, B17R, and B14R. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 3 genes selected from the group consisting of B15R, B17R, and B14R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the recombinant orthopoxvirus vector has a deletion of at least 1 gene selected from the group consisting of K ORF A, K ORF B, B ORF E, B ORF F, and B ORF G. In some embodiments, the vector has a deletion of at least 2, 3, or 4 genes selected from the group consisting of K ORF A, K ORF B, B ORF E, B ORF F, and B ORF G. In some embodiments, the deletion includes each of K ORF A, K ORF B, B ORF E, B ORF F, and B ORF G. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the recombinant orthopoxvirus vector has a deletion of at least 1 gene selected from the group of ITR genes consisting of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In some embodiments, the deletion includes at least 2, 3, 4, 5, 6, 7 or 8 genes, each independently selected from the group of ITR genes consisting of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In some embodiments, the deletion includes each of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. In any one of the embodiments, disclosed herein, the recombinant orthopoxvirus genome may further include a B8R deletion.

In some embodiments, the orthopoxvirus is a vaccinia virus.

In some embodiments, the vector further includes a transgene encoding a tumor-associated antigen. In some embodiments, the tumor-associated antigen is a tumor-associated antigen listed in any one of Tables 3-30 herein. In some embodiments, the tumor-associated antigen is a tumor-associated antigen selected from the group consisting of CD19, CD33, EpCAM, CEA, PSMA, EGFRvIII, CD133, EGFR, CDH19, ENPP3, DLL3, MSLN, ROR1, HER2, HLAA2, EpHA2, EpHA3, MC SP, CSPG4, NG2, RON, FLT3, BCMA, CD20, FAPα, FRα, CA-9, PDGFRα, PDGFRβ, FSP1, S100A4, ADAM12m, RET, MET, FGFR, INSR, and NTRK. In some embodiments, the tumor-associated antigen includes MAGE-A3, or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes NY-ESO-1, or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes one or more human papillomavirus (HPV) proteins, or fragments thereof. In some embodiments, the tumor-associated antigen includes (i) E6 and E7 proteins, or fragments thereof, of HPV16 and (ii) E6 and E7 proteins, or fragments thereof, of HPV18. In some embodiments, the tumor-associated antigen includes brachyury or one or more fragments thereof. In some embodiments, the tumor-associated antigen includes prostatic acid phosphatase, or one or more fragments thereof.

In some embodiments, the vector further includes a transgene encoding an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of OX40 ligand, ICOS ligand, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD40/CD40L antibody or antigen-binding fragment thereof, anti-Lag3 antibody or antigen-binding fragment thereof, anti-CTLA-4 antibody or antigen-binding fragment thereof, anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD1 antibody or antigen-binding fragment thereof, and anti-Tim-3 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof or an anti-CTLA-4 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

As described above, antibodies or antigen-binding fragments thereof described herein may be full-length antibodies or antibody fragments, such as a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab′)₂molecule, and a tandem scFv (taFv). In some embodiments, the antibody or antigen-binding fragment thereof contains two or more CDRs covalently bound to one another, e.g., by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge, or by a linker, such as a linker described herein. In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain polypeptide. In some embodiments, the antibody or antigen-binding fragment thereof has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

In some embodiments, the vector further includes a transgene encoding an interleukin. In some embodiments, the interleukin (IL) is selected from the group consisting of IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-7, IL-10, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, IL-21, and IL-23. In some embodiments, the interleukin is selected from the group consisting of IL-12 p35, IL-12 p40, and IL-12 p70. In some embodiments, the interleukin is membrane-bound.

In some embodiments, the vector further includes a transgene encoding an interferon. In some embodiments, the interferon is selected from the group consisting of IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, IFN-zeta, and IFN-gamma.

In some embodiments, the vector further includes a transgene encoding a TNF superfamily member protein. In some embodiments, the TNF superfamily member protein is selected from the group consisting of TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.

In some embodiments, the vector further includes a transgene encoding a cytokine. In some embodiments, the cytokine selected from the group consisting of GM-CSF, FMS-like tyrosine kinase 3 ligand (Flt3 ligand), CD40 ligand, anti-TGF-beta, anti-VEGF-R2, and guanyl adenylate cyclase (cGAS). In some embodiments, the cytokine is Flt3 ligand.

In some embodiments, upon contacting a population of mammalian cells (e.g., human cells, such as human cancer cells) with the nucleic acid or the recombinant orthopoxvirus vector, the cells exhibit increased syncytia formation relative to a population of mammalian cells of the same type contacted with a form of the orthopoxvirus vector that does not include the deletions, as assessed, for instance, by visual inspection using microscopy techniques described herein or known in the art.

In some embodiments, upon contacting a population of mammalian cells (e.g., human cells, such as human cancer cells) with the nucleic acid or the recombinant orthopoxvirus vector, the cells exhibit increased spreading of the orthopoxvirus vector relative to a population of mammalian cells of the same type contacted with a form of the orthopoxvirus vector that does not include the deletions, as assessed, for instance, using plaque-forming assays described herein or known in the art.

In some embodiments, the nucleic acid or the recombinant orthopoxvirus vector exerts an increased cytotoxic effect on a population of mammalian cells (e.g., human cells, such as human cancer cells) relative to that of a form of the orthopoxvirus vector that does not include the deletions, as assessed, for instance, using cell death assays descried herein or known in the art.

In some embodiments, the mammalian cells are from a cell line selected from the group consisting of U2OS, 293, 293T, Vero, HeLa, A549, BHK, BSC40, CHO, OVCAR-8, 786-0, NCI-H23, U251, SF-295, T-47D, SKMEL2, BT-549, SK-MEL-28, MDA-MB-231, SK-OV-3, MCF7, M14, SF-268, CAKI-1, HPAV, OVCAR-4, HCT15, K-562, and HCT-116.

In another aspect, the invention features a packaging cell line that contains the nucleic acid or the recombinant orthopoxvirus vector of any of the aspects or embodiments described herein.

In another aspect, the invention features a method of treating cancer in a mammalian patient by administering a therapeutically effective amount of the nucleic acid or the recombinant orthopoxvirus vector to the patient.

In some embodiments, the mammalian patient is a human patient.

In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer.

In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

In some embodiments, the method further includes administering to the patient an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from a group consisting of OX40 ligand, ICOS ligand, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD40/CD40L antibody or antigen-binding fragment thereof, anti-Lag3 antibody or antigen-binding fragment thereof, anti-CTLA-4 antibody or antigen-binding fragment thereof, anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD1 antibody or antigen-binding fragment thereof, and anti-Tim-3 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof or an anti-CTLA-4 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or antigen-binding fragment thereof.

In some embodiments, the method further includes administering to the patient an interleukin. In some embodiments, the interleukin is selected from a group consisting of IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-7, IL-10, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, IL-21, and IL-23. In some embodiments, the interleukin is selected from a group consisting of IL-12 p35, IL-12 p40, and IL-12 p70. In some embodiments, the interleukin is membrane-bound.

In some embodiments, the method further includes administering to the patient an interferon. In some embodiments, the interferon is selected from a group consisting of IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, IFN-zeta, and IFN-gamma.

In some embodiments, the method further includes administering to the patient a TNF superfamily member protein. In some embodiments, the TNF superfamily member protein is selected from a group consisting of TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.

In some embodiments, the method further includes administering to the patient a cytokine. In some embodiments, the cytokine is selected from a group consisting of GM-CSF, Flt3 ligand, CD40 ligand, anti-TGF-beta, anti-VEGF-R2, and cGAS (guanyl adenylate cyclase).

In another aspect, the invention features a kit containing the nucleic acid or vector of any of the aspects or embodiments described herein and a package insert instructing a user of the kit to express the nucleic acid or vector in a host cell.

In another aspect, the invention features a kit containing the nucleic acid or recombinant orthopoxvirus vector of any of the aspects or embodiments described herein and a package insert instructing a user to administer a therapeutically effective amount of the nucleic acid or recombinant orthopoxvirus vector to a mammalian patient (e.g., a human patient) having cancer, thereby treating the cancer.

Definitions

As used herein, the term “about” refers to a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab′, F(ab′)2, Fab, Fv, rlgG, and scFv fragments. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab′)2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24:316, 1 983; incorporated herein by reference).

The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments can be a Fab, F(ab′)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1 989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.

As used herein, the term “bispecific antibodies” refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens.

As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.

As used herein, the term “chimeric” antibody refers to an antibody having variable sequences derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397; incorporated herein by reference.

As used herein, the term “complementarity determining region” (CDR) refers to a hypervariable region found both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). As is appreciated in the art, the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).

As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated.

As used herein, the terms “conservative mutation,” “conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in table 1 below. From this table it is appreciated that the conservative amino acid families include (i) G, A, V, L and I; (ii) D and E; (iii) C, Sand T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg).

TABLE 1

Representative physicochemical properties

of naturally occurring amino acids

Electrostatic

3
1
Side-
character at

Letter
Letter
chain
physiological
Steric

Amino Acid
Code
Code
Polarity
pH (7.4)
Volume^†

Alanine
Ala
A
nonpolar
neutral
small

Arginine
Arg
R
polar
cationic
large

Asparagine
Asn
N
polar
neutral
intermediate

Aspartic acid
Asp
D
polar
anionic
intermediate

Cysteine
Cys
C
nonpolar
neutral
intermediate

Glutamic acid
Glu
E
polar
anionic
intermediate

Glutamine
Gln
Q
polar
neutral
intermediate

Glycine
Gly
G
nonpolar
neutral
small

Histidine
His
H
polar
Both neutral
large

and cationic

forms in

equilibrium

at pH 7.4

Isoleucine
Ile
I
nonpolar
neutral
large

Leucine
Leu
L
nonpolar
neutral
large

Lysine
Lys
K
polar
cationic
large

Methionine
Met
M
nonpolar
neutral
large

Phenylalanine
Phe
F
nonpolar
neutral
large

Proline
Pro
P
non-polar
neutral
intermediate

Serine
Ser
S
polar
neutral
small

Threonine
Thr
T
polar
neutral
intermediate

Tryptophan
Trp
W
nonpolar
neutral
bulky

Tyrosine
Tyr
Y
polar
neutral
large

Valine
Val
V
nonpolar
neutral
intermediate

^†based on volume in A³: 50-100 is small, 100-150 is intermediate, 150-200 is large, and >200 is bulky

As used herein, the terms “delete,” “deletion,” and the like refer to modifications to a gene or a regulatory element associated therewith or operatively linked thereto (e.g., a transcription factor-binding site, such as a promoter or enhancer element) that remove the gene or otherwise render the gene nonfunctional. Exemplary deletions, as described herein, include the removal of the entirety of a nucleic acid encoding a gene of interest, from the start codon to the stop codon of the target gene. Other examples of deletions as described herein include the removal of a portion of the nucleic acid encoding the target gene (e.g., one or more codons, or a portion thereof, such as a single nucleotide deletion) such that, upon expression of the partially-deleted target gene, the product is nonfunctional or less functional then a wild-type form of the target gene. Exemplary deletions as described herein include the removal of all or a portion of the regulatory element(s) associated with a gene of interest, such as all or a portion of the promoter and/or enhancer nucleic acids that regulate expression of the target gene.

As used herein, the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction so as to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of known chemical protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. using established procedures. Additionally, the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., U.S. Pat. No. 6,964,859; incorporated herein by reference).

As used herein, the term “diabodies” refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure. Accordingly, the term “triabodies” refers to trivalent antibodies comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain. In order to fold into their native structure, peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993; incorporated herein by reference).

As used herein, a “dual variable domain immunoglobulin” (“DVD-lg”) refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent. (Gu et al., Meth. Enzymol., 502:25-41, 2012; incorporated by reference herein).

As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).

As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from.

As used herein, the term “framework region” or “FW region” includes amino acid residues that are adjacent to the CDRs. FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.

As used herein, the term “heterospecific antibodies” refers to monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Traditionally, the recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537, 1 983). Similar procedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos. 6,210,668; 6,193,967; 6,132,992; 6,106,833; 6,060,285; 6,037,453; 6,010,902; 5,989,530; 5,959,084; 5,959,083; 5,932,448; 5,833,985; 5,821,333; 5,807,706; 5,643,759, 5,601,819; 5,582,996, 5,496,549, 4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:21 0 (1986); incorporated herein by reference. Heterospecific antibodies can include Fc mutations that enforce correct chain association in multi-specific antibodies, as described by Klein et al, mAbs 4(6):653-663, 2012; incorporated herein by reference.

As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C_L, C_Hdomains (e.g., C_H1, C_H2, C_H3), hinge, (V_L, V_H)) is substantially non-immunogenic in humans, with only minor sequence changes or variations. A human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single-chain antibody, it can include a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 1998/46645; WO 1998/50433; WO 1998/24893; WO 1998/16654; WO 1996/34096; WO 1996/33735; and WO 1991/10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; incorporated by reference herein.

As used herein, the term “humanized” antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂or other target-binding subdomains of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FR regions may also be those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., Nature 332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370 to Queen et al; EP239400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539; EP592106; and EP519596; incorporated herein by reference.

As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, the term “multi-specific antibodies” refers to antibodies that exhibit affinity for more than one target antigen. Multi-specific antibodies can have structures similar to full immunoglobulin molecules and include Fc regions, for example IgG Fc regions. Such structures can include, but not limited to, IgG-Fv, lgG-(scFv)2, DVD-1g, (scFv)2-(scFv)2-Fc and (scFv)2-Fc-(scFv)2. In case of lgG-(scFv)2, the scFv can be attached to either the N-terminal or the C-terminal end of either the heavy chain or the light chain. Exemplary multi-specific molecules have been reviewed by Kontermann, 2012, mAbs 4(2):182-197, Yazaki et al., 2013, Protein Engineering, Design & Selection 26(3):1 87-1 93, and Grote et al., 2012, in Proetzel & Ebersbach (eds.), Antibody Methods and Protocols, Methods in Molecular Biology vol. 901, chapter 16:247-263; incorporated herein by reference. Exemplary multi-specific molecules that lack Fc regions and into which antibodies or antibody fragments can be incorporated include scFv dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated), are described by Hudson and Souriau, 2003, Nature Medicine 9:129-134; incorporated herein by reference.

As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software, such as BLAST, ALIGN, or Megalign (ONASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may be, for example, at least 30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a 5 position in the candidate sequence is occupied by the same amino acid residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

As used herein, the term “primatized antibody” refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are known in the art. See e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference.

As used herein, the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame.

As used herein, the terms “regulatory element” and the like refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, C A, 1990); incorporated herein by reference.

As used herein, the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as cancer or an infectious disease). Examples of subjects and patients include mammals, such as humans, receiving treatment for diseases or conditions, for example, cell proliferation disorders, such as cancer.

As used herein, the term “scFv” refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain. scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and/or CDR-H3) separated by a linker. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). scFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019, Flo et al., (Gene 77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51:6363, 1991); and Takkinen et al., (Protein Engineering 4:837, 1991). The VL and VH domains of a scFv molecule can be derived from one or more antibody molecules. It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules of the invention can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, in some embodiments, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues). Alternatively or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. scFv fragments are described, for example, in WO 2011/084714; incorporated herein by reference.

As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen may bind to the antigen with a K D of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen may bind to the antigen with a K D of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof may exhibit a K D of greater than 100 nM (e.g., greater than 500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

As used herein, the terms “treat” or “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a cell proliferation disorder, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

As used herein, the term “vector” refers to a nucleic acid vector, e.g., a DNA vector, such as a plasmid, a RNA vector, virus or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026; incorporated herein by reference. Expression vectors of the invention may contain one or more additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a host cell, such as a mammalian cell (e.g., a human cell). Exemplary vectors that can be used for the expression of antibodies and antibody fragments described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Vectors may contain nucleic acids that modulate the rate of translation of a target gene or that improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, e.g., 5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

As used herein, the term “VII” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 1 50,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (VH) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (VL) and a constant domain at the carboxy terminus.

Gene Definitions

As used herein, “C2L” refers to a orthopoxvirus gene, such as a gene that encodes a kelch-like protein. Non-limiting examples of protein sequences encoding the C2L gene are listed in tables 31-35 below. The term “C2L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “C1L” refers to a orthopoxvirus gene. Non-limiting examples of protein sequences encoding the C2L gene are listed in tables 31-35 below. The term “C1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “N1L” refers to a orthopoxvirus gene, such as a gene that encodes a BCL-2 inhibitor. Non-limiting examples of protein sequences encoding the N1L gene are listed in tables 31-35 below. The term “N1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “N2L” refers to a orthopoxvirus gene, such as a gene that encodes a TLR signaling inhibitor. Non-limiting examples of protein sequences encoding the N2L gene are listed in tables 31-35 below. The term “N2L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “M1L” refers to a orthopoxvirus gene, such as a gene that encodes an Ankyrin repeat protein. Non-limiting examples of protein sequences encoding the M1L gene are listed in tables 31-35 below. The term “M1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “M2L” refers to a orthopoxvirus gene, such as a gene that encodes an NF-κB inhibitor. Non-limiting examples of protein sequences encoding the M2L gene are listed in tables 31-35 below. The term “M2L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K1L” refers to a orthopoxvirus gene, such as a gene that encodes an NF-κB inhibitor. Non-limiting examples of protein sequences encoding the K1L gene are listed in tables 31-35 below. The term “K1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K2L” refers to a orthopoxvirus gene, such as a gene that encodes an Ankyrin repeat protein. Non-limiting examples of protein sequences encoding the K2L gene are listed in tables 31-35 below. The term “K2L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K3L” refers to a orthopoxvirus gene, such as a gene that encodes a PKR inhibitor. Non-limiting examples of protein sequences encoding the K3L gene are listed in tables 31-35 below. The term “K3L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K4L” refers to a orthopoxvirus gene, such as a gene that encodes a phospholipase-D. Non-limiting examples of protein sequences encoding the K4L gene are listed in tables 31-35 below. The term “K4L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K5L” refers to a orthopoxvirus gene, such as a gene that encodes a monoglyceride lipase. Non-limiting examples of protein sequences encoding the K5L gene are listed in tables 31-35 below. The term “K5L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K6L” refers to a orthopoxvirus gene, such as a gene that encodes a monoglyceride lipase. Non-limiting examples of protein sequences encoding the K6L gene are listed in tables 31-35 below. The term “K6L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “K7R” refers to a orthopoxvirus gene, such as a gene that encodes an NF-κB inhibitor. Non-limiting examples of protein sequences encoding the K7R gene are listed in tables 31-35 below. The term “K7R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “F1L” refers to a orthopoxvirus gene, such as a gene that encodes a caspase-9 inhibitor. Non-limiting examples of protein sequences encoding the F1L gene are listed in tables 31-35 below. The term “F1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “F2L” refers to a orthopoxvirus gene, such as a gene that encodes a dUTPase. Non-limiting examples of protein sequences encoding the F2L gene are listed in tables 31-35 below. The term “F2L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “F3L” refers to a orthopoxvirus gene, such as a gene that encodes a kelch-like protein. Non-limiting examples of protein sequences encoding the F3L gene are listed in tables 31-35 below. The term “F1L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B14R” refers to a orthopoxvirus gene. Non-limiting examples of protein sequences encoding the B14R gene are listed in tables 36-40 below. The term “B14R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B15R” refers to a orthopoxvirus gene. Non-limiting examples of protein sequences encoding the B15R gene are listed in tables 36-40 below. The term “B15R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B16R” refers to a orthopoxvirus gene, such as a gene that encodes a IL-1-beta inhibitor. Non-limiting examples of protein sequences encoding the B16R gene are listed in tables 31-35 below. The term “B16R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B17L” refers to a orthopoxvirus gene. Non-limiting examples of protein sequences encoding the B17L gene are listed in tables 36-40 below. The term “B17L” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B18R” refers to a orthopoxvirus gene, such as a gene that encodes an Ankyrin repeat protein. Non-limiting examples of protein sequences encoding the B18R gene are listed in tables 36-40 below. The term “B18R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B19R” refers to a orthopoxvirus gene, such as a gene that encodes a IFN-alpha-beta-receptor-like secreted glycoprotein. Non-limiting examples of protein sequences encoding the B19R gene are listed in tables 36-40 below. The term “B19R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B20R” refers to a orthopoxvirus gene, such as a gene that encodes an Ankyrin repeat protein. Non-limiting examples of protein sequences encoding the B20R gene are listed in tables 36-40 below. The term “B20R” may also include fragments or variants of the proteins listed in the tables below, or homologous genes from another orthopoxvirus strain.

As used herein, “B8R” refers to a orthopoxvirus gene, such as a gene that encodes a secreted protein with homology to the gamma interferon (IFN-7). A nonlimiting example of a protein sequence encoded by an exemplary B8R gene in a Copenhagen strain of the vaccinia virus is given in UniProtKB database entry P21004 and is reproduced below:

(SEQ ID NO: 209)

MRYIIILAVLFINSIHAKITSYKFESVNFDSKIEWTGDGLYNISLKNYGI

KTWQTMYTNVPEGTYDISAFPKNDFVSFWVKFEQGDYKVEEYCTGLCVEV

KIGPPTVTLTEYDDHINLYIEHPYATRGSKKIPIYKRGDMCDIYLLYTAN

FTFGDSEEPVTYDIDDYDCTSTGCSIDFATTEKVCVTAQGATEGFLEKIT

PWSSEVCLTPKKNVYTCAIRKEDVPNFKDKMARVIKRKFNKQSQSYLTKF

LGSTSNDVTTFLSMLNLTKYS

The term “B8R” may also include fragments or variants of the proteins listed above, or homologous genes from another orthopoxvirus strain. Variants include without limitation those sequences having 85 percent or greater identity to the sequences disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the phylogenetic analysis of 59 poxvirus strains, including the Orthopoxvirus virus strains.

FIG. 2 shows the abundance of different viral strains after passaging 5 Vaccinia viruses in different tumor types.

FIG. 3 shows the ability to replicate in various different patient tumor cores of Vaccinia wild-type strains.

FIG. 4 shows plaque size measurements of different Vaccinia wild-type strains.

FIG. 5A shows the number of TTAA sites across 1 kb regions in Vaccinia Copenhagen genome.

FIG. 5B shows the frequency of Transposon Insertions across Vaccinia Copenhagen genome. Each dot represents a transposon knockout of a particular gene. The position of the dot on the y-axis is determined by the frequency of the knockout.

FIG. 5C shows Poxvirus gene conservation in 59 viruses. Higher conservation indicates the gene is present in a larger amount of species.

FIG. 6 shows the frequency of various transposon knockouts after passaging in permissive cancer cells.

FIG. 7 shows plaque size measurements of purified transposons.

FIG. 8 shows the genomic structure of a 5p deletion (CopMD5p) and a 3p deletion (CopMD3p). CopMD5p and CopMD3p were crossed to generate CopMD5p3p.

FIG. 9 shows a heatmap showing cancer cell death following infection with either Copenhagen or CopMD5p3p at various doses.

FIG. 10 shows the growth curves of Copenhagen and CopMD5p3p replication in 4 different cancer cell lines.

FIG. 11 shows the ability of Copenhagen and CopMD5p3p to replicate in patient ex vivo samples as shown by tittering.

FIG. 12 shows that the modified CopMD5p3p virus forms different plaques than the parental virus. CopMD5p3p plaques are much clearer in the middle and we can see syncytia (cell fusion).

FIG. 13 shows CopMD5p3p induces syncytia (cell fusion) in 786-O cells.

FIG. 14 shows that CopMD5p3p is able to control tumour growth similarly to Copenhagen wild-type but does not cause weight loss.

FIG. 15 shows that CopMD5p3p does not cause pox lesion formation when compared to two other Vaccinia strains (Copenhagen and Wyeth) harboring the oncolytic knockout of thymidine kinase.

FIG. 16 shows the IVIS bio-distribution of Vaccinia after systemic administration in nude CD-1 mice. Luciferase encoding CopMD5p3p (TK KO) is tumor specific and does not replicate in off target tissues.

FIG. 17 shows the bio-distribution of Vaccinia after systemic administration. CopMD5p3p replicates similarly to other oncolytic Vaccinia in the tumour but replicates less in off target tissues/organs.

FIG. 18 shows the immunogenicity of Vaccinia in Human PBMCs. The ability of CopMD5p3p to induce human innate immune cell activation is stronger than that of wild-type Copenhagen. Data was acquired through flow cytometric analysis.

FIG. 19 shows the immunogenicity of Vaccinia in Mouse Splenocytes. The ability of CopMD5p3p to induce mouse innate immune cell activation is stronger than that of Copenhagen. Data was acquired through flow cytometric analysis.

FIG. 20 shows the immunogenicity of Vaccinia in Human cells. The ability of CopMD5p3p to activate NF-kB immune transcription factor is stronger than that of Copenhagen or VVdd but similar to that of MG-1. Data shown are western blots.

FIG. 21 shows the synergy with immune checkpoint inhibitor Anti-CTLA-4 (100 μg) in an aggressive melanoma model (B16-F10). In vivo efficacy measured by survival in an immune competent murine model treated with Vaccinia and Immune Checkpoint Inhibitors Anti-CTLA4.

FIG. 22 shows the synergy with immune checkpoint inhibitor Anti-CTLA4 (100 μg). In vivo efficacy measured by tumor growth (top row) and survival (bottom row) in an immune competent murine model treated with Vaccinia and Immune Checkpoint Inhibitor Anti-CTLA4. CopMD5p3p (left column) is compared to oncolytic Copenhagen TK KO (right column).

FIG. 23 shows the synergy with immune checkpoint inhibitor Anti-PD1 (100 μg). In vivo efficacy measured by tumor growth (top row) and survival (bottom row) in an immune competent murine model treated with Vaccinia and Immune Checkpoint Inhibitor Anti-PD1. CopMD5p3p (left column) is compared to oncolytic Copenhagen TK KO (right column).

FIG. 24 shows the synergy with immune checkpoint inhibitor Anti-PD1 (25 μg) and Anti-CTLA-4 (25 μg). In vivo efficacy measured by tumor growth (top row) and survival (bottom row) in an immune competent murine model treated with Vaccinia and Immune Checkpoint Inhibitors Anti-PD1 and Anti-CTLA4. CopMD5p3p (left column) is compared to oncolytic Copenhagen TK KO (right column).

FIG. 25 shows a schematic representation of the homologous recombination targeting strategy employed to generate denovo 5p (left) and 3p (right) major deletions in various vaccinia strains.

FIG. 26 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to proliferate in various cell lines.

FIG. 27 shows the cytotoxic effects of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions on various cell lines, as assessed by coomassie blue (upper panel) and an Alamar Blue assay (lower panel). The order of strains listed for each cell line along the x-axis of the chart shown in the lower panel is as follows: from left to right, CopMD5p, CopMD5p3p, CopMD3p, and CopWT.

FIG. 28 shows the distribution of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions upon administration to mice.

FIG. 29 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to activate Natural Killer (NK) cells and stimulate an immune response.

FIG. 30 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to enhance NK cell-mediated degranulation against HT29 cells, a measure of NK cell activity and stimulate an immune response.

FIG. 31 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to prime T-cells to initiate an anti-tumor immune response.

FIG. 32 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to spread to distant locations from the initial point of infection.

FIG. 33 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to form plaques, a measure of viral proliferation.

FIG. 34 shows the ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to form plaques in 786-O cells.

FIG. 35 shows the percentage of genes deleted in CopMD5p3p in various poxvirus genomes.

FIG. 36 shows infection of normal versus cancer cell lines of SKV-B8R+ virus.

FIG. 37 shows SKV-B8R+ does not impair interferon signaling.

FIG. 38 shows SKV (CopMD5p3-B8R−) has similar efficacy in tumour control compared to SKV-B8R+.

FIG. 39 shows SKV engineered to express 2 immunotherapeutic transgenes and an antibody.

FIG. 40 shows SKV expressing murine IL-12 p35 membrane bound has greater efficacy in controlling murine tumours.

FIG. 41 shows major double deletions engineered in various vaccinia strains enhance cancer cell killing in vitro.

FIG. 42 shows the phenotypic characterization of HeLa cells infected with various vaccinia strains.

FIG. 43 shows 5p3p vaccinia strains do not induce weight loss compared to wildtype strains.

FIG. 44 shows 5p3p vaccinia strains do not induce pox lesions compared to wildtype strains.

DETAILED DESCRIPTION

The present invention features genetically modified orthopoxviruses, such as vaccinia viruses (e.g. Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, LC16m8, CV-1, modified vaccinia Ankara (MVA), Dairen I, GLV-1h68, IHD-J, L-IVP, LC16m8, LC16mO, Tashkent, Tian Tan, and WAU86/88-1 viruses), as well as the use of the same for the treatment of various cancers. The invention is based in part on the surprising discovery that orthopoxviruses, such as Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, LC16m8, CV-1, modified vaccinia Ankara (MVA), Dairen I, GLV-1h68, IHD-J, L-IVP, LC16m8, LC16mO, Tashkent, Tian Tan, and WAU86/88-1 viruses, exhibit markedly improved oncolytic activity, replication in tumors, infectivity, immune evasion, tumor persistence, capacity for incorporation of exogenous DNA sequences, and amenability for large scale manufacturing when the viruses are engineered to contain deletions in one or more, or all, of the C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, B20R, K ORF A, K ORF B, B ORF E, B ORF F, B ORF G, B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R genes. In various embodiments of the invention, the modified orthopox viruses contain a deletion of the B8R gene. While inactive in mice, the B8R gene neutralizes antiviral activity of human IFN-γ. In various embodiments, at least one transgene is subsequently inserted into locus of the B8R gene (now deleted) through a homologous recombination targeting strategy. In various embodiments, the modified orthopoxvirus expresses at least one of three transgenes: IL-12-TM, FLT3-L and anti-CLTA4 antibody.

The orthopoxviruses described herein can be administered to a patient, such as a mammalian patient (e.g., a human patient) to treat a variety of cell proliferation disorders, including a wide range of cancers. The sections that follow describe orthopoxviruses and genetic modifications thereto, as well as methods of producing and propagating genetically modified orthopoxviruses and techniques for administering the same to a patient.

Poxvirus

Generally, a poxvirus viral particle is oval or brick-shaped, measuring some 200-400 nm long. The external surface is ridged in parallel rows, sometimes arranged helically. Such particles are extremely complex, containing over 100 distinct proteins. The extracellular forms contain two membranes (EEV: extracellular enveloped virions), whereas intracellular particles only have an inner membrane (IMV: intracellular mature virions). The outer surface is composed of lipid and protein that surrounds the core, which is composed of a tightly compressed nucleoprotein. Antigenically, poxviruses are also very complex, inducing both specific and cross-reacting antibodies. There are at least ten enzymes present in the particle, mostly concerned with nucleic acid metabolism/genome replication.

The genome of the wild-type poxvirus is linear double-stranded DNA of 130-300 Kbp. The ends of the genome have a terminal hairpin loop with several tandem repeat sequences. Several poxvirus genomes have been sequenced, with most of the essential genes being located in the central part of the genome, while non-essential genes are located at the ends. There are about 250 genes in the poxvirus genome. Replication takes place in the cytoplasm, as the virus is sufficiently complex to have acquired all the functions necessary for genome replication. There is some contribution by the cell, but the nature of this contribution is not clear. However, even though poxvirus gene expression and genome replication occur in enucleated cells, maturation is blocked, indicating some role by the cell.

Once into the cell cytoplasm, gene expression is carried out by viral enzymes associated with the core. Expression is divided into 2 phases: early genes: which represent about of 50% genome, and are expressed before genome replication, and late genes, which are expressed after genome replication. The temporal control of expression is provided by the late promoters, which are dependent on DNA replication for activity. Genome replication is believed to involve self-priming, leading to the formation of high molecular weight concatemers, which are subsequently cleaved and repaired to make virus genomes. Viral assembly occurs in the cytoskeleton and probably involves interactions with the cytoskeletal proteins (e.g., actin-binding proteins). Inclusions form in the cytoplasm that mature into virus particles. Cell to cell spread may provide an alternative mechanism for spread of infection. Overall, replication of this large, complex virus is rather quick, taking just 12 hours on average. At least nine different poxviruses cause disease in humans, but variola virus and vaccinia are the best known. Variola strains are divided into variola major (25-30% fatalities) and variola minor (same symptoms but less than 1% death rate). Infection with both viruses occurs naturally by the respiratory route and is systemic, producing a variety of symptoms, but most notably with variola characteristic pustules and scarring of the skin.

Vaccinia Virus as a Species of Orthopoxvirus

Vaccinia virus is a large, complex enveloped virus having a linear double-stranded DNA genome of about 190K by and encoding for approximately 250 genes. Vaccinia is well-known for its role as a vaccine that eradicated smallpox. Post-eradication of smallpox, scientists have been exploring the use of vaccinia as a tool for delivering genes into biological tissues (gene therapy and genetic engineering). Vaccinia virus is unique among DNA viruses as it replicates only in the cytoplasm of the host cell. Therefore, the large genome is required to code for various enzymes and proteins needed for viral DNA replication. During replication, vaccinia produces several infectious forms, which differ in their outer membranes: the intracellular mature virion (IMV), the intracellular enveloped virion (IEV), the cell-associated enveloped virion (CEV), and the extracellular enveloped virion (EEV). IMV is the most abundant infectious form and is thought to be responsible for spread between hosts. On the other hand, the CEV is believed to play a role in cell-to-cell spread and the EEV is thought to be important for long-range dissemination within the host organism.

Vaccinia virus is closely related to the virus that causes cowpox. The precise origin of vaccinia is unknown, but the most common view is that vaccinia virus, cowpox virus, and variola virus (the causative agent for smallpox) were all derived from a common ancestral virus. There is also speculation that vaccinia virus was originally isolated from horses. A vaccinia virus infection is mild and typically asymptomatic in healthy individuals, but it may cause a mild rash and fever, with an extremely low rate of fatality. An immune response generated against a vaccinia virus infection protects that person against a lethal smallpox infection. For this reason, vaccinia virus was used as a live-virus vaccine against smallpox. The vaccinia virus vaccine is safe because it does not contain the smallpox virus, but occasionally certain complications and/or vaccine adverse effects may arise, especially if the vaccine is immunocompromised.

Exemplary strains of the vaccinia virus include, but are not limited to, Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, LC16m8, CV-1, modified vaccinia Ankara (MVA), Dairen I, GLV-1h68, IHD-J, L-IVP, LC16m8, LC16mO, Tashkent, Tian Tan, and WAU86/88-1.

Thymidine Kinase Mutants

Several current clinical studies testing vaccinia virus as an oncolytic virus harbor deletions in the viral Thymidine Kinase (TK) gene. This deletion attenuates the virus, rendering the virus dependent upon the activity of cellular thymidine kinase for DNA replication and, thus, viral propagation. Cellular thymidine kinase is expressed at a low level in most normal tissues and at elevated levels in many cancer cells. Through metabolic targeting, TK-viruses can grow in cells that have a high metabolic rate (e.g., healthy cells or tumor cells) and will not grow well in cells that have low levels of thymidine kinase. Since there exist quiescent tumour cells (e.g., cancer stem cells), TK-viruses are likely compromised in their ability to kill this population of cancer cells just as chemotherapy is largely ineffective. The modified viral vectors described in this disclosure retains virus synthetic machinery (including TK) and may propagate in quiescent cancer cells. The viral modifications of this disclosure may allow the virus to be highly selective without deleting TK or other DNA metabolizing enzymes (e.g., ribonucleotide reductase) and could be more effective in tumors with a low metabolic rate.

Virus Propagation

The present invention features poxviruses, including those constructed with one or more gene deletions compared to wild-type, such that the virus exhibits desirable properties for use against cancer cells, while being less toxic or non-toxic to non-cancer cells. This section summarizes various protocols, by way of example, for producing recombinant poxviruses described herein, such as methods for generating mutated viruses through the use of recombinant DNA technology.

For example, to generate mutations in the poxvirus genome, native and modified polypeptides may be encoded by a nucleic acid molecule comprised in a vector. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., (1989) and Ausubel et al., 1994, both incorporated herein by reference. In addition to encoding a modified polypeptide such as modified gelonin, a vector may encode non-modified polypeptide sequences such as a tag or targeting molecule.

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses (which does not qualify as a vector if it expresses no exogenous polypeptides). A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a modified protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. Host cells may be derived from prokaryotes or eukaryotes, including yeast cells, insect cells, and mammalian cells, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5α, JM109, and KCB, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla, Calif.). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris. Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

Genetic Modifications to the Orthopoxvirus Genome
Methods of Genetic Modification.

Methods for the insertion or deletion of nucleic acids from a target genome include those described herein and known in the art. One such method that can be used for incorporating polynucleotides encoding target genes into a target genome involves the use of transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5′ and 3′ excision sites. Once a transposon has been delivered to a target nucleic acid (e.g., in a host cell), expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In certain cases, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the target genome by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the target genome completes the incorporation process. In certain cases, the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the mammalian cell genome. Transposon systems include the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US2005/0112764), the disclosures of each of which are incorporated herein by reference.

Additional methods for nucleic acid delivery to effect expression of compositions of the present invention are believed to include virtually any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

CopMD5p, CopMD3p, and CopMD5p3p Deletions.

In various embodiments, various genes are deleted to enhance the oncolytic activity of the orthopoxvirus. Most of the deletions described herein are either involved in blocking a host response to viral infection or otherwise have an unknown function. In various embodiments, at least one of the genes depicted in Table 2 are deleted from the recombinant orthopoxvirus genome. In various embodiments, at least 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 or 31 of the genes depicted in Table 2 are deleted from the recombinant orthopox genome. In various embodiments, all of the genes depicted in Table 2 are deleted from the recombinant orthopoxvirus genome. Three exemplary embodiments of the present invention, CopMD5p, CopMD3p and CopMD5p3p, are described herein. Depicted in Table 2 below are clusters of deleted genes and their function in CopMD5p, CopMD3p, and CopMD5p3p virus. In various embodiments, where two copies of an ITR exist, only the right ITR of the genome is deleted and the left ITR remains intact. Deletions are confirmed by whole genome sequencing.

TABLE 2

Deleted genes in Orthopoxviruses

Name
Category
Function
Virus Deletions

C2L
Host interaction
Inhibits NFkB
CopMD5p
CopMD5p3p

C1L
Unknown
Unknown

N1L
Host interaction
Inhibits NFkB and Apoptosis

N2L
Host interaction
Inhibits IRF3

M1L
Unknown
Unknown

M2L
Host interaction
Inhibits NFkB and Apoptosis

K1L
Host interaction
Inhibits PKR and NF-kB

K2L
Host interaction
Prevents cell fusion

K3L
Host interaction
Inhibits PKR

K4L
DNA replication
DNA modifying nuclease

K5L
Pseudogene
Pseudogene

K6L
Pseudogene
Pseudogene

K7R
Host interaction
Inhibits NFkB and IRF3

F1L
Host interaction
Inhibits Apoptosis

F2L
DNA replication
Deoxyuridine triphosphatase

F3L
Host interaction
Virulence factor

B14R
Pseudogene
Pseudogene
CopMD3p

B15R
Unknown
Unknown

B16R
Host interaction
IL-1-beta-inhibitor

B17L
Unknown
Unknown

B18R
Unknown
Ankyrin-like

B19R
Host interaction
Secreted IFNa sequestor

B20R
Unknown
Ankyrin-like

B21R-ITR*
Unknown
Unknown

B22R-ITR*
Unknown
Unknown

B23R-ITR*
Unknown
Unknown

B24R-ITR*
Unknown
Unknown

B25R-ITR*
Unknown
Unknown

B26R-ITR*
Unknown
Unknown

B27R-ITR*
Unknown
Unknown

B28R-ITR*
Pseudogene
TNF-a receptor

B29R-ITR*
Host interaction
Secreted CC-chemokine sequestor

B8R Gene Deletions.

In various embodiments, the orthopox viruses are further genetically modified to contain deletions in the B8R gene. The vaccinia virus B8R gene encodes a secreted protein with homology to gamma interferon receptor (IFN-γ). In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of gamma interferon including human and rat gamma interferon; it does not, however, bind significantly to murine IFN-γ. Deleting the B8R gene prevents the impairment of IFN-γ in humans. Deletion of the B8R gene results in enhanced safety without a concomitant reduction in immunogenicity.

Transgene Insertions

In various embodiments, additional transgenes may be inserted into the vector. In various embodiments, one, two or three transgenes are inserted into the locus of the deleted B8R gene. In some strains, in addition to the transgene(s) present at the site of the B8R deletion, the strain also has, at least one transgene is inserted into an additional locus on the orthopox virus that is not the locus of the deleted B8R gene. In various embodiments, at least one transgene is inserted into boundaries of the 5p deletions, at least one transgene is inserted into the boundaries of the 3p deletions or both. In various, embodiments at least three, four, five or more transgenes are inserted into the modified orthopox virus genome.

In various embodiments, the recombinant orthopoxvirus vector can include at least one transgene encoding an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to OX40 ligand, ICOS ligand, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD40/CD40L antibody or antigen-binding fragment thereof, anti-Lag3 antibody or antigen-binding fragment thereof, anti-CTLA-4 antibody or antigen-binding fragment thereof, anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD1 antibody or antigen-binding fragment thereof, and anti-Tim-3 antibody or antigen-binding fragment thereof.

In various embodiments, the recombinant orthopoxvirus vector can include at least one transgene encoding at least one interleukin protein. Exemplary interleukin proteins for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-7, IL-10, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, IL-21, and IL-23.

In various embodiments, recombinant orthopoxvirus vector can include a transgene encoding an interferon. Exemplary interferons for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, IFN-zeta, and IFN-gamma.

In various embodiments, the recombinant orthopoxvirus vector can include a transgene encoding a TNF superfamily member protein. Exemplary TNF superfamily member proteins for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.

In various embodiments, the recombinant orthopoxvirus vector can include a transgene encoding a cytokine. Exemplary cytokines for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to GM-CSF, Flt3 ligand, CD40 ligand, anti-TGF-beta, anti-VEGF-R2, and cGAS (guanyl adenylate cyclase).

In various embodiments, the recombinant orthopoxvirus vector can include a transgene encoding a tumor-associated antigen. Exemplary tumor-associated antigens for expression by the orthopoxvirus of the compositions and methods of the invention include but are not limited to CD19, CD33, EpCAM, CEA, PSMA, EGFRvIII, CD133, EGFR, CDH19, ENPP3, DLL3, MSLN, ROR1, HER2, HLAA2, EpHA2, EpHA3, MCSP, CSPG4, NG2, RON, FLT3, BCMA, CD20, FAPα, FRα, CA-9, PDGFRα, PDGFRβ, FSP1, S100A4, ADAM12m, RET, MET, FGFR, INSR, NTRK, MAGE-A3, NY-ESO-1, one or more human papillomavirus (HPV) proteins, E6 and E7 proteins of HPV16, E6 and E7 proteins of HPV18, brachyury, or prostatic acid phosphatase, or one or more fragments thereof. Additional examples of tumor-associated antigens for use in conjunction with the compositions and methods described herein include, but are not limited to, those listed in tables 3-30.

Methods of Treatment
Pharmaceutical Composition, Administration, and Doses

Therapeutic compositions containing recombinant orthopoxvirus vectors of the invention can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

To induce oncolysis, kill cells, inhibit growth, inhibit metastases, decrease tumor size and otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one may contact a tumor with the modified orthopoxvirus, e.g., by administration of the orthopoxvirus to a patient having cancer by way of, for instance, one or more of the routes of administration described herein. The route of administration may vary with the location and nature of the cancer, and may include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulation.

The term “intravascular” is understood to refer to delivery into the vasculature of a patient, meaning into, within, or in a vessel or vessels of the patient. In certain embodiments, the administration is into a vessel considered to be a vein (intravenous), while in others administration is into a vessel considered to be an artery. Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein. Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.

Intratumoral injection, or injection directly into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. The viral particles may advantageously be contacted by administering multiple injections to the tumor, spaced, for example, at approximately 1 cm intervals. In the case of surgical intervention, the present invention may be used preoperatively, such as to render an inoperable tumor subject to resection. Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature. Such continuous perfusion may take place, for example, for a period of from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, or about 12-24 hours following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion may be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.

Treatment regimens may vary, and often depend on tumor type, tumor location, disease progression, and health and age of the patient. Certain types of tumor will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations. In certain embodiments, the tumor being treated may not, at least initially, be resectable. Treatments with the therapeutic agent of the disclosure may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection will serve to eliminate microscopic residual disease at the tumor site.

The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) for a viral construct. Unit doses may range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², to 10¹³pfu and higher. Additionally or alternatively, depending on the kind of virus and the titer attainable, one may deliver 1 to 100, 10 to 50, 100-1000, or up to about or at least about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, or 1×10¹⁵or higher infectious viral particles (vp), including all values and ranges there between, to the tumor or tumor site.

Another method of delivery of the recombinant orthopoxvirus genome disclosed herein to cancer or tumor cells may be via intratumoral injection. However, the pharmaceutical compositions disclosed herein may alternatively be administered parenterally, intravenously, intradermally, intramuscularly, transdermally or even intraperitoneally as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety). Injection of nucleic acid constructs may be delivered by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection. An exemplary needleless injection system that may be used for the administration of recombinant orthopoxviruses described herein is exemplified in U.S. Pat. No. 5,846,233. This system features a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery. Another exemplary syringe system is one that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Pat. No. 5,846,225).

Mixtures of the viral particles or nucleic acids described herein may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form may be sterile and may be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.

As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically acceptable” or “pharmacologically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

Cancer

The recombinant orthopoxvirus disclosed herein can be administered to a mammalian subject, such as a human, suffering from a cell proliferation disorder, such as cancer, e.g., to kill cancer cells directly by oncolysis and/or to enhance the effectiveness of the adaptive immune response against the target cancer cells. In some embodiments, the cell proliferation disorder is a cancer, such as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, or throat cancer. In particular cases, the cell proliferation disorder may be a cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T-cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sezary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

A physician having ordinary skill in the art can readily determine an effective amount of the recombinant orthopoxvirus vector for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician may start prescribing doses of recombinant orthopoxvirus vector at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a dose of recombinant orthopoxvirus vector and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in the volume of one or more tumors). In general, a suitable daily dose of a recombinant orthopoxvirus vector of the invention will be an amount of the recombinant orthopoxvirus vector which is the lowest dose effective to produce a therapeutic effect. A daily dose of a therapeutic composition of the recombinant orthopoxvirus vector of the invention may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for the recombinant orthopoxvirus vector of the invention to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.

Recombinant orthopoxvirus vectors of the invention can be monitored for their ability to attenuate the progression of a cell proliferation disease, such as cancer, by any of a variety of methods known in the art. For instance, a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with recombinant orthopoxvirus vector of the invention by analyzing the volume of one or more tumors in the patient. Alternatively, a physician may monitor the responsiveness of a subject (e.g., a human) t to treatment with recombinant orthopoxvirus vector of the invention by analyzing the T-reg cell population in the lymph of a particular subject. For instance, a physician may withdraw a sample from a mammalian subject (e.g., a human) and determine the quantity or density of cancer cells using established procedures, such as fluorescence activated cell sorting. A finding that the quantity of cancer cells in the sample has decreased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more) relative to the quantity of cancer cells in a sample obtained from the subject prior to administration of the recombinant orthopoxvirus may be an indication that the orthopoxvirus administration is effectively treating the cancer.

Combination Therapy

In various embodiments, the recombinant orthopoxvirus may be co-administered with other cancer therapeutics. Furthermore, in various embodiments, the recombinant orthopoxviruses described herein are administered in conjunction with other cancer treatment therapies, e.g., radiotherapy, chemotherapy, surgery, and/or immunotherapy. In some aspects of this invention, the recombinant orthopoxvirus described herein are administered in conjunction with checkpoint inhibitors. In various embodiments, the recombinant orthopoxvirus may be administered in conjunction with treatment with another immunoncology product. The recombinant orthopoxviruses of the present invention and other therapies or therapeutic agents can be administered simultaneously or sequentially by the same or different routes of administration. The determination of the identity and amount of therapeutic agent(s) for use in the methods of the present invention can be readily made by ordinarily skilled medical practitioners using standard techniques known in the art.

The recombinant orthopoxvirus vectors described herein may be administered with one or more additional agents, such as an immune checkpoint inhibitor. For instance, the recombinant orthopoxvirus vector can be administered simultaneously with, admixed with, or administered separately from an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors for use in conjunction with the compositions and methods of the invention include but are not limited to OX40 ligand, ICOS ligand, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD40/CD40L antibody or antigen-binding fragment thereof, anti-Lag3 antibody or antigen-binding fragment thereof, anti-CTLA-4 antibody or antigen-binding fragment thereof, anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD1 antibody or antigen-binding fragment thereof, and anti-Tim-3 antibody or antigen-binding fragment thereof.

Additionally or alternatively, a vector of the invention can be administered simultaneously with, admixed with, or administered separately from an interleukin (IL). For instance, the recombinant orthopoxvirus vector can be administered simultaneously with, admixed with, or administered separately from an interleukin. Exemplary interleukins for use in conjunction with the compositions and methods of the invention include but are not limited to IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-7, IL-10, IL-12 p35, IL-12 p40, IL-12 p70, IL-15, IL-18, IL-21, and IL-23.

Additionally or alternatively, a vector of the invention can be administered simultaneously with, admixed with, or administered separately from an interferon. For instance, the recombinant orthopoxvirus vector can be administered simultaneously with, admixed with, or administered separately from an interferon. Exemplary interferons for use in conjunction with the compositions and methods of the invention include but are not limited to IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, IFN-zeta, and IFN-gamma.

Additionally or alternatively, a vector of the invention can be administered simultaneously with, admixed with, or administered separately from a TNF superfamily member protein. For instance, the recombinant orthopoxvirus vector can be administered simultaneously with, admixed with, or administered separately from a TNF superfamily member protein. Exemplary TNF superfamily member proteins for use in conjunction with the compositions and methods of the invention include but are not limited to TRAIL, Fas ligand, LIGHT (TNFSF-14), TNF-alpha, and 4-1BB ligand.

Additionally or alternatively, a vector of the invention can be administered simultaneously with, admixed with, or administered separately from a cytokine. For instance, the recombinant orthopoxvirus vector can be administered simultaneously with, admixed with, or administered separately from a cytokine. Exemplary cytokines for use in conjunction with the compositions and methods of the invention includes but are not limited to GM-CSF, Flt3 ligand, CD40 ligand, anti-TGF-beta, anti-VEGF-R2, and cGAS (guanyl adenylate cyclase).

TABLE 3

Ovarian cancer

Tumor-associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
Kallikrein 4
FLGYLILGV;
Wilkinson et al. Cancer Immunol.

SVSESDTIRSISIAS;
Immunother. 61(2): 169-79 (2012).

LLANGRMPTVLQCVN;
Hural et al. J. Immunol. 169(1): 557-

and
65 (2002).

RMPTVLQCVNVSVVS

2
PBF
CTACRWKKACQR
Tsukahara et al. Cancer Res.

64(15): 5442-8 (2004).

3
PRAME
VLDGLDVLL;
Kessler et al. J. Exp. Med.

SLYSFPEPEA;
193(1): 73-88 (2001).

ALYVDSLFFL;
Ikeda et al. Immunity 6(2): 199-208

SLLQHLIGL;
(1997).

and

LYVDSLFFL

4
WT1
TSEKRPFMCAY;
Asemissen et al. Clin. Cancer Res.

CMTWNQMNL;
12(24): 7476-82 (2006)

LSHLQMHSRKH;
Ohminami et al. Blood. 95(1): 286-

KRYFKLSHLQMHSRKH;
93 (2000).

and
Guo et al. Blood. 106(4): 1415-8

KRYFKLSHLQMHSRKH
2005).

Lin et al. J. Immunother. 36(3): 159-

70 (2013).

Fujiki et al. J. Immunother.

30(3): 282-93 (2007).

5
HSDL1
CYMEAVAL
Wick et al. Clin. Cancer Res.

20(5): 1125-34 (2014).

6
Mesothelin
SLLFLLFSL
Hassan et al. Appl.

VLPLTVAEV
Immunohistochem. Mol. Morphol.

ALQGGGPPY
13(3): 243-7 (2005).

LYPKARLAF
Thomas et al J Exp Med. 2004 Aug

AFLPWHRLF
2; 200(3): 297-306.

7
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAE
Knights et al. Cancer Immunol

L-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jager et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIR
(2002).

L-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAA
Mandic et al. J Immunol.

E-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIR
101(25): 9363-8 (2004).

LT
Ayyoub et al. Clin Cancer Res.

VLLKEFTVSG
16(18): 4607-15 (2010).

AADHRQLQLSISSCLQQL
Slager et al. J Immunol.

LKEFTVSGNILTIRL
172(8): 5095-102 (2004).

PGVLLKEFTVSGNILTIR
Mizote et al. Vaccine. 28(32): 5338-

L-TAADHR
46 (2010).

LLEFYLAMPFATPMEAE
Jager et al. J Exp Med. 191(4): 625-

L-ARRSLAQ
30 (2000).

KEFTVSGNILT
Zarour et al. Cancer Res.

LLEFYLAMPFATPM
60(17): 4946-52 (2000).

AGATGGRGPRGAGA
Zeng et at. J Immunol. 165(2): 1153-

9(2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

8
CEA
TYYRPGVNLSLSC
Galanis et al. Cancer Res. 70(3): 875-

EIIYPNASLLIQN
82 (2010).

YACFVSNLATGRNNS
Bast et al. Am. J. Obstet. Gynecol.

LWWVNNQSLPVSP
149(5): 553-9 (1984).

LWWVNNQSLPVSP
Crosti et al. J Immunol.

LWWVNNQSLPVSP
176(8): 5093-9 (2006).

EIIYPNASLLIQN
Kobayashi et al. Clin Cancer Res.

NSIVKSITVSASG
8(10): 3219-25 (2002).

KTWGQYWQV
Campi et al. Cancer Res.

(A)MLGTHTMEV
63(23): 8481-6 (2003).

ITDQVPFSV
Bakker et al. Int J Cancer. 62(1): 97-

YLEPGPVTA
102 (1995).

LLDGTATLRL
Tsai et al. J Immunol. 158(4): 1796-

VLYRYGSFSV
802 (1997).

SLADTNSLAV
Kawakami et al. J Immunol.

RLMKQDFSV
154(8): 3961-8 (1995).

RLPRIFCSC
Cox et al. Science. 264(5159): 716-9

LIYRRRLMK
(1994).

ALLAVGATK
Kawakami et al. J Immunol.

IALNFPGSQK
154(8): 3961-8 (1995).

RSYVPLAHR
Kawakami et al. J Immunol.

161(12): 6985-92 (1998).

Skipper et al. J Immunol.

157(11): 5027-33 (1996).

Michaux et al. J Immunol.

192(4): 1962-71 (2014).

9
p53
VVPCEPPEV
Hung et al. Immunol. Rev. 222: 43-

69 (2008).

10
Her2/Neu
HLYQGCQVV
Nakatsuka et al. Mod. Pathol.

YLVPQQGFFC
19(6): 804-814 (2006).

PLQPEQLQV
Pils et al. Br. J Cancer 96(3): 485-91

TLEEITGYL
(2007).

ALIHHNTHL
Scardino et al. Eur J Immunol.

PLTSIISAV
31(11): 3261-70 (2001).

VLRENTSPK
Scardino et al. J Immunol.

TYLPTNASL
168(11): 5900-6 (2002).

Kawashima et al. Cancer Res.

59(2): 431-5 (1999).

Okugawa et al. Eur J Immunol.

30(11): 3338-46 (2000).

11
EpCAM
RYQLDPKFI
Spizzo et al. Gynecol. Oncol.

103(2): 483-8 (2006).

Tajima et al. Tissue Antigens.

64(6): 650-9 (2004).

12
CA125
ILFTINFTI
Bast et al. Cancer 116(12): 2850-

VLFTINFTI
2853 (2010).

TLNFTITNL

VLQGLLKPL

VLQGLLRPV

RLDPKSPGV

QLYWELSKL

KLTRGIVEL

QLTNGITEL

QLTHNITEL

TLDRNSLYV

13
Folate
FLLSLALML
Bagnoli et al. Gynecol. Oncol.

receptor α
NLGPWIQQV
88: S140-4 (2003).

Pampeno et al. (2016) High-ranking

In Silico epitopes [determined by 3

algorithms: BISMAS, IEDB,

RANKPEP] unpublished

14
Sperm
ILDSSEEDK
Chiriva-Inernati et al. J.

protein 17

Immunother. 31(8): 693-703 (2008).

15
TADG-12
YLPKSWTIQV
Bellone et al. Cancer 115(4): 800-11

WIHEQMERDLKT
(2009).

Underwood et al. BBA Mol. Basis of

Disease. 1502(3): 337-350 (2000).

16
MUC-16
ILFTINFTI
Chekmasova et al. Clin. Cancer Res.

VLFTINFTI
16(14): 3594-606 (2010).

TLNFTITNL

VLQGLLKPL

VLQGLLRPV

RLDPKSPGV

QLYWELSKL

KLTRGIVEL

QLTNGITEL

QLTHNITEL

TLDRNSLYV

17
L1CAM
LLANAYIYV
Hong et al. J. Immunother. 37(2): 93-

YLLCKAFGA
104 (2014).

KLSPYVHYT
Pampeno et al. (2016) High-ranking

In Silico epitopes [determined by 3

algorithms: BISMAS, IEDB,

RANKPEP] unpublished

18
Mannan-MUC-1
PDTRPAPGSTAPPAHGV
Loveland et al. Clin. Cancer Res.

TSA
12(3 Pt 1): 869-77 (2006).

STAPPVHNV
Godelaine et al. Cancer Immunol

LLLLTVLTV
Immunother. 56(6): 753-9 (2007).

PGSTAPPAHGVT
Ma et al. Int J Cancer. 129(10): 2427-

34 (2011).

Wen et al. Cancer Sci. 102(8): 1455-

61 (2011).

Jerome et al. J Immunol.

151(3): 1654-62 (1993).

Brossart et al. Blood. 93(12): 4309-

17 (1999).

Hiltbold et al. Cancer Res.

58(22): 5066-70 (1998).

19
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

20
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

21
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(19

Pt 1): 6047-57 (2004).

22
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAA
Sun et al. Cancer Immunol

EVP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

23
MAGE-A4
EVDPASNTY
Kobayashi et al. Tissue Antigens.

GVYDGREHTV
62(5): 426-32 (2003).

NYKRCFPVI
Duffour et al. Eur J Immunol.

SESLICMIF
29(10): 3329-37 (1999).

Miyahara et al. Clin Cancer Res.

11(15): 5581-9 (2005).

Ottaviani et al. Cancer Immunol

Immunother. 55(7): 867-72 (2006)

Zang et al. Tissue Antigens.

60(5): 365-71 (2002).

24
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

25
SSX-4
INKTSGPKRGKHAWTHR
Ayyoub et al. Clin Immunol.

LRE
114(1): 70-8 (2005).

YFSKKEWEKMKSSEKIV
Valmori et al. Clin Cancer Res.

YVY
12(2): 398-404 (2006).

MKLNYEVMTKLGFKVT

LPPF

KHAWTHRLRERKQLVV

YEEI

LGFKVTLPPFMRSKRAA

DFH

KSSEKIVYVYMKLNYEV

MTK

KHAWTHRLRERKQLVV

YEEI

26
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-

LSRLSNRLL
17 (2008).

27
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-

17 (2008).

TABLE 4

Breast cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
ENAH(hMena)
TMNGSKSPV
Di Modugno et al. Int. J. Cancer.

109(6): 909-18 (2004).

2
mammaglobin-A
PLLENVISK
Jaramillo et al. Int. J. Cancer.

102(5): 499-506 (2002).

3
NY-BR-1
SLSKILDTV
Wang et al. Cancer Res. 66(13): 6826-

33 (2006).

4
EpCAM
RYQLDPKFI
Gastl et al. Lancet 356(9246): 1981-2

(2000).

Tajima, 2004

5
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer. 132(2): 345-

APRGPHGGAASGL
54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer. 132(2): 345-

SLLMWITQCFLPVF
54 (2013).

LLEFYLAMPFATPMEAE
Knights et al. Cancer Immunol

L-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLICEFTVSGNILTIR
(2002).

L-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAA
Mandic et al. J Immunol. 174(3): 1751-

E-VPR
9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIR
101(25): 9363-8 (2004).

LT
Ayyoub et al. Clin Cancer Res.

VLLKEFTVSG
16(18): 4607-15 (2010).

AADHRQLQLSISSCLQQL
Slager et al. J Immunol. 172(8): 5095-

LKEFTVSGNILTIRL
102 (2004).

PGVLLKEFTVSGNILTIR
Mizote et al. Vaccine. 28(32): 5338-

L-TAADHR
46 (2010).

LLEFYLAMPFATPMEAE
Jager et al. J Exp Med. 191(4): 625-

L-ARRSLAQ
30 (2000).

KEFTVSGNILT
Zarour et al. Cancer Res. 60(17): 4946-

LLEFYLAMPFATPM
52 (2000).

AGATGGRGPRGAGA
Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009)

Zarour et al. Cancer Res. 62(1): 213-8

(2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

6
BAGE-1
AARAVFLAL
Boel et al. Immunity. 2(2): 167-

75 (1995).

7
HERV-K-
MLAVISCAV
Schiavetti et al. Cancer Res.

MEL

62(19): 5510-6 (2002).

8
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

9
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18 Pt

1): 6047-57 (2004).

10
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAA
Sun et al. Cancer Immunol

EVP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWICRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol. 172(8): 5095-

102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol. 170(3): 1490-

7(2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

11
MAGE-A1
EADPTGHSY
Traversari et al. J Exp Med.

KVLEYVIKV
176(5): 1453-7 (1992).

SLFRAVITK
Ottaviani et al. Cancer Immunol

EVYDGREHSA
Immunother. 54(12): 1214-20 (2005).

RVRFFFPSL
Pascolo et al. Cancer Res.

EADPTGHSY
61(10): 4072-7 (2001).

REPVTKAEML
Chaux et al. J Immunol. 163(5): 2928-

KEADPTGHSY
36 (1999).

DPARYEFLW
Luiten et al. Tissue Anitgens.

ITKKVADLVGF
55(2): 49-52 (2000).

SAFPTTINF
Luiten et al. Tissue Antigens.

SAYGEPRKL
56(1): 77-81 (2000).

RVRFFFPSL
Tanzarella et al. Cancer Res.

TSCILESLFRAVITK
59(11): 2668-74 (1999).

PRALAETSYVKVLEY
Stroobant et al. Eur J Immunol.

FLLLKYRAREPVTKAE
42(6): 1417-28 (2012).

EYVIKVSARVRF
Corbière et al. Tissue Antigens.

63(5): 453-7 (2004).

Goodyear et al. Cancer Immunol

Immunother. 60(12): 1751-61 (2011).

van der Bruggen et al. Eur J Immunol.

24(9): 2134-40 (1994).

Wang et al. Cancer Immunol

Immunother. 56(6): 807-18 (2007).

Chaux et al. J Exp Med. 189(5): 767-78

(1999).

Chaux et al. Eur J Immunol. 31(6):

1910-6 (2001).

12
MAGE-A2
YLQLVFGIEV
Kawashima et al. Hum Immunol.

EYLQLVFGI
59(1): 1-14 (1998).

REPVTKAEML
Tahara et al. Clin Cancer Res.

EGDCAPEEK
5(8): 2236-41 (1999).

LLKYRAREPVTKAE
Tanzarella et al. Cancer Res.

59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Chaux et al. J Exp Med. 89(5): 767-78

(1999).

13
mucink
PDTRPAPGSTAPPAHGV
Jerome et al. J Immunol. 151(3): 1654-

TSA
62 (1993).

14
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

15
SSX-2
KASEKIFYV
Ayyoub et al. J Immunol.

EKIQKAFDDIAKYFSK
168(4): 1717-22 (2002).

FGRLQGISPKI
Ayyoub et al. J Immunol.

WEKMKASEKIFYVYMK
172(11): 7206-11 (2004).

RK
Neumann et al. Cancer Immunol

KIFYVYMKRKYEAMT
Immunother. 60(9): 1333-46 (2011).

KIFYVYMKRKYEAM
Ayyoub et al. Clin Immunol.

114(1): 70-8 (2005).

Neumann et al. Int J Cancer.

112(4): 661-8 (2004).

Ayyoub et al. J Clin Invest.

113(8): 1225-33 (2004).

16
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-17

LSRLSNRLL
(2008).

17
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-17

(2008).

18
TRAG-3
CEFHACWPAFTVLGE
Janjic et al. J Immunol. 177(4): 2717-

27 (2006).

19
Her2/Neu
HLYQGCQVV
Nakatsuka et al. Mod. Pathol.

YLVPQQGFFC
19(6): 804-814 (2006).

PLQPEQLQV
Pils et al. Br. J. Cancer 96(3): 485-91

TLEEITGYL
(2007).

ALIHHNTHL
Scardino et al. Eur J Immunol.

PLTSIISAV
31(11): 3261-70 (2001).

VLRENTSPK
Scardino et al. J Immunol.

TYLPTNASL
168(11): 5900-6 (2002).

Kawashima et al. Cancer Res.

59(2): 431-5 (1999).

Okugawa et al. Eur J Immunol.

30(11): 3338-46 (2000).

20
c-myc

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

21
cyclin B1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

22
MUC1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

23
p53
VVPCEPPEV
Hung et al. Immunol. Rev. 222:43-69

(2008).

http://cancerimmunity.org/peptide/mutations/

24
p62

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

25
Survivin

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

TABLE 5

Testicular cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
CD45
KFLDALISL
Tomita et al. Cancer Sci.

102(4): 697-705 (2011).

2
DKK1
ALGGHPLLGV
Qian et al. Blood. (5): 1587-94

(2007).

3
PRAME
VLDGLDVLL,
Kessler et al. J Exp Med.

SLYSFPEPEA,
193(1): 73-88 (2001).

ALYVDSLFFL,
Ikeda et al. Immunity 6(2): 199-

SLLQHLIGL,
208 (1997).

LYVDSLFFL

4
RU2AS
LPRWPPPQL
Van Den Eynde et al. J. Exp.

Med. 190(12): 1793-800 (1999).

5
Telomerase
ILAKFLHWL;
Vonderheide et al. Immunity

RLVDDFLLV;
10(6): 673-9 (1999).

RPGLLGASVLGLDDI;
Miney et al. Proc. Natl. Acad.

and
Sci. U.S.A. 97(9): 4796-801

LTDLQPYMRQFVAHL
(2000).

Schroers et al. Cancer Res.

62(9): 2600-5 (2002).

Schroers et al. Clin. Cancer Res.

9(13): 4743-55 (2003).

TABLE 6

Pancreatic cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
ENAH (hMena)
TMNGSKSPV
Di Modugno et al. Int. J.

Cancer. 109(6): 909-18

(2004).

2
PBF
CTACRWKKACQR
Tsukahara et al. Cancer Res.

64(15): 5442-8 (2004).

3
K-ras
VVVGAVGVG
Gjertsen et al. Int. J. Cancer.

72(5): 784-90 (1997).

4
Mesothelin
SLLFLLFSL
Le et al. Clin. Cancer Res.

VLPLTVAEV
18(3): 858-68 (2012).

ALQGGGPPY
Hassan et al. Appl.

LYPKARLAF
Immunohistochem. Mol.

AFLPWHRLF
Morphol. 13(3): 243-7 (2005).

Thomas et al J Exp Med.

2004 Aug. 2; 200(3): 297-306.

5
mucink
PDTRPAPGSTAPPAHGVTSA
Jerome et al. J Immunol.

151(3): 1654-62 (1993).

TABLE 7

Liver cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
G250/
HLSTAFAR;
Vissers et al. Cancer Res. 59(21): 5554-9

MN/
KIFGSLAFL;
(1999).

CAIX
IISAVVGIL;
Fisk et al. J Exp Med. 181(6): 2109-17

ALCRWGLLL;
(1995).

ILHNGAYSL;
Brossart et al. Cancer Res. 58(4): 732-6

RLLQETELV;
(1998).

VVKGVVFGI; and
Kawashima et al. Hum Immunol.

YMIMVKCWMI
59(1): 1-14 (1998).

Rongcun et al. J Immunol. 163(2): 1037-

44 (1999).

2
Hepsin
SLLSGDWVL;
Guo et al. Scand J Immunol. 78(3): 248-

GLQLGVQAV;
57 (2013).

and

PLTEYIQPV

3
Intestinal
SPRWWPTCL
Ronsin et al. J Immunol. 163(1): 483-90

carboxyl

(1999).

esterase

4
alpha-
GVALQTMKQ;
Butterfield et al. Cancer Res.

foetoprotein
FMNICFIYEI;
59(13): 3134-42 (1999).

and QLAVSVILRV
Pichard et al. J Immunother. 31(3): 246-

53 (2008)

Alisa et al. Clin. Cancer Res.

11(18): 6686-94 (2005).

5
M-CSF
LPAVVGLSPGEQEY
Probst-Kepper et al. J Exp Med.

193(10): 1189-98 (2001).

6
PBF
CTACRWKKACQR
Tsukahara et al. Cancer Res.

64(15): 5442-8 (2004).

7
PSMA
NYARTEDFF
Horiguchi et al. Clin Cancer Res.

8(12): 3885-92 (2002).

8
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res. 60(16): 4499-

YLAMPFATPME
506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer. 132(2): 345-

APRGPHGGAASGL
54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer. 132(2): 345-

SLLMWITQCFLPVF
54 (2013).

LLEFYLAMPFATPMEAEL-
Knights et al. Cancer Immunol

ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12 (2002).

PGVLLKEFTVSGNILTIRL-
Zeng et al. Proc Natl Acad Sci USA.

TAADHR
98(7): 3964-9 (2001).

RLLEFYLAMPFA
Mandic et al. J Immunol. 174(3): 1751-

QGAMLAAQERRVPRAAE-
9 (2005).

VPR
Chen et al. Proc Natl Acad Sci USA.

PFATPMEAELARR
101(25): 9363-8 (2004).

PGVLLKEFTVSGNILTIRLT
Ayyoub et al. Clin Cancer Res.

VLLKEFTVSG
16(18): 4607-15 (2010).

AADHRQLQLSISSCLQQL
Slager et al. J Immunol. 172(8): 5095-

LKEFTVSGNILTIRL
102 (2004).

PGVLLKEFTVSGNILTIRL-
Mizote et al. Vaccine. 28(32): 5338-

TAADHR
46 (2010).

LLEFYLAMPFATPMEAEL-
Jager et al. J Exp Med. 191(4): 625-

ARRSLAQ
30 (2000).

KEFTVSGNILT
Zarour et al. Cancer Res. 60(17): 4946-

LLEFYLAMPFATPM
52 (2000).

AGATGGRGPRGAGA
Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-8

(2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

9
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol Immunother.

VP-R
55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Stager et al. J Immunol. 172(8): 5095-

102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Stager et al. J Immunol. 170(3): 1490-

7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

10
HERV-K-
MLAVISCAV
Schiavetti et al. Cancer Res.

MEL

62(19): 5510-6 (2002).

11
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

12
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

EYSKECLKEF
Monji et al. Clin Cancer Res. 10(18 Pt

1): 6047-57 (2004).

EYLSLSDKI

13
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

14
c-myc

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

15
cyclin B1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

16
p53
VVPCEPPEV
Hung et al. Immunol. Rev. 222:43-69

(2008).

http://cancerimmunity.org/peptide/mutations/

17
p62

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

18
Survivin

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

TABLE 8

Colorectal cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
ENAH (hMena)
TMNGSKSPV
Di Modugno et al. Int. J Cancer.

109(6): 909-18 (2004).

2
Intestinal
SPRWWPTCL
Ronsin et al. J Immunol. 163(1): 483-

carboxyl

90 (1999).

esterase

3
CASP-5
FLIIWQNTM
Schwitalle et al. Cancer Immun. 4: 14

(2004).

4
COA-1
TLYQDDTLTLQAAG
Maccalli et al. Cancer Res.

63(20): 6735-43 (2003).

5
OGT
SLYKFSPFPL
Ripberger. J Clin Immunol. 23(5): 415-

23 (2003).

6
OS-9
KELEGILLL
Vigneron et al. Cancer Immun. 2: 9

(2002).

7
TGF-betaRII
RLSSCVPVA
Linnebacher et al. Int. J. Cancer.

93(1): 6-11 (2001).

8
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer. 132(2): 345-

APRGPHGGAASGL
54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer. 132(2): 345-

SLLMWITQCFLPVF
54 (2013).

LLEFYLAMPFATPMEAE
Knights et al. Cancer Immunol

L-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2:12

PGVLLKEFTVSGNILTLR
(2002).

L-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAA
Mandic et al. J Immunol. 174(3): 1751-

E-VPR
9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIR
101(25): 9363-8 (2004).

LT
Ayyoub et al. Clin Cancer Res.

VLLKEFTVSG
16(18): 4607-15 (2010).

AADHRQLQLSISSCLQQL
Slager et al. J Immunol. 172(8): 5095-

LKEFTVSGNILTIRL
102 (2004).

PGVLLKEFTVSGNILTIR
Mizote et al. Vaccine. 28(32): 5338-

L-TAADHR
46 (2010).

LLEFYLAMPFATPMEAE
Jager et al. J Exp Med. 191(4): 625-

L-ARRSLAQ
30 (2000).

KEFTVSGNILT
Zarour et al. Cancer Res. 60(17): 4946-

LLEFYLAMPFATPM
52 (2000).

AGATGGRGPRGAGA
Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15 (13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-8

(2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

9
CEA
TYYRPGVNLSLSC
Duffy, Clin. Chem. 47(4): 624-30

EIIYPNASLLIQN
(2001).

YACFVSNLATGRNNS
Parkhurst et al. Mol. Ther. 19(3): 620-6

LWWVNNQSLPVSP
(2011).

LWWVNNQSLPVSP
Galanis et al. Cancer Res. 70(3): 875-

LWWVNNQSLPVSP
82 (2010).

EIIYPNASLLIQN
Bast et al. Am. J. Obstet. Gynecol.

NSIVKSITVSASG
149(5): 553-9 (1984).

KTWGQYWQV
Crosti et al. J Immunol. 176(8): 5093-9

(A)MLGTHTMEV
(2006).

ITDQVPFSV
Kobayashi et al. Clin Cancer Res.

YLEPGPVTA
8(10): 3219-25 (2002).

LLDGTATLRL
Campi et al. Cancer Res. 63(23): 8481-

VLYRYGSFSV
6 (2003).

SLADTNSLAV
Bakker et at. Int J Cancer. 62(1): 97-

RLMKQDFSV
102 (1995).

RLPRIFCSC
Tsai et al. J Immunol. 158(4): 1796-

LIYRRRLMK
802 (1997).

ALLAVGATK
Kawakami et al. J Immunol.

IALNFPGSQK
154(8): 3961-8 (1995).

RSYVPLAHR
Cox et al. Science. 264(5159): 716-9

(1994).

Kawakami et al. J Immunol.

154(8): 3961-8 (1995).

Kawakami et al. J Immunol.

161(12): 6985-92 (1998).

Skipper et al. J Immunol.

157(11): 5027-33 (1996).

Michaux et al. J Immunol.

192(4): 1962-71 (2014).

10
HERV-K-
MLAVISCAV
Schiavetti et al. Cancer Res.

MEL

62(19): 5510-6 (2002).

11
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

12
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18 Pt

1): 6047-57 (2004).

13
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAA
Sun et at. Cancer Immunol

EVP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol. 172(8): 5095-

102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol. 170(3): 1490-

7(2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

14
MAGE-A2
YLQLVFGIEV
Kawashima et al. Hum Immunol.

EYLQLVFGI
59(1): 1-14 (1998).

REPVTKAEML
Tahara et al. Clin Cancer Res.

EGDCAPEEK
5(8): 2236-41 (1999).

LLKYRAREPVTKAE
Tanzarella et al. Cancer Res.

59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Chaux et al. J Exp Med. 89(5): 767-78

(1999).

15
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

16
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-17

LSRLSNRLL
(2008).

17
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-17

(2008).

18
c-myc

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

19
cyclin B1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

20
MUC1

Reuschenbach et al. Cancer Imnumol.

Immunother. 58: 1535-1544 (2009)

21
p53
VVPCEPPEV
Hung et al. Immunol. Rev. 222:43-69

(2008).

http://cancerimmunity.org/peptide/mutations/

22
p62

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

23
Survivin

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

24
gp70

Castle et al., BMC Genomics 15: 190

(2014)

TABLE 9

Thyroid cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
CALCA
VLLQAGSLHA
El Hage et al. Proc. Natl.

Acad. Sci. U.S.A.

105(29): 10119-24 (2008).

2
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad.

p157-165 (SLLMWITQC),
Scie. U.S.A. 103(39): 14453-8

HLA-Cw3-
(2006).

restricted p92-100 (LAMP-
Gnjatic et al. PNAS

FATPM) and
Sep. 26, 2000 vol. 97

HLA-Cw6-restricted p80-88
no. 20 p. 10919

(ARGPESRLL)
Jager et al. J Exp Med.

SLLMWITQC
187(2): 265-70 (1998).

MLMAQEALAFL
Chen et al. J Immunol.

YLAMPFATPME
165(2): 948-55 (2000).

ASGPGGGAPR
Valmori et al. Cancer Res.

LAAQERRVPR
60(16): 4499-506 (2000).

TVSGNILTIR
Aarnoudse et al. Int J Cancer.

APRGPHGGAASGL
82(3): 442-8 (1999).

MPFATPMEAEL
Eikawa et al. Int J Cancer.

KEFTVSGNILTI
132(2): 345-54 (2013).

MPFATPMEA
Wang et al. J Immunol.

FATPMEAEL
161(7): 3598-606 (1998).

FATPMEAELAR
Matsuzaki et al. Cancer

LAMPFATPM
Immunol Immunother.

ARGPESRLL
57(8)1185-95 (2008).

SLLMWITQCFLPVF
Ebert et al. Cancer Res.

LLEFYLAMPFATPMEAEL-
69(3): 1046-54 (2009).

ARRSLAQ
Eikawa et al. Int J Cancer.

EFYLAMPFATPM
132(2): 345-54 (2013).

PGVLLKEFTVSGNILTIRL-
Knights et al. Cancer

TAADIAR
Immunol Immunother.

RLLEFYLAMPFA
58(3): 325-38 (2009).

QGAMLAAQERRVPRAAE-
Jäger et al. Cancer Immun.

VPR
2: 12 (2002).

PFATPMEAELARR
Zeng et al. Proc Natl Acad Sci

PGVLLKEFTVSGNILTIRLT
USA. 98(7): 3964-9 (2001).

VLLKEFTVSG
Mandic et al. J Immunol.

AADHRQLQLSISSCLQQL
174(3): 1751-9 (2005).

LKEFTVSGNILTIRL
Chen et al. Proc Natl Acad

PGVLLKEFTVSGNILTIRL-
Sci USA. 101(25): 9363-

TAADHR
8 (2004).

LLEFYLAMPFATPMEAEL-
Ayyoub et al. Clin Cancer

ARRSLAQ
Res. 16(18): 4607-15 (2010).

KEFTVSGNILT
Slager et al. J Immunol.

LLEFYLAMPFATPM
172(8): 5095-102 (2004).

AGATGGRGPRGAGA
Mizote et al. Vaccine.

28(32): 5338-46 (2010).

Jager et al. J Exp Med.

191(4): 625-30 (2000).

Zarour et al. Cancer Res.

60(17): 4946-52 (2000).

Zeng et al. J Immunol.

165(2): 1153-9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res.

62(1): 213-8 (2002).

Hasegawa et al. Clin Cancer

Res. 12(6): 1921-7 (2006).

3
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

4
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

5
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res.

10(18 Pt 1): 6047-57 (2004).

6
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol.

SLLMWITQCFLPVF
161(7): 3598-606 (1998).

QGAMLAAQERRVPRAAEVP-R
Sun et al. Cancer Immunol

AADHRQLQLSISSCLQQL
Immunother. 55(6): 644-52

CLSRRPWKRSWSAGSCPG-
(2006).

MPHL
Slager et al. Cancer Gene

ILSRDAAPLPRPG
Ther. 11(3): 227-36 (2004).

AGATGGRGPRGAGA
Zeng et al. Proc Natl Acad Sci

USA. 98(7): 3964-9 (2001).

Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med.

191(4): 625-30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity.

20(1): 107-18 (2004).

Hasegawa et al. Clin Cancer

Res. 12(6): 1921-7 (2006).

7
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J

Cancer. 107(5): 863-5 (2003).

TABLE 10

Lung cancer

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
CD274
LLNAFTVTV
Munir et al. Cancer Res. 73(6): 1764-76

(2013).

2
mdm-2
VLFYLGQY
Asai et al. Cancer Immun. 2: 3 (2002).

3
alpha-
FIASNGVKLV
Echchakir et al. Cancer Res.

actinin-4

61(10): 4078-83 (2001).

4
Elongation
ETVSEQSNV
Hogan et al. Cancer Res. 58(22): 5144-

factor 2

50 (1998).

(squamous

cell

carcinoma of

the lung)

5
ME1(non-
FLDEFMEGV
Karanikas et al. Cancer Res.

small cell

61(9): 3718-24 (2001).

lung

carcinoma)

6
NFYC
QQITKTEV
Takenoyama et al. Int. J Cancer.

(squamous

118(8): 1992-7 (2006).

cell

carcinoma of

the lung)

7
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer. 132(2):

APRGPHGGAASGL
345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer. 132(2):

SLLMWITQCFLPVF
345-54 (2013).

LLEFYLAMPFATPMEAE
Knights et al. Cancer Immunol

L-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAA
Mandic et al. J Immunol. 174(3): 1751-

E-VPR
9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol. 172(8): 5095-

PGVLLKEFTVSGNILTIRL
102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAE
46 (2010).

L-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res. 60(17): 4946-

AGATGGRGPRGAGA
52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-8

(2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

8
GAGE-1, 2, 8
YRPRPRRY
Van den Eynde et al. J Exp Med.

182(3): 689-98 (1995).

9
HERV-K-
MLAVISCAV
Schiavetti et al. Cancer Res.

MEL

62(19): 5510-6 (2002).

10
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

11
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18 Pt

1): 6047-57 (2004).

12
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAA
Sun et al. Cancer Immunol

EVP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol. 172(8): 5095-

102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol. 170(3): 1490-

7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

13
MAGE-A2
YLQLVFGIEV
Kawashima et al. Hum Immunol.

EYLQLVFGI
59(1): 1-14 (1998).

REPVTKAEML
Tahara et al. Clin Cancer Res.

EGDCAPEEK
5(8): 2236-41 (1999).

LLKYRAREPVTKAE
Tanzarella et al. Cancer Res.

59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Chaux et al. J Exp Med. 89(5): 767-78

(1999).

14
MAGE-A6
MVKISGGPR
Zorn et al. Eur J Immunol. 29(2): 602-7

(squamous
EVDPIGHVY
(1999).

cell lung
REPVTKAEML
Benlalam et al. J Immunol.

carcinoma)
EGDCAPEEK
171(11): 6283-9 (2003).

ISGGPRISY
Tanzarella et al. Cancer Res.

LLKYRAREPVTKAE
59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Vantomme et al. Cancer Immun.

3: 17 (2003).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

15
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

16
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-17

LSRLSNRLL
(2008).

17
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-17

(2008).

18
TRAG-3
CEFHACWPAFTVLGE
Janjic et al. J Immunol. 177(4): 2717-27

(2006).

19
XAGE-
RQKKIRIQL
Ohue et al. Int J Cancer. 131(5): E649-

1b/GAGED2a
HLGSRQKKIRIQLRSQ
58 (2012).

(non-small
CATWKVICKSCISQTPG
Shimono et al. Int J Oncol. 30(4): 835-

cell lung

40 (2007).

cancer)

20
c-myc

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

21
cyclin B1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

22
Her2/Neu
HLYQGCQVV
Nakatsuka et al. Mod. Pathol.

YLVPQQGFFC
19(6): 804-814 (2006).

PLQPEQLQV
Pils et al. Br. J. Cancer 96(3): 485-91

TLEEITGYL
(2007).

ALIHHNTHL
Scardino et al. Eur J Immunol.

PLTSIISAV
31(11): 3261-70 (2001).

VLRENTSPK
Scardino et al. J Immunol.

TYLPTNASL
168(11): 5900-6 (2002).

Kawashima et al. Cancer Res.

59(2): 431-5 (1999).

Okugawa et al. Eur J Immunol.

30(11): 3338-46 (2000).

23
MUC1

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

24
p53
VVPCEPPEV
Hung et al. Immunol. Rev. 222: 43-69

(2008).

http://cancerimmunity.org/peptide/muta-

tions/

25
p62

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

26
Survivin

Reuschenbach et al. Cancer Immunol.

Immunother. 58: 1535-1544 (2009)

TABLE 11

Prostate cancer

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
DKK1
ALGGHPLLGV
Qian et al. Blood.

110(5): 1587-94 (2007).

2
ENAH (hMena)
TMNGSKSPV
Di Modugno et al. Int. J.

Cancer. 109(6): 909-18

(2004).

3
Kallikrein 4
FLGYLILGV;
Wilkinson et al. Cancer

SVSESDTIRSISIAS;
Immunol Immunother.

LLANGRMPTVLQCVN;
61(2): 169-79 (2012).

and
Hural et al. J. Immunol.

RMPTVLQCVNVSVVS
169(1): 557-65 (2002).

4
PSMA
NYARTEDFF
Horiguchi et al. Clin Cancer

Res. 8(12): 3885-92 (2002).

5
STEAP1
MIAVFLPIV and
Rodeberg et al. Clin. Cancer

HQQYFYKIPILVINK
Res. 11(12): 4545-52 (2005).

Kobayashi et al. Cancer

Res. 67(11): 5498-504

(2007).

6
PAP
FLFLLFFWL;
Olson et al. Cancer

TLMSAMTNL;
Immunol Immunother.

and
59(6): 943-53 (2010).

ALDVYNGLL

7
PSA (prostate
FLTPKKLQCV and
Correale et al. J Natl.

carcinoma)
VISNDVCAQV
Cancer Inst. 89(4): 293-300

(1997).

8
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad.

p157-165 (SLLMWITQC),
Scie. U.S.A. 103(39): 14453-

HLA-Cw3-
8 (2006).

restricted p92-100 (LAMP-
Gnjatic et al. PNAS

FATPM) and
Sep. 26, 2000 vol. 97

HLA-Cw6-restricted p80-88
no. 20 p. 10919

(ARGPESRLL)
Jager et al. J Exp Med.

SLLMWITQC
187(2): 265-70 (1998).

MLMAQEALAFL
Chen et al. J Immunol.

YLAMPFATPME
165(2): 948-55 (2000).

ASGPGGGAPR
Valmori et al. Cancer Res.

LAAQERRVPR
60(16): 4499-506 (2000).

TVSGNILTIR
Aarnoudse et al. Int J

APRGPHGGAASGL
Cancer. 82(3): 442-8 (1999).

MPFATPMEAEL
Eikawa et al. Int J Cancer.

KEFTVSGNILTI
132(2): 345-54 (2013).

MPFATPMEA
Wang et al. J Immunol.

FATPMEAEL
161(7): 3598-606(1998).

FATPMEAELAR
Matsuzaki et al. Cancer

LAMPFATPM
Immunol Immunother.

ARGPESRLL
57(8)1185-95 (2008).

SLLMWITQCFLPVF
Ebert et al. Cancer Res.

LLEFYLAMPFATPMEAEL-
69(3): 1046-54 (2009).

ARRSLAQ
Eikawa et al. Int J Cancer.

EFYLAMPFATPM
132(2): 345-54 (2013).

PGVLLKEFTVSGNILTLRL-
Knights et al. Cancer

TAADHR)
Immunol Immunother.

RLLEFYLAMPFA
58(3): 325-38 (2009).

QGAMLAAQERRVPRAAE-
Jäger et al. Cancer Immun.

VPR
2: 12 (2002).

PFATPMEAELARR
Zeng et al. Proc Natl Acad

PGVLLKEFTVSGNILTIRLT
Sci USA. 98(7): 3964-

VLLKEFTVSG
9 (2001).

AADHRQLQLSISSCLQQL
Mandic et al. J Immunol.

LKEFTVSGNILTIRL
174(3): 1751-9 (2005).

PGVLLICEFTVSGNILTIRL-
Chen et al. Proc Natl Acad

TAADHR
Sci USA. 101(25): 9363-

LLEFYLAMPFATPMEAEL-
8 (2004).

ARRSLAQ
Ayyoub et al. Clin Cancer

KEFTVSGNILT
Res. 16(18): 4607-15 (2010).

LLEFYLAMPFATPM
Slager et al. J Immunol.

AGATGGRGPRGAGA
172(8): 5095-102 (2004).

Mizote et al. Vaccine.

28(32): 5338-46 (2010).

Jager et al. J Exp Med.

191(4): 625-30 (2000).

Zarour et al. Cancer Res.

60(17): 4946-52 (2000).

Zeng et al. J Immunol.

165(2): 1153-9 (2000).

Bioley et al. Clin Cancer

Res. 15(13): 4467-74 (2009).

Zarour et al. Cancer Res.

62(1): 213-8 (2002).

Hasegawa et al. Clin Cancer

Res. 12(6): 1921-7 (2006).

9
BAGE-1 (non-
AARAVFLAL
Boel et al. Immunity.

small cell lung

2(2): 167-75 (1995).

carcinoma)

10
GAGE-1, 2, 8
YRPRPRRY
Van den Eynde et al. J Exp

(non-small cell

Med. 182(3): 689-98 (1995).

lunch carcinoma)

11
GAGE-3, 4, 5, 6, 7
YYWPRPRRY
De Backer et al. Cancer

(lung squamous

Res. 59(13): 3157-65 (1999).

cell carcinoma

and lung

adenocarcinoma)

12
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

13
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

14
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer

Res. 10(18 Pt 1): 6047-57

(2004).

15
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J

SLLMWITQC
Cancer. 82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol.

SLLMWITQCFLPVF
161(7): 3598-606 (1998).

QGAMLAAQERRVPRAAEVP-R
Sun et al. Cancer Immunol

AADHRQLQLSISSCLQQL
Immunother. 55(6): 644-52

CLSRRPWKRSWSAGSCPG-
(2006).

MPHL
Slager et al. Cancer Gene

ILSRDAAPLPRPG
Ther. 11(3): 227-36 (2004).

AGATGGRGPRGAGA
Zeng et al. Proc Natl Acad

Sci USA. 98(7): 3964-

9(2001).

Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med.

191(4): 625-30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity.

20(1): 107-18 (2004).

Hasegawa et al. Clin Cancer

Res. 12(6): 1921-7 (2006).

16
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J

Cancer. 107(5): 863-5

(2003).

TABLE 12

Kidney cancer

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
FGF5
NTYASPRFK
Hanada et al. Nature.

427(6971): 252-6 (2004).

2
Hepsin
SLLSGDWVL;
Guo et al. Scand J Immunol.

GLQLGVQAV;
78(3): 248-57 (2013).

and

PLTEYIQPV

3
Intestinal
SPRWWPTCL
Ronsin et al. J Immunol.

carboxyl

163(1): 483-90 (1999).

esterase

4
M-CSF
LPAVVGLSPGEQEY
Probst-Kepper et al. J Exp

Med. 193(10): 1189-98 (2001).

5
RU2AS
LPRWPPPQL
Van Den Eynde et al. J. Exp.

Med. 190(12): 1793-800

(1999).

6
hsp70-2 (renal
SLFEGIDIYT
Gaudin et al. J. Immunol.

cell carcinoma)

162(3): 1730-8 (1999).

7
Mannan-MUC-1
PDTRPAPGSTAPPAHGVTSA
Loveland et al. Clin. Cancer

(renal cell
STAPPVHNV
Res. 12(3 Pt 1): 869-77 (2006).

carcinoma)
LLLLTVLTV
Loveland et al. Clin. Cancer

PGSTAPPAHGVT
Res. 12(3 Pt 1): 869-77 (2006).

Godelaine et al. Cancer

Immunol Immunother.

56(6): 753-9 (2007).

Ma et al. Int J Cancer.

129(10): 2427-34 (2011).

Wen et al. Cancer Sci.

102(8): 1455-61 (2011).

Jerome et al. J Immunol.

151(3): 1654-62 (1993).

Brossart et al. Blood.

93(12): 4309-17 (1999).

Hiltbold et al. Cancer Res.

58(22): 5066-70 (1998).

8
MAGE-A9 (renal
ALSVMGVYV
Oehlrich et al. Int J Cancer.

cell carcinoma)

117(2): 256-64 (2005).

TABLE 13

Melanoma

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
Hepsin
SLLSGDWVL;
Guo et at. Scand J Immunol.

GLQLGVQA;
78(3): 248-57 (2013).

and

PLTEYIQPV

2
ARTC1
YSVYFNLPADTIYTN
Wang et al J Immunol. 174(5): 2661-

70 (2005).

3
B-RAF
EDLTVKIGDFGLATEKSR
Sharkey et at. Cancer Res.

WSGSHQFEQLS
64(5): 1595-9 (2004).

4
beta-catenin
SYLDSGIHF
Robbins et al. J. Exp. Med.

183(3): 1185-92 (1996).

5
Cdc27
FSWAMDLDPKGA
Wang et al. Science.

284(5418): 1351-4 (1999).

6
CDK4
ACDPHSGHFV
Wölfel et al. Science.

269(5228): 1281-4 (1995).

7
CDK12
CILGKLFTK
Robbins et al. Nat Med. 19(6): 747-

52. (2013).

8
CDKN2A
AVCPWTWLR
Huang et al. J Immunol.

172(10): 6057-64 (2004).

9
CLPP
ILDKVLVHL
Corbière et al. Cancer Res.

71(4): 1253-62 (2011).

10
CSNK1A1
GLFGDIYLA
Robbins et al. Nat Med. 19(6): 747-

52 (2013).

11
FN1
MIFEKHGFRRTTPP
Wang et al. J Exp Med.

195(11): 1397-406 (2003).

12
GAS7
SLADEAEVYL
Robbins, et al. Nat Med. 19(6): 747-

52 (2013).

13
GPNMB
TLDWLLQTPK
Lennerz et al. Proc. Natl. Acad. Sci.

U.S.A. 102(44): 16013-8 (2005).

14
HAUS3
ILNAMIAKI
Robbins et al. Nat Med. 19(6): 747-

52 (2013).

15
LDLR-
WRRAPAPGA
Wang et al. J Exp Med.

fucosyltransferase
and
189(10): 1659-68 (1999).

PVTWRRAPA

16
MART2
FLEGNEVGKTY
Kawakami et al. J Immunol.

166(4): 2871-7 (2001).

17
MATN
KTLTSVFQK
Robbins et al. Nat Med. 19(6): 747-

52 (2013).

18
MUM-1
EEKLIVVLF
Coulie et al. Proc. Natl. Acad. Sci.

U.S.A. 92(17): 7976-80 (1995).

19
MUM-2
SELFRSGLDSY
Chiari et al. Cancer Res.

and
59(22): 5785-92 (1999).

FRSGLDSYV

20
MUM-3
EAFIQPITR
Baurain et al. J. Immunol.

164(11): 6057-66 (2000).

21
neo-PAP
RVIKNSIRLTL
Topalian et al. Cancer Res.

62(19): 5505-9 (2002).

22
Myosin class I
KINKNPKYK
Zorn, et al. Eur. J. Immunol.

29(2): 592-601 (1999).

23
PPP1R3B
YTDFHCQYV
Robbins et al. Nat Med. 19(6): 747-

52 (2013).

Lu et al. J Immunol. 190(12): 6034-

42 (2013).

24
PRDX5
LLLDDLLVSI
Sensi et al. Cancer Res. 65(2): 632-

40 (2005).

25
PTPRK
PYYFAAELPPRNLPEP
Novellino et al. J. Immunol.

170(12): 6363-70 (2003).

26
N-ras
ILDTAGREEY
Linard et al. J. Immunol.

168(9): 4802-8 (2002).

27
RBAF600
RPHVPESAF
Lennerz et al. Proc. Natl. Acad. Sci.

U.S.A. 102(44): 16013-8 (2005).

28
SIRT2
KIFSEVTLK
Lennerz et al. Proc. Natl. Acad. Sci.

U.S.A. 102(44): 16013-8 (2005).

29
SNRPD1
SHETVIIEL
Lennerz et al. Proc. Natl. Acad. Sci.

U.S.A. 102(44): 16013-8 (2005).

30
Triosephosphate
GELIGILNAAKVPAD
Pieper et al. J Exp Med. 189(5): 757-

isomerase

66 (1999).

31
OA1
LYSACFWWL
Touloukian et al. J. Immunol.

170(3): 1579-85 (2003).

32
RAB38/NY-MEL-1
VLHWDPETV
Walton et al. J Immunol.

177(11): 8212-8 (2006).

33
TRP-1/gp75
MSLQRQFLR;
Touloukian et al. Cancer Res.

ISPNSVFSQWRVVCDSLE
62(18): 5144-7 (2002).

DY;
Robbins et al. J. Immunol.

SLPYWNFATG;
(10): 6036-47 (2002).

and
Osen et al. PLoS One. 5(11): e14137

SQWRVVCDSLEDYDT
(2010).

34
TRP-2
SVYDFFVWL;
Parkhurst et al. Cancer Res.

TLDSQVMSL;
58(21): 4895-901 (1998).

LLGPGRPYR;
Noppen et al. Int. J. Cancer.

ANDPIFVVL;
87(2): 241-6 (2000).

QCTEVRADTRPWSGP;
Wang et al. J. Exp. Med.

and
1184(6): 2207-16 (1996).

ALPYWNFATG
Wang et al. J. Immunol. 160(2): 890-

7(1998).

Castelli et al. J. Immunol.

162(3): 1739-48 (1999).

Paschen et al. Clin. Cancer Res.

(14): 5241-7 (2005).

Robbins et al. J. Immunol.

169(10): 6036-47 (2002).

35
tyrosinase
KCDICTDEY;
Kittlesen et al. J. Immunol.

SSDYVIPIGTY;
160(5): 2099-106 (1998).

MLLAVLYCL;
Kawakami et al. J. Immunol.

CLLWSFQTSA;
(12): 6985-92 (1998).

YMDGTMSQV;
Wölfel et al. Eur. J. Immunol.

AFLPWHRLF;
24(3): 759-64 (1994).

IYMDGTADFSF;
Riley et al. J. Immunother.

QCSGNFMGF;
24(3): 212-20 (2001).

TPRLPSSADVEF;
Skipper et al. J. Exp. Med.

LPSSADVEF;
183(2): 527-34 (1996).

LHHAFVDSIF;
Kang et al. J. Immunol.

SEIWRDIDF;
155(3): 1343-8 (1995).

QNILLSNAPLGPQFP;
Dalet et al. Proc. Natl. Acad. Sci.

SYLQDSDPDSFQD;
U.S.A. 108(29): E323-31 (2011)

and
Lennerz et al. Proc. Natl. Acad. Sci.

FLLHHAFVDSIFEQWLQR
U.S.A. 102(44): 16013-8 (2005).

HRP
Benlalam et al. J. Immunol.

171(11): 6283-9 (2003).

Morel et al. Int. J. Cancer.

83(6): 755-9 (1999).

Brichard et al. Eur. J. Immunol.

26(1): 224-30 (1996).

Topalian et al. J. Exp. Med.

(5): 1965-71 (1996).

Kobayashi et al. Cancer Res.

58(2): 296-301 (1998).

36
Melan-A/MART-1
YTTAEEAAGIGILTVILGV
Meng et al. J. Immunother. 23: 525-

LLLIGCWYCRR
534(2011)

37
gp100/Pmel17
ALNFPGSQK
El Hage et al. Proc. Natl. Acad. Sci.

ALNFPGSQK
U.S.A. 105(29): 10119-24 (2008).

VYFFLPDHL
Kawashima et al. Hum Immunol.

RTKQLYPEW
59(1): 1-14 (1998).

HTMEVTVYHR
Robbins et al. J Immunol.

SSPGCQPPA
159(1): 303-8 (1997).

VPLDCVLYRY
Sensi et al. Tissue Antigens.

LPHSSSHWL
59(4): 273-9 (2002).

SNDGPTLI
Lennerz et al. Proc Natl Acad Sci

GRAMLGTHTMEVTVY
USA. 102(44): 16013-8 (2005).

WNRQLYPEWTEAQRLD
Benlalam et al. J Immunol.

TTEWVETTARELPIPEPE
171(11): 6283-9 (2003).

TGRAMLGTHTMEVTVYH
Vigneron et al. Tissue Antigens.

GRAMLGTHTMEVTVY
65(2): 156-62 (2005).

Castelli et al. J Immunol.

162(3): 1739-48 (1999).

Touloukian et al. J Immunol.

164(7): 3535-42 (2000).

Parkhurst et al. J Immunother.

27(2): 79-91 (2004).

Lapointe et al. J Immunol.

167(8): 4758-64 (2001).

Kobayashi et al. Cancer Res.

61(12): 4773-8 (2001).

38
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9(2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

39
BAGE-1
AARAVFLAL
Boel et al. Immunity. 2(2): 167-

75 (1995).

40
GAGE-1, 2, 8
YRPRPRRY
Van den Eynde et al. J Exp Med.

182(3): 689-98 (1995).

41
GAGE-3, 4, 5, 6, 7
YYWPRPRRY
De Backer et al. Cancer Res.

(cutaneous

59(13): 3157-65 (1999).

melanoma)

42
GnTVf
VLPDVFIRC(V)
Guilloux et al. J Exp Med.

183(3): 1173-83 (1996).

43
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

44
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

45
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

46
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Stager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

47
LY6K
RYCNLEGPPI
Suda et al. Cancer Sci. 98(11): 1803-

KWTEPYCVIAAVKIFPRF
8 (2007).

FMV-AKQ
Tomita et al. Oncoimmunology.

KCCKIRYCNLEGPPINSSVF
3: e28100 (2014).

48
MAGE-A1
EADPTGHSY
Traversari et al. J Exp Med.

KVLEYVIKV
176(5): 1453-7 (1992).

SLFRAVITK
Ottaviani et al. Cancer Immunol

EVYDGREHSA
Immunother. 54(12): 1214-20 (2005).

RVRFFFPSL
Pascolo et al. Cancer Res.

EADPTGHSY
61(10): 4072-7 (2001).

REPVTKAEML
Chaux et al. J Immunol.

KEADPTGHSY
163(5): 2928-36 (1999).

DPARYEFLW
Luiten et al. Tissue Antigens.

ITKKVADLVGF
55(2): 149-52 (2000).

SAFPTTINF
Luiten et al. Tissue Antigens.

SAYGEPRKL
56(1): 77-81 (2000).

RVRFFFPSL
Tanzarella et al. Cancer Res.

TSCILESLFRAVITK
59(11): 2668-74 (1999).

PRALAETSYVKVLEY
Stroobant et al. Eur J Immunol.

FLLIKYRAREPVTKAE
42(6): 1417-28 (2012).

EYVIKVSARVRF
Corbière et al. Tissue Antigens.

63(5): 453-7 (2004).

Goodyear et al. Cancer Immunol

Immunother. 60(12): 1751-61 (2011).

van der Bruggen et al. Eur J

Immunol. 24(9): 2134-40 (1994).

Wang et al. Cancer Immunol

Immunother. 56(6): 807-18 (2007).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

Chaux et al. Eur J Immunol.

31(6): 1910-6 (2001).

49
MAGE-A6
MVKISGGPR
Zorn et al. Eur J Immunol.

EVDPIGHVY
29(2): 602-7 (1999).

REPVTKAEML
Benlalam et al. J Immunol.

EGDCAPEEK
171(11): 6283-9 (2003).

ISGGPRISY
Tanzarella et al. Cancer Res.

LLKYRAREPVTKAE
59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Vantomme et al. Cancer Immun.

3:17 (2003).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

50
MAGE-A10
GLYDGMEHL
Huang et al. J Immunol.

DPARYEFLW
162(11): 6849-54 (1999).

Chaux et al. J Immunol.

163(5): 2928-36 (1999).

51
MAGE-A12
FLWGPRALV
van der Bruggen et al. Eur J

VRIGHLYIL
Immunol. 24(12): 3038-43 (1994).

EGDCAPEEK
Heidecker et al. J Immunol.

REPFTKAEMLGSVIR
164(11): 6041-5 (2000).

AELVHFLLLKYRAR
Panelli et al. J Immunol.

164(8): 4382-92 (2000).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Wang et al. Cancer Immunol

Immunother. 56(6): 807-18 (2007).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

52
MAGE-C2
LLFGLALIEV
Ma et al. Int J Cancer. 109(5): 698-

ALKDVEERV
702 (2004).

SESIKKKVL
Godelaine et al. Cancer Immunol

ASSTLYLVF
Immunother. 56(6): 753-9 (2007).

SSTLYLVFSPSSFST
Ma et al. Int J Cancer. 129(10): 2427-

34 (2011).

Wen et al. Cancer Sci. 102(8): 1455-

61 (2011).

53
NA88-A
QGQHFLQKV
Moreau-Aubry et al. J Exp Med.

191(9): 1617-24 (2000).

54
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

55
SSX-2
KASEKIFYV
Ayyoub et al. J Immunol.

EKIQKAFDDIAKYFSK
168(4): 1717-22 (2002).

FGRLQGISPKI
Ayyoub et al. J Immunol.

WEKMKASEKIFYVYMKRK
172(11): 7206-11 (2004).

KIFYVYMKRKYEAMT
Neumann et al. Cancer Immunol

KIFYVYMKRKYEAM
Immunother. 60(9): 1333-46 (2011).

Ayyoub et al. Clin Immunol.

114(1): 70-8 (2005).

Neumann et al. Int J Cancer.

112(4): 661-8 (2004).

Ayyoub et al. J Clin Invest.

113(8): 1225-33 (2004).

56
SSX-4
INKTSGPKRGKHAWTHR
Ayyoub et al. J Immunol.

LRE
174(8): 5092-9 (2005).

YFSKKEWEKMKSSEKIV
Valmori et al. Clin Cancer Res.

YVY
12(2): 398-404 (2006).

MKLNYEVMTKLGFKVTL

PPF

KHAWTHRLRERKQLVV

YEEI

LGFKVTLPPFMRSKRAA

DFH

KSSEKIVYVYMKLNYEV

MTK

KHAWTHRLRERKQLVV

YEEI

57
TRAG-3
CEFHACWPAFTVLGE
Janjic et al. J Immunol. 177(4): 2717-

27 (2006).

58
TRP2-INT2g
EVISCKLIKR
Lupetti et al. J Exp Med.

188(6): 1005-16 (1998).

59
pgk

Morgan et al., J. Immunol.

171: 3287-3295 (2003)

TABLE 14

Squamous cell carcinoma

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
CASP-8
FPSDSWCYF
Mandruzzato et al. J. Exp. Med.

186(5): 785-93 (1997).

2
p53
VVPCEPPEV
Ito et al. Int. J. Cancer.

120(12): 2618-24 (2007).

3
SAGE
LYATVIHDI
Miyahara et al. Clin Cancer Res.

11(15): 5581-9 (2005).

TABLE 15

Chronic myeloid leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
BCR-ABL
SSKALQRPV;
Yotnda et al. J. Clin. Invest.

GFKQSSKAL;
101(10): 2290-6 (1998).

ATGFKQSSKALQRPVAS;
Bosch et al. Blood. 88(9): 3522-7

and
(1996).

ATGFKQSSKALQRPVAS
Makita et al. Leukemia.

16(12): 2400-7 (2002).

2
dek-can
TMKQICKKEIRRLHQY
Makita et al. Leukemia.

16(12): 2400-7 (2002).

3
EFTUD2
KILDAVVAQK
Lennerz et al. Proc. Natl. Acad.

Sci. U.S.A. 102(44): 16013-8

(2005).

4
GAGE-3, 4, 5,
YYWPRPRRY
De Backer et al. Cancer Res.

6, 7

59(13): 3157-65 (1999).

TABLE 16

Acute lymphoblastic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
ETV6-AML1
RIAECILGM
Yotnda et al. J. Clin. Invest.

and
(2): 455-62 (1998).

IGRIAECILGMNPSR
Yun et al. Tissue Antigens.

54(2): 153-61 (1999).

2
GAGE-3, 4, 5,
YYWPRPRRY
De Backer et al. Cancer Res.

6, 7

59(13): 3157-65 (1999).

TABLE 17

Acute myelogenous leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
FLT3-ITD
YVDFREYEYY
Graf et al. Blood. 109(7): 2985-8

(2007).

2
Cyclin-A1
FLDRFLSCM
Ochsenreither et al. Blood.

and
119(23): 5492-501 (2012).

SLIAAAAFCLA

3
GAGE-3, 4, 5,
YYWPRPRRY
De Backer et al. Cancer Res.

6, 7

59(13): 3157-65 (1999).

TABLE 18

Chronic lymphocytic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
FNDC3B
VVMSWAPPV
Rajasagi et al. Blood. 124(3): 453-

62 (2014).

2
GAGE-3, 4, 5,
YYWPRPRRY
De Backer et al. Cancer Res.

6, 7

59(13): 3157-65 (1999).

TABLE 19

Promyelocytic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
pml-RARalpha
NSNHVASGAGEAAIETQS
Gambacorti-Passerini et al. Blood.

SSSEEIV
81(5): 1369-75 (1993).

2
GAGE-3, 4, 5,
YYWPRPRRY
De Backer et al. Cancer Res.

6, 7

59(13): 3157-65 (1999).

TABLE 20

Multiple myeloma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
MAGE-C1
ILFGISLREV
Anderson et al. Cancer Immunol

KVVEFLAML
Immunother. 60(7): 985-97 (2011).

SSALLSIFQSSPE
Nuber et al. Proc Natl Acad Sci USA.

SFSYTLLSL
107(34): 15187-92 (2010).

VSSFFSYTL

2
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9(2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

3
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLMMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

4
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

5
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

6
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

7
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

TABLE 21

B-cell lymphoma

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
D393-CD20
KPLFRRMSSLELVIA
Vauchy et al. Int

J Cancer. 137(1):

116-26 (2015).

TABLE 22

Bladder carcinoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
BAGE-1
AARAVFLAL
Boel et al. Immunity. 2(2): 167-

75 (1995).

2
GAGE-1, 2, 8
YRPRPRRY
Van den Eynde et al. J Exp Med.

182(3): 689-98 (1995).

3
GAGE-3, 4, 5, 6, 7
YYWPRPRRY
De Backer et al. Cancer Res.

59(13): 3157-65 (1999).

4
MAGE-A4
EVDPASNTY
Kobayashi et al. Tissue Antigens.

(transitional cell
GVYDGREHTV
62(5): 426-32 (2003).

carcinoma of
NYKRCFPVI
Duffour et al. Eur J Immunol.

urinary bladder)
SESLKMIF
29(10): 3329-37 (1999).

Miyahara et al. Clin Cancer Res.

11(15): 5581-9 (2005).

Ottaviani et al. Cancer Immunol

Immunother. 55(7): 867-72 (2006).

Zhang et al. Tissue Antigens.

60(5): 365-71 (2002).

5
MAGE-A6
MVKISGGPR
Zorn et al. Eur J Immunol.

EVDPIGHVY
29(2): 602-7 (1999).

REPVTKAEML
Benlalam et al. J Immunol.

EGDCAPEEK
171(11): 6283-9 (2003).

ISGGPRISY
Tanzarella et al. Cancer Res.

LLKYRAREPVTKAE
59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Vantomme et al. Cancer Immun.

3: 17 (2003).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

6
SAGE
LYATVIHDI
Miyahara et al. Clin Cancer Res.

11(15): 5581-9 (2005).

7
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matusuzki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

8
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

9
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

10
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

11
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

12
SP17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

TABLE 23

Head and neck cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
BAGE-1 (head and
AARAVFLAL
Boel et al. Immunity. 2(2): 167-

neck squamous cell

75 (1995).

carcinoma)

2
GAGE-1, 2, 8
YRPRPRRY
Van den Eynde et al. J Exp Med.

182(3): 689-98 (1995).

3
GAGE-3, 4, 5, 6, 7
YYWPRPRRY
De Backer et al. Cancer Res.

59(13): 3157-65 (1999).

4
LY6K
RYCNLEGPPI
Suda et al. Cancer Sci. 98(11): 1803-

KWTEPYCVIAAVKIFPRF
8 (2007).

FMV-AKQ
Tomita et al. Oncoimmunology.

KCCKIRYCNLEGPPINSSVF
3: e28100 (2014).

5
MAGE-A3 (head
EVDPIGHLY
Gaugler et al. J Exp Med.

and neck squamous
FLWGPRALV
179(3): 921-30 (1994).

cell carcinoma)
KVAELVHFL
van der Bruggen et al. Eur J

TFPDLESEF
Immunol. 24(12): 3038-43 (1994).

VAELVHFLL
Kawashima et al. Hum Immunol.

MEVDPIGHLY
59(1): 1-14 (1998).

EVDPIGHLY
Oiso et al. Int J Cancer. 81(3): 387-

REPVTKAEML
94 (1999).

AELVHFLLL
Miyagawa et al. Oncology. 70(1): 54-

MEVDPIGHLY
62 (2006).

WQYFFPVIF
Bilsborough et al. Tissue Antigens.

EGDCAPEEK
60(1): 16-24 (2002).

KKLLTQHFVQENYLEY
Schultz et al. Tissue Antigens.

RKVAELVHFLLLKYR
57(2): 103-9 (2001).

KKLLTQHFVQENYLEY
Tanzarella et al. Cancer Res.

ACYEFLWGPRALVETS
59(11): 2668-74 (1999).

RKVAELVHFLLLKYR
Schultz et al. J Exp Med.

VIFSKASSSLQL
195(4): 391-9 (2002).

VFGIELMEVDPIGHL
Herman et al. Immunogenetics.

GDNQIMPKAGLLIIV
43(6): 377-83 (1996).

TSYVKVLHHMVKISG
Russo et al. Proc Natl Acad Sci USA.

RKVAELVHFLLLKYRA
97(5): 2185-90 (2000).

FLLLKYRAREPVTKAE
Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Schultz et al. Cancer Res.

60(22): 6272-5 (2000).

Cesson et al. Cancer Immunol

Immunother. 60(1): 23-35 (2011).

Schultz et al. J Immunol.

172(2): 1304-10 (2004).

Zhang et al. J Immunol. 171(1): 219-

25 (2003).

Cesson et al. Cancer Immunol

Immunother. 60(1): 23-35 (2010).

Kobayashi et al. Cancer Res.

61(12): 4773-8 (2001).

Cesson et al. Cancer Immunol

Immunother. 60(1): 23-35 (2011).

Consogno et al. Blood. 101(3): 1038-

44 (2003).

Manici et al. J Exp Med. 189(5): 871-

6 (1999).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

6
MAGE-A6
MVKISGGPR
Zorn et al. Eur J Immunol.

EVDPIGHVY
29(2): 602-7 (1999).

REPVTKAEML
Benlalam et al. J Immunol.

EGDCAPEEK
171(11): 6283-9 (2003).

ISGGPRISY
Tanzarella et al. Cancer Res.

LLKYRAREPVTKAE
59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Vantomme et al. Cancer Immun.

3: 17 (2003).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

7
SAGE
LYATVIHDI
Miyahara et al. Clin Cancer Res.

11(15): 5581-9 (2005).

TABLE 24

Esophageal cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
GAGE-3, 4, 5, 6, 7
YYWPRPRRY
De Backer et al. Cancer Res.

(Esophageal

59(13): 3157-65 (1999).

squamous cell

carcinoma and

esophageal

adenocarcinoma)

2
MAGE-A2
YLQLVFGIEV
Kawashima et al. Hum Immunol.

EYLQLVFGI
59(1): 1-14 (1998).

REPVTKAEML
Tahara et al. Clin Cancer Res.

EGDCAPEEK
5(8): 2236-41 (1999).

LLKYRAREPVTKAE
Tanzarella et al. Cancer Res.

59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

3
MAGE-A6
MVKISGGPR
Zorn et al. Eur J Immunol.

EVDPIGHVY
29(2): 602-7 (1999).

REPVTKAEML
Benlalam et al. J Immunol.

EGDCAPEEK
171(11): 6283-9 (2003).

ISGGPRISY
Tanzarella et al. Cancer Res.

LLKYRAREPVTKAE
59(11): 2668-74 (1999).

Breckpot et al. J Immunol.

172(4): 2232-7 (2004).

Vantomme et al. Cancer Immun.

3: 17 (2003).

Chaux et al. J Exp Med. 189(5): 767-

78 (1999).

4
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int. J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol 161(7): 3598-

KEFTVSGNILTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

5
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

6
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

7
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

8
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

9
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

TABLE 25

Brain cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-

LSRLSNRLL
17 (2008).

2
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-

17 (2008).

TABLE 26

Pharynx cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-

LSRLSNRLL
17 (2008).

2
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-

17 (2008).

TABLE 27

Tumors of the tongue

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL
Adair et al. J Immunother. 31(1): 7-

LSRLSNRLL
17 (2008).

2
TAG-2
LSRLSNRLL
Adair et al. J Immunother. 31(1): 7-

17 (2008).

TABLE 28

Synovial cell sarcoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol.

SLLMWITQCFLPVF
161(7): 3598-606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

2
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20

(LAMP-FATPM)
p. 10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol.

KEFTVSGNILTI
161(7): 3598-606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Immunother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

3
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

4
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

5
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI

6
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

TABLE 29

Neuroblastoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

2
NY-ESO-1
HLA-A2-restricted peptide
Jager et al. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-
Gnjatic et al. PNAS

Cw3-restricted p92-100
Sep. 26, 2000 vol. 97 no. 20 p.

(LAMP-FATPM)
10919

and HLA-Cw6-
Jager et al. J Exp Med. 187(2): 265-

restricted p80-88
70 (1998).

(ARGPESRLL)
Chen et al. J Immunol. 165(2): 948-

SLLMWITQC
55 (2000).

MLMAQEALAFL
Valmori et al. Cancer Res.

YLAMPFATPME
60(16): 4499-506 (2000).

ASGPGGGAPR
Aarnoudse et al. Int J Cancer.

LAAQERRVPR
82(3): 442-8 (1999).

TVSGNILTIR
Eikawa et al. Int J Cancer.

APRGPHGGAASGL
132(2): 345-54 (2013).

MPFATPMEAEL
Wang et al. J Immunol. 161(7): 3598-

KEFTVSGNLLTI
606 (1998).

MPFATPMEA
Matsuzaki et al. Cancer Immunol

FATPMEAEL
Immunother. 57(8)1185-95 (2008).

FATPMEAELAR
Ebert et al. Cancer Res. 69(3): 1046-

LAMPFATPM
54 (2009).

ARGPESRLL
Eikawa et al. Int J Cancer.

SLLMWITQCFLPVF
132(2): 345-54 (2013).

LLEFYLAMPFATPMEAEL
Knights et al. Cancer Immunol

-ARRSLAQ
Imununother. 58(3): 325-38 (2009).

EFYLAMPFATPM
Jäger et al. Cancer Immun. 2: 12

PGVLLKEFTVSGNILTIRL
(2002).

-TAADHR
Zeng et al. Proc Natl Acad Sci USA.

RLLEFYLAMPFA
98(7): 3964-9 (2001).

QGAMLAAQERRVPRAAE
Mandic et al. J Immunol.

-VPR
174(3): 1751-9 (2005).

PFATPMEAELARR
Chen et al. Proc Natl Acad Sci USA.

PGVLLKEFTVSGNILTIRLT
101(25): 9363-8 (2004).

VLLKEFTVSG
Ayyoub et al. Clin Cancer Res.

AADHRQLQLSISSCLQQL
16(18): 4607-15 (2010).

LKEFTVSGNILTIRL
Slager et al. J Immunol.

PGVLLKEFTVSGNILTIRL
172(8): 5095-102 (2004).

-TAADHR
Mizote et al. Vaccine. 28(32): 5338-

LLEFYLAMPFATPMEAEL
46 (2010).

-ARRSLAQ
Jager et al. J Exp Med. 191(4): 625-

KEFTVSGNILT
30 (2000).

LLEFYLAMPFATPM
Zarour et al. Cancer Res.

AGATGGRGPRGAGA
60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et al. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

3
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

4
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

5
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

6
Sp17
ILDSSEEDK
Chiriva-Internati et al. Int J Cancer.

107(5): 863-5 (2003).

TABLE 30

Uterine cancer

Tumor-associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL
Aarnoudse et al. Int J Cancer.

SLLMWITQC
82(3): 442-8 (1999).

LAAQERRVPR
Rimoldi et al. J Immunol.

ELVRRILSR
165(12): 7253-61 (2000).

APRGVRMAV
Wang et al. J Immunol. 161(7): 3598-

SLLMWITQCFLPVF
606 (1998).

QGAMLAAQERRVPRAAE
Sun et al. Cancer Immunol

VP-R
Immunother. 55(6): 644-52 (2006).

AADHRQLQLSISSCLQQL
Slager et al. Cancer Gene Ther.

CLSRRPWKRSWSAGSCP
11(3): 227-36 (2004).

G-MPHL
Zeng et al. Proc Natl Acad Sci USA.

ILSRDAAPLPRPG
98(7): 3964-9 (2001).

AGATGGRGPRGAGA
Slager et al. J Immunol.

172(8): 5095-102 (2004).

Jager et al. J Exp Med. 191(4): 625-

30 (2000).

Slager et al. J Immunol.

170(3): 1490-7 (2003).

Wang et al. Immunity. 20(1): 107-18

(2004).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

2
NY-ESO-1
HLA-A2-restricted peptide
Jager et at. Proc. Natl. Acad. Scie.

p157-165 (SLLMWITQC),
U.S.A. 103(39): 14453-8 (2006).

HLA-Cw3-restricted p92-
Gnjatic et al. PNAS

100 (LAMP-FATPM) and
Sep. 26, 2000 vol. 97 no. 20 p.

HLA-Cw6-restricted p80-88
10919

(ARGPESRLL)
Jager et al. J Exp Med. 187(2): 265-

SLLMWITQC
70 (1998).

MLMAQEALAFL
Chen et al. J Immunol. 165(2): 948-

YLAMPFATPME
55 (2000).

ASGPGGGAPR
Valmori et al. Cancer Res.

LAAQERRVPR
60(16): 4499-506 (2000).

TVSGNILTIR
Aarnoudse et al. Int J Cancer.

APRGPHGGAASGL
82(3): 442-8 (1999).

MPFATPMEAEL
Eikawa et al. Int J Cancer.

KEFTVSGNILTI
132(2): 345-54 (2013).

MPFATPMEA
Wang et al. J Immunol. 161(7): 3598-

FATPMEAEL
606 (1998).

FATPMEAELAR
Matsuzaki et al. Cancer Immunol

LAMPFATPM
Immunother. 57(8)1185-95 (2008).

ARGPESRLL
Ebert et al. Cancer Res. 69(3): 1046-

SLLMWITQCFLPVF
54 (2009).

LLEFYLAMPFATPMEAEL-
Eikawa et al. Int J Cancer.

ARRSLAQ
132(2): 345-54 (2013).

EFYLAMPFATPM
Knights et al. Cancer Immunol

PGVLLKEFTVSGNILTIRL-
Immunother. 58(3): 325-38 (2009).

TAADHR
Jäger et al. Cancer Immun. 2: 12

RLLEFYLAMPFA
(2002).

QGAMLAAQERRVPRAAE-
Zeng et al. Proc Natl Acad Sci USA.

VPR
98(7): 3964-9 (2001).

PFATPMEAELARR
Mandic et al. J Immunol.

PGVLLKEFTVSGNILTIRLT
174(3): 1751-9 (2005).

VLLKEFTVSG
Chen et al. Proc Natl Acad Sci USA.

AADHRQLQLSISSCLQQL
101(25): 9363-8 (2004).

LKEFTVSGNILTIRL
Ayyoub et al. Clin Cancer Res.

PGVLLKEFTVSGNILTIRL-
16(18): 4607-15 (2010).

TAADHR
Slager et al. J Immunol.

LLEFYLAMPFATPMEAEL-
172(8): 5095-102 (2004).

ARRSLAQ
Mizote et al. Vaccine. 28(32): 5338-

KEFTVSGNILT
46 (2010).

LLEFYLAMPFATPM
Jager et al. J Exp Med. 191(4): 625-

AGATGGRGPRGAGA
30 (2000).

Zarour et al. Cancer Res.

60(17): 4946-52 (2000).

Zeng et al. J Immunol. 165(2): 1153-

9 (2000).

Bioley et at. Clin Cancer Res.

15(13): 4467-74 (2009).

Zarour et al. Cancer Res. 62(1): 213-

8 (2002).

Hasegawa et al. Clin Cancer Res.

12(6): 1921-7 (2006).

3
HERV-K-MEL
MLAVISCAV
Schiavetti et al. Cancer Res.

62(19): 5510-6 (2002).

4
KK-LC-1
RQKRILVNL
Fukuyama et al. Cancer Res.

66(9): 4922-8 (2006).

5
KM-HN-1
NYNNFYRFL
Fukuyama et al. Cancer Res.

EYSKECLKEF
66(9): 4922-8 (2006).

EYLSLSDKI
Monji et al. Clin Cancer Res. 10(18

Pt 1): 6047-57 (2004).

6
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5): 863-5 (2003).

Gene Alignment

An exemplary alignment of select orthopoxvirus genes is shown below. Various genes of 5 vaccinia virus strains, Copenhagen (“cop”), Western Reserver (“WR”), Tian Tan (“Tian”), Wyeth, and Lister, align as follows:

C2L

CLUSTAL O(1.2.4) multiple sequence alignment

cop
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESLLDYIRW
60

WR
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESILDYIRW
60

Tian
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESLLDYIRW
60

Wyeth
MESVTFSINGEIIQVNKEIITASPYNFFKRIQEHHINDEVIILNGINYHAFESLLDYMRW
60

Lister
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESLLDYMRW
60

**** ***************************:**::**.*****************:**

cop
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGIKKLYNA
120

WR
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGIKKLYNA
120

Tian
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKQYGIKKLYNA
120

Wyeth
KKINITINNVEMILVAAVIIDVTPVVDLCVKTMIHNINSTNCIRMFNESKRYGIKKLYNA
120

Lister
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINFTNCIRMFNFSKRYGIKKLYNA
120

*****************:**** *************** ***********:*********

cop
SMSEIINNITAVTSDPEFGKLSKDELTTILSHENVNVNHEDVTAMILLKWIHKNPNDVDI
180

WR
SMSEIINNITAVTSDPEFGKLSKDELTTILSHENVNVNHEDVTAMILLKWIHKNPNDVDI
180

Tian
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

Wyeth
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

Lister
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

*********************************:**************************

cop
INILHPKFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIKNSDYISTITHYSPRTE
240

WR
INILHPKFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIKNSDYISTITHYSPRTE
240

Tian
INILHPKFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIKNSDYISTITHYSPRTE
240

Wyeth
INILHPKFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIKNSDYISTITHYSPRTE
240

Lister
INILHPKFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIKNSDYISTITHYSPRTE
240

************************************************************

cop
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

WR
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Tian
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Wyeth
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Lister
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNRHVYSVSRVDLKTKKWKTVTNMSSLKSEV
300

******************************************:*****************

cop
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

WR
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Tian
STCVNNGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Wyeth
STCVNNGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Lister
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

*****:******************************************************

cop
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

WR
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

Tian
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

Wyeth
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYAITGITHETRNYLY
420

Lister
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

**********************************************.*************

cop
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

WR
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

Tian
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

Wyeth
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

Lister
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

************************************************************

cop
YDMFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

WR
YDMFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

Tian
YDMFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

Wyeth
YDMFTKDET------HKSLPSFLSNCEKQFLQ
506

Lister
YDMFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

********* *****************

C1L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MVKNNKI-----SNSCRMIMSTNPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
55

WR
MVKNNKIQKNKISNSCRMIMSTDPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
60

Tian
MVKNNKI-----SNSCRMIMSTDPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
55

Wyeth
MVKNNKI-----SNSCRMIMSTNPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDRDYTS
55

Lister
MVKNNKI-----SNSCRMIMSTNPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
55

******* ************************************************

Cop
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKMTEDIK
115

WR
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKMTEDIK
120

Tian
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKMTEDIK
115

Wyeth
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKMTEDIK
115

Lister
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKMTADIK
115

*************************************:****************** ***

Cop
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

WR
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
180

Tian
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

Wyeth
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

Lister
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINRYSKELGLATEYFNKYGHLMFYTLPI
175

************************************************************

Cop
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

WR
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
229

Tian
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

Wyeth
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

Lister
PYNRFFCRNSIGFLAVLSPTIGHVKAFYRFIEYVSIDDRRKFKKELMSK
224

*************************************************

N1L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

WR
MRTLLIRYILWRNDNDQTYYNDNFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

Tian
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

Wyeth
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

Lister
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

**********************:*************************************

Cop
QPVNNIEDAKRMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

WR
QPVNNIEDAKRMIAISARVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Tian
QPVNNIEDAKRMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Wyeth
QPVNNIEDAKRMIAISARVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Lister
QPVNNIEDAKRMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

*********************************************************

N2L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILNRF
60

WR
MTSSANDNNEPKVLEMVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILDRF
60

Tian
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPYIMDCINRHINMCIQRTYSSSIIAILDRF
60

Wyeth
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIIDCINRHINMCIQRTYSSSIIAILDRF
60

Lister
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILDRF
60

******************************* *:***********************:**

Cop
LTMNKDELNNTQCHIIKEFMTYEQMAIDHYGEYVNAILYQIRKRPNQHHTIDLFKKIKRT
120

WR
LMMNKDELNNTQCHIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQHHTIDLFKRIKRT
120

Tian
LMMNKDELNNTQCHIIKNL-----------------------------------------
79

Wyeth
LTMNKDELNNTQCHIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQHHTIDLFKKIKRT
120

Lister
LTMNRDELNNTQCHIIKEFMTYEQMAIDHYGEYVNAILYQIRKRPNQHHTIDLFKKIKRT
120

* ***************::

Cop
PYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFINYVETKYF
175

WR
RYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFINYVETKYF
175

Tian
-------------------------------------------------------

Wyeth
RYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFIDYVETKYF
175

Lister
RYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFIDYVETKYF
175

M1L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
60

WR
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
60

Tian
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
60

Wyeth
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
60

Lister
MIFVIESKLLQIYRN--RNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
58

*************** *******************************************

Cop
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
120

WR
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
120

Tian
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
120

Wyeth
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
120

Lister
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
118

************************************************************

Cop
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER
180

WR
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER
180

Tian
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER
180

Wyeth
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINN-------------------
161

Lister
GADPNACDKQHKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER
178

*********::******************************

Cop
VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWMMKLGISPSKPDHDGNTPLHI
240

WR
VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWMMKLGISPSKPDHDGNTPLHI
240

Tian
VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWMMKLGISPSKPDHDGNTPLHI
240

Wyeth
------------------------------------------------------------

Lister
VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWMMKLGISPSKPDHDGNTPLHI
238

Cop
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLSTSNVITDQTV
300

WR
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLSTSNVITDQTV
300

Tian
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLSTSNVITDQTV
300

Wyeth
------------------------------------------------------------

Lister
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLSTSNVITDQTV
298

Cop
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
360

WR
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
360

Tian
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
360

Wyeth
------------------------------------------------------------

Lister
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
358

Cop
YETMVDYLLFNHFSVDSVVNGHTCMSECVRLNNPVILSKLMLHNPTSETMYLTMKAIEKD
420

WR
YETMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVILSKLMLHNPTSETMYLTMKAIEKD
420

Tian
YETMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVILSKLMLHNLTSETMYLTMKAIEKD
420

Wyeth
------------------------------------------------------------

Lister
YETMVDYLLENHFSVDSVVNGHTCMSECVRLNNPVILSKLMLHNPTSETMYLTMKAIEKD
418

Cop
KLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
472

WR
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
472

Tian
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
472

Wyeth
----------------------------------------------------

Lister
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
470

M2L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

WR
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

Tian
------------------------MSSSTRLPVLVLAAELTIGVNYDINSTIIGECHMSE
36

Wyeth
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

Lister
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

:************************

Cop
SYIDRNANIVLIGYGLEINMTIMDTDQREVAAAEGVGKDNKLSVLLFTTQRLDKVHHNIS
120

WR
SYIDRNANIVLTGYGLEINNTIMDTDQRFVAAAEGVGKDNKLSVLLFTTQRLDKVHHNIS
120

Tian
SYIDRNANIVLTGYGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFTTQRLDKVHHNIS
96

Wyeth
SYIDRNANIVLTGYGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFTTQRLDKVHHNIS
120

Lister
SYIDRNANIVLTGYGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFTTQRLDKVHHNIS
120

************************************************************

Cop
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
180

WR
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
180

Tian
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
156

Wyeth
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
180

Lister
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
180

************************************************************

Cop
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

WR
YLYHNSEYSMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

Tian
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
196

Wyeth
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

Lister
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

********.*******************************

K1L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLE
60

WR
MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLE
60

Tian
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALYYAIADNNVRLVCTLLNSGALKNLLE
60

Wyeth
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLE
60

Lister
MDLSRINTWKSKQLKSFLSSKDAFKADINGHSALYYAIADNNVRLVCTLLNAGALKNLLE
60

**********************:****::**********************:********

Cop
NEFPLHQAATLEDTKIVKILLFSGMDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRL
120

WR
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRL
120

Tian
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRL
120

Wyeth
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRL
120

Lister
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWRL
120

************************:***********************************

Cop
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHTTIKNGHVDMMILLL
180

WR
MFYGKTGWETSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

Tian
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

Wyeth
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

Lister
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

********************************************* **************

Cop
DYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSVNLENVLLDDAEITKMII
240

WR
DYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMII
240

Tian
DYMTVDKHQ---------------------------------------------------
189

Wyeth
DYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMII
240

Lister
DYMTSTNTNNSLLFIPDIKLAIDNKDIEMIQALFKYDINIYSANLENVLLDDAEIAKMII
240

**** : :

Cop
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN
284

WR
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN
284

Tian
--------------------------------------------

Wyeth
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN
284

Lister
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELKLMYVNCVKKN
284

K2L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTR
60

WR
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTR
60

Tian
MIALLILSLACSASAYRLQGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTR
60

Wyeth
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTR
60

Lister
------------------------------------------------------------

Cop
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPLYYQQYHR
120

WR
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Tian
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Wyeth
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Lister
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
60

**************************************************** *******

Cop
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGTWQYPFDIT
180

WR
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDIT
180

Tian
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGTWQYPFDIT
180

Wyeth
-----LNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDIT
175

Lister
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDIT
120

********************************************** ********

Cop
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMT
240

WR
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMT
240

Tian
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMT
240

Wyeth
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDKEYDMVRLPYKDANISMYLAIGDNMT
235

Lister
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMT
180

**********************************:*************************

Cop
HFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
300

WR
HFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMENPDNASFKHM
300

Tian
HFTDSITAA-KDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
299

Wyeth
HFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
295

Lister
HFTDSITAAKLDYWSSQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
240

********* **** ********************************************

Cop
TRDPLYIYKMFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
360

WR
TRDPLYIYKMFQNAKIDVDEQGTVARASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
360

Tian
TRDPLYIYKMFQNAKIDVDEQGTVAEASTIMVATARSSPEELEFNTPFVFIIRHDITGFI
359

Wyeth
TRDPLYIYKMFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
355

Lister
TRDPLYIYKMFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
300

****************************************:*******************

Cop
LFMGKVESP
369

WR
LFMGKVESP
369

Tian
LFMGKVESP
368

Wyeth
LFMGKVESP
364

Lister
LFMGKVESP
309

*********

K ORF A

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MGHIITYCQVHTNISILIRKAHHIIFFVIDCDCISLQFSNYVHHGNRFRTVLISKTSIAC
60

Tian
MGHIITYCQVHTNISILIRKAYHIIFFVIDCDCISLQFSNYVHHGNRFRTVLISKTSIAC
60

*********************:**************************************

Cop
FSDIKRILPCTFKIYSINDCP
81

Tian
FSDIKRILPCTFKIYSINDCP
81

*********************

K3L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHSEAILAESVKMHMDRYVEYRDKLVG
60

WR
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHFEAILAESVKMHMDRYVEYRDKLVG
60

Tian
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHSEAILAESVKMHMDRYVEYRDKLVG
60

Wyeth
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPHFEAILAESVKMHMDRYVEYRDKLVG
60

Lister
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPHSEAILAESVKMHMDRYVEYRDKLVG
60

*********************:************* ************************

Cop
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

WR
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

Tian
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

Wyeth
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

Lister
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

****************************

K4L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

WR
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

Wyeth
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

Lister
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

************************************************************

Cop
TIILNEIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

WR
TIILNKIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

Wyeth
TIILNEIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

Lister
TIILNEIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

*****:******************************************************

Cop
SDNTHIYLGSANMDWRSLTQVKELGIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKNF
180

WR
SDNTHIYLGSANMDWRSLTQVKELGIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKNF
180

Wyeth
SDNTHIYLGSANMDWRSLTQVKELGIAIFNNRNLAADLIQIFEVYWYLGVNNLPYNWKNF
180

Lister
SDNTHIYLGSANMDWRSLTQVKELGIAIFNNRNLAADLIQIFEVYWYLGVNNLPYNWKNF
180

************************************************************

Cop
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

WR
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

Wyeth
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

Lister
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

************************************************************

Cop
FIPIIYSKAGKILFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSK
300

WR
FIPIIYSKAGNILFWPYIEDELRRAAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSK
300

Wyeth
FIPIIYSKAGKILFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSK
300

Lister
FIPIIYSKAGKILFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSK
300

**********:*************:***********************************

Cop
NIDIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

WR
NINIEVKLFIVPDADPPIPYSRVNEAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

Wyeth
NINIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

Lister
NINIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

**:*********************************************************

Cop
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMKQCTNDIYCDEIQPEKEIPE
420

WR
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCRLLKNMKQCTNDIYCDEIQPEKEIPE
420

Wyeth
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMKQCTNDIYCDEIQPEKEIPE
420

Lister
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMKQCTNDIYCDEIQPEKEIPE
420

**********************************:*************************
420

Cop
YSLE
424

WR
YSLE
424

Wyeth
YSLE
424

Lister
YSLE
424

****

K5L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
-------------MGATISILASYDNPNLFTAMILMSPLVNADAVSRLNLLAAKLMGTIT
47

WR
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKINGTIT
60

Tian
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKLMGTIT
60

Wyeth
-------------MGATISILASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKLMGTIT
47

Lister
---------MGHSMGATISILASYDNPNLFTAMILMSPLVNADAVSRINLLAAKLMGTIT
51

. **************************:*************

Cop
PNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKINTPR
107

WR
LNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKINTPR
120

Tian
LNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKINTPR
120

Wyeth
PNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKINTPP
107

Lister
PNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKINTPP
111

**********************************************************

Cop
LSYSREQTMRL-----VMFQVHIISCNMQIVIE---------------------------
135

WR
LSYSREQTIRL-----AMF-----------------------------------------
134

Tian
LSYSREQTIRL-----AMF-----------------------------------------
134

Wyeth
TLILQGTNNEISDVLGAYYFMQHANCNREIKIYEGAKHHLHKETDEVKKSVMKEIETWIF
167

Lister
TLILQGTNNKISDVLGAYYFMQHANCNREIKIYEGAKHHLHKETDEVKKSVMKEIETWIF
171

:..: .:

Cop
----

WR
----

Tian
----

Wyeth
NRVK
171

Lister
NRVK
175

K6L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

WR
MSANCMFNIDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

Wyeth
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

Lister
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

************************************************************

Cop
HGRSNGEKMMIDDFGTARGNY
81

WR
HGRSNGEKMMIDDFGTARGNY
81

Wyeth
HGRSNGEKMMIDDFGTARGNY
81

Lister
HGRSNGEKMMIDDFGTARGNY
81

*********************

K7R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

WR
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

Tian
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

Wyeth
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

Lister
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

************************************************************

Cop
DDVAIRYYGIDKINEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITEHWGYKKISES
120

WR
DDVAIRYYGIDKINEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITEHWGYKKISES
120

Tian
DDVAIRYYGIDKINEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITEHWGYKKISES
120

Wyeth
DDVAIRYYGIDKINEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITEHWGYKKISES
120

Lister
DDVAIRYYGIDKINEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITEHWGYKKISES
120

************************************************************

Cop
RFQSLGNITDLMTDDNINILILFLEKKLN
149

WR
RFQSLGNITDLMTDDNINILILFLEKKLN
149

Tian
RFQSLGNITDLMTDDNINILILFLEKKLN
149

Wyeth
RFQSLGNITDLMTDDNINILILFLEKKLN
149

Lister
RFQSLGNITDLMTDDNINILILFLEKKLN
149

*****************************

F1L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

WR
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

Tian
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDRDYVYPLPENMVYRFDKSTNILDYLS
60

Wyeth
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

Lister
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

**********************************:*************************

Cop
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

WR
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Tian
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Wyeth
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Lister
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

************************************************************

Cop
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHTICDD
180

WR
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRCMNPVETIKMFTLLSHTICDD
180

Tian
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRUMNPVKTIKMFTLLSHTICDD
180

Wyeth
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITKLGRRCMNPVKTIKMFTLLSHTICDD
180

Lister
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHTICDD
180

**********************************:*********:***************

Cop
CFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

WR
YFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

Tian
CFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

Wyeth
CFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

Lister
CFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

**********************************************

F2L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

WR
MFNMNINSPVRFVKETNRAKSPTRQSPYAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

Tian
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

Wyeth
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

Lister
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

************************************************************

Cop
ICYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

WR
FCYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

Tian
ICYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

Wyeth
ICYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

Lister
FCYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

:***********************************************************

Cop
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

WR
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

Tian
YYPELEEVQSLDSTDRGDQGFGSTGLR
147

Wyeth
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

Lister
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

**************:************

F3L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNSTILKKLSPYFRTHLRQKYTKNKD
60

WR
MPIFTNTVYCKNILALSMTKKFKTIIDAIGGNIIVNSTILKKLSPYFRTHLRQKYTKNKD
60

Tian
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNSTILKKLSPYFRTHLRQKYTKNKD
60

Wyeth
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNSTILKKLSPYFRTHLRQKYTKNKD
60

Lister
MPIFVNTVYCKNILALSMTKKFRTIIDAIGGNSIVNSTILKKLSPYFRTHLRQKYTKNKD
60

************************************************************

Cop
PVTRVCLDLDIHSLTSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCINFILRDF
120

WR
PVTWVCLDLDIHSLTSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCINFILRDF
120

Tian
PVTRVCLDLDIHSLTSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCINFILRDF
120

Wyeth
PVTRVCLDLDIHSLTSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCINFILRDF
120

Lister
PVTRVCLDLDIHSLTSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCINFILRDF
120

*** ********************************************************

Cop
RKEYCVECYMMGIEYGLSNLLCHTKNFIAKHFLELEDDIIDNFDYLSMKLILESDELNVP
180

WR
RKEYCVECYMMGIEYGLSNLLCHTKNFIAKHFLELEDDIIDNFDYLSMKLILESDELNVP
180

Tian
RKEYCVECYMMGIEYGLSNLLCHTKNFIAKHFLELEDDIIDNFDYLSMELILESDELNVP
180

Wyeth
RKEYCVECYMMGIEYGLSNLLCHTKNFIAKHFLELEDDIIDNFDYLSMKLILESDELNVP
180

Lister
RKEYCVECYMMGIEYGLSNLLCHTKNFIAKHFLELEDDIIDNFDYLSIKLILESDELNVP
180

***********************************************:************

Cop
DEDYVVDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

WR
DEDYVVDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

Tian
DEDYVVDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

Wyeth
DEDYVVDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

Lister
DEDYVVDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

************************************************************

Cop
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

WR
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

Tian
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

Wyeth
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

Lister
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

************************************************************

Cop
PIPPMNSPRLYATGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

WR
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Tian
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Wyeth
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Lister
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

************:***********************************************

Cop
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

WR
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

Tian
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

Wyeth
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

Lister
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

************************************************************

Cop
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRGSYIDTIEVYNHHTYSWNIWDGK
480

WR
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNINDGK
480

Tian
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNINDGK
480

Wyeth
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNIWDGK
480

Lister
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNIWDGK
480

************************************* **********************

B14R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
------------------------------------------------------------

WR
MDIFREIASSMKGENVFISPASISSVLTILYYGANGSTAEQLSKYVEKEENMDKVSAQNI
60

Tian
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
------------------------------------------------------------

WR
SFKSINKVYGRYSAVFKDSFLRKIGDKFQTVDFTDCRTIDAINKCVDIFTEGKINPLLDE
120

Tian
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
---MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGELFNHASVK
57

WR
PLSPDTCLLAISAVYFKAKNLTPFEKEFTSDYPFYVSPTEMVDVSMNSMYGKAFNHASVK
180

Tian
---MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVK
57

Wyeth
---MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVK
57

: *********************************************: *******

Cop
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCNSLDAMFIDVHIPK
117

WR
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCNSLEATFIDVHIPK
240

Tian
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFMDAMFIDVHIPK
117

Wyeth
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFMDAMFIDVHIPK
117

**********************************************: ::* ********

Cop
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAAT
177

WR
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNSDVSVDAMIHKTYIDVNEEYTEAAAAT
300

Tian
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAAT
177

Wyeth
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTFAAAAT
177

********************************* **************************

Cop
CALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRYCSPTTNC
222

WR
CALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRYCSPTTNC
345

Tian
CALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRYCSPTTNC
222

Wyeth
CALVSDCASTVTNEFCADHPFIYVIRHVDGKILFVGRYCSPTTNC
222

**********:*****.****************************

B15R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYWSSYAYRNRQCAGQLYSTLLSFRDD
60

WR
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYWSSYAYANRQCAGQLYSTLLSFRDD
60

Tian
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYWSSYAYRNRQCAGQLYSTLLSFRDD
60

Wyeth
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYWSSYAYRNRQCAGQLYSTLLSFRDD
60

Lister
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYWSSYAYRNRQCAGQLYSTLLSFRDD
60

**************************:*********************************

Cop
AELVFIDIRELVKNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

WR
AELVFIDIRELVKNMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Tian
AELVFIDIRELVKNMEWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Wyeth
AELVFIDIRELVKHMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Lister
AELVFIDIRELVKNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

*************:*********:************************************

Cop
PTSNSLNALFVMLEMLNYVDYNIIFRRMN
149

WR
PTSNSLNALFVMLEMLNYVDYNIIFRRMN
149

Tian
PTSNSLNALFVMLEMLNYVDYNIIFRRMN
149

Wyeth
PTSNSLNALFVMLEMLNYVDYNIIFRRMN
149

Lister
PTSNSLNALFVMLEMLNYVDYNIIFRRMN
149

*****************************

B ORF E

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMVFTSPVSSSICTKSDDGRNLSDGFL
60

Tian
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMVFTSPVSSSICTKSDDGRNLSDGFL
60

************************************************************

Cop
LIRYITTDDFCTIFDIIPRHIFYQLANVDEH
91

Tian
LIRYITTDDFCTIFDIIPRHIFYQLANVDEH
91

*******************************

B16R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MSILPVIFLPIFFYSSFVQTFNASECIDKGXYFASFMELENEPVILPCPQINTISSGYNI
60

WR
MSILPVIFLSIFFYSSFVQTFNAPECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

Tian
------------------------------------MELENEPVILPCPQINTLSSGYNI
24

Wyeth
MSILPVIFLSIFFYSSFVQTFNASECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

Lister
MSILPVIFLPIFFYSSFVQTFNAPECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

************************

Cop
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

WR
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

Tian
LDILWEKAGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVL
84

Wyeth
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

Lister
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

************************************************************

Cop
ESNIDFISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLRNKRLKQRTPGIIT
180

WR
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLRNKRLKQRTPGIIT
180

Tian
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLRNKRLKQRTPGIIT
144

Wyeth
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLRNKRLKQRTPGIIT
180

Lister
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLRNKRLKQRTPGIIT
180

*****:******************************************************

Cop
IEDVRKNDAGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIHPTMQLPEGVVTSIGSNLTI
240

WR
IEDVRKNDAGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVTSIGSNLTI
240

Tian
IEDVRKNDAGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVTSIGSNLTI
204

Wyeth
IEDVRKNDAGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVTSIGSNLTI
240

Lister
IEDVRKNDAGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVTSIGSNLTI
240

******************** ******************* *****:*:**********

Cop
ACRVSLRPPTTDADVFWISNGMYYEEDDGDGDGRISVANKIYMTDKRRVITSRLNINPVK
300

WR
ACRVSLRPPTTDADVFWISNGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNINPVK
300

Tian
ACRVSLRPPTTDADVFWISNGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNINPVK
264

Wyeth
ACRVSLRPPTTDTDVFWISNGMYYEEDDGDGDGRISVANKIYMIDKRRVITSRLNINPVK
300

Lister
ACRVSLRPPTTDADVFWISNGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNINPVK
300

************:******************:****************************

Cop
EEDATTFTCMAFTIPSISKTVTVSIT
326

WR
EEDATTFTCMAFTIPSISKTVTVSIT
326

Tian
EEDATTFTCMAFTIPSISKTVTVSI-
289

Wyeth
EEDATTFTCMAFTIPSISKTVTVSIT
326

Lister
EEDATTFTCMAFTIPSISKTVTVSIT
326

*************************

B ORF F

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MVIIPGVRCLSLLFLRRRCPLHIISAFTLLAINALILGHTISPVDLSFTICGYEIKSIFD
60

Tian
MVIIPGVRCLSLLFLAARCPLHIISAFTLLAINALILGHTISPVDLSFTICGYEIRSIFD
60

*******************************************************:****

Cop
SETDTIVKFNDIMSQ
75

Tian
SKTDTIVKFNDIMSQ
75

*:*************

B17L

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYYSAEKYMCRYTTLNHNCVNVRRCAL
60

WR
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYYSAEKYMCRYTTLNHNCINVRRCAL
60

Tian
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYYSAEKYMCRYTTLNHNCVNVRRCAL
60

Wyeth
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYYSAEKYMCRYTTLNHNCVNVRRCAL
60

Lister
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYYSAEKYMCRYTTLNHNCINVRRCAL
60

****************************************************:*******

Cop
DSKLLHDIITNCKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTRNL
120

WR
DSKLLHDIITNCKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTRNL
120

Tian
DSKLLHDIITNCKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTRNL
120

Wyeth
DSKLLHDIITNCKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTRNL
120

Lister
DSKLLHDIITNCKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTRNL
120

************************************************************

Cop
DKVSVKTYKGVKVKKLNRCADHAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITPVE
180

WR
DKVSVKTYKGVKVKKLNRCADHAIVINPFVKFKLTLPNKTSHAKVLVTFCKLKTDITPVE
180

Tian
DKVSVKTYKGVKVKKLNRCADHAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQIE
180

Wyeth
DKVSVKTYKGVKVKKLNRCADHAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQIE
180

Lister
DKVSVKTYKGVKVKKLNRCADHAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQIE
180

****************************************************:**** :*

Cop
APLPGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINSSKFACVLKLHRSMYRIPPFPI
240

WR
APLPGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINSSKFACVLKLHRSMYRIPPFPI
240

Tian
APLSGNVLVYTFPNINKRIPGYIHVNIEGCIDGMIYINSSKFACVLKLHRSMYRIPPFPI
240

Wyeth
AFISGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINSSKFACVLKLHRSMYRIPPFPI
240

Lister
APLSGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINSSKFACVLKLHRSMYRIPPFPI
240

*** *********:**********:***********************************

Cop
DICSCCSQYTNDDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

WR
DICSCCSQYINYDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

Tian
DICSCCSQYTNGDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

Wyeth
DICSCCSQYTNDDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

Lister
DICSCCSQYTNDDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

********* * ************************************************

Cop
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

WR
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

Tian
HEYVKIALGIVCKLMINNMHSIVGVNRSNTFVNCLLEDNV
340

Wyeth
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

Lister
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

****************************************

B18R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MSRRLIYVLNINRKSTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQHVTGYTA
60

WR
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQPVTGYTA
60

Tian
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRHPVTGYTA
60

Wyeth
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQPVTGYTA
60

*************:**************************************: ******

Cop
LHCYLYNNYFTNDVLKILLNHDVNVTMKTSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

WR
LHCYLYNNYFTNDVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

Tian
LHCYLYNNYFTNDVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

Wyeth
LHCYLYNNYFTNDVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

*********************.*:************************************

Cop
HLSHRDYSNLLLEYIKSRYMLLKEEDIDENIVSTLLDKGIDPNETQDGYTALHYYYLCLA
180

WR
HLLHRDYSNLLLEYIKSRYMLLKEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCLA
180

Tian
HLLHRDYSNLLLEYIKSRYMLLKEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCLA
180

Wyeth
HLSHRDYSNLLLEYIKSRYMLLKEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCLA
180

** *********************************************************

Cop
HVYKPGECRKPITIKKAKRIISLFIQHGANLNALDNCGNTPFHLYLSIEMCNNIHMTKML
240

WR
HVYKPGECRKPITIKKAKRIISLFIQHGANLNALDNCGNTPFHLYLSIEMCNNIHMTKML
240

Tian
HVYKPGECRKPITIKKAKRIISLFIQHGANLNALDNCGNTPFHLYLSIEMCNNIHMTKML
240

Wyeth
HVYKPGECRKPITIKKAKRIISLFIQHGANLNALDNCGNTPFHLYLSIEMCNNIHMTKML
240

************************************************************

Cop
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRMIVFEFIK
300

WR
LTFNPNFEICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRIIVFEFIK
300

Tian
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRIIVFEFIK
300

Wyeth
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRIIVFEFIK
300

*******:********************************************:*******

Cop
TYSTRPADSITYLMNRFKNINIYTRYEGKTLLHVACEYNNTQVIDYLIRINGDINALTDN
360

WR
TYSTRPADSITYLMNRFKNIDIYTRYEGKTLLHVACEYNNTHVIDYLIRINGDINALTDN
360

Tian
TYSTRPADSITYLMNRFKNINIYTRYEGKTLLHVACEYNNTQVIDYLIRINGDINALTDN
360

Wyeth
TYSTRPADSITYLMNRFKNINIYTRYEGKTLLHVACEYNNTHVIDYLIRINGDINALTDN
360

********************:********************:******************

Cop
NKHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQLPSLPIFDIKSFEKFISYC
420

WR
NKHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQLPSLPIFDIKSFEKFISYC
420

Tian
NKHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDOLPSLPIFDIKSFEKFISYC
420

Wyeth
NKHAIQLIIDNKENSPYTIDCLLYILRYIVDKNVIRSLVDQLPSLPIFDIKSFEKFISYC
420

**** **************:****************************************

Cop
ILLDDTFYDRHVKNRDSKTYRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDTTLYAV
480

WR
ILIDDTFYNRHVRNRDSKTYRYAFSKYMSFDKYDGIITKCHKETILLKLSTVLDTTLYAV
480

Tian
ILLDDTFYDRHVKNRNSKTYRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDTTLYAV
480

Wyeth
ILLDDTFYNRHVRNRNSKTYRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDTTLYAV
480

********:***:**:*************************.**:***************

Cop
LRCHNSRKLRRYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

WR
LRCHNSKKLRRYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

Tian
LRCHNSRKLRRYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

Wyeth
LRCHNSKKLRRYLNELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

******:******.**********************************************

Cop
NCLLTLLPSEIIYEILYMLTINDLYNISYPPTKV
574

WR
NCLLTILPSEIIYEILYMLTINDLYNISYPPTKV
574

Tian
NCLLTLLPSEIIYEILYMLTINDLYNISYPPTKV
574

Wyeth
NCLLTLLPSEIIYEILYMLTINDLYNISYPPTKV
574

**********************************

B19R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGT
60

WR
MTMKMMVHIYFVSL--LLLLFHSYAIDIENEITEFFNKMRDTIPAKDSKWLNPACMFGGT
58

Tian
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGT
60

Wyeth
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGT
60

************** ********************************************

Cop
MNDMATLGEPFSAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNKRVKHGDLWIANY
120

WR
MNDIAALGEPFSAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNKRVKHGDLWIANY
118

Tian
MNDIAALGEPFSAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNKRVKHGDLWIANY
120

Wyeth
MNDIATLGEPFSAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNKRVKHGDLWIANY
120

***:*:******************************************************

Cop
TSKFSNRRYLCTVTTKNGDCVQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

WR
TSKFSNRRYLCTVTTKNGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAK
178

Tian
TSKFSNRRYLCTVTTKNGDCVQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

Wyeth
TSKFSNRRYLCTVTTKNGDCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

*****************************:******************************

Cop
HYNNITWYKDNKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
240

WR
HYNNITWYKDNKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
238

Tian
HYNNITWYKDNKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
240

Wyeth
HYNNITWYKDNKEINIDDIKYSQTGKKLIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
240

**************************:*********************************

Cop
CKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
300

WR
CKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
298

Tian
CKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
300

Wyeth
CKILVTIPSQDHRFKLKRNCGYASN----------------------------------
265

**************** : .

Cop
DFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYFEKTLTTTVVLE
353

WR
DFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYFEKTLTTTVVLE
351

Tian
DFDVYSVLTSRGGITEATLYFENVTEEYIGNTYKCRGHNYYFEKTLTTTVVLE
353

Wyeth
-----------------------------------------------------

B21R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MSLESFIITTFNNNSSTNIDNMCHLYVKVCPSSLLFRLFVECCDINKLVEGTTPLHCYLM
60

Wyeth
MSLESFIITTFNNNSSTNIDNMCHLYVKVCPSSLLFRLFVECCDINKLVEGTTPLHCYLM
60

************************************************************

Cop
NEGFESSVLKNLLKEYVMNTFNVHDIHYTNI
91

Wyeth
NEGFESSVLKNLLKEYVMTSITQIFNS----
87

******************.::.

B22R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCAQFRPCHCHATKDSLNTVADVRHC
60

Wyeth
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCAQFRPCHCHATKDSLNTVADVRHC
60

Lister
-------------------------------MASPCAKFRPCHCHATKDSLNTVADVRHC
29

** ***:**********************

Cop
LTEYILWVSHRWTHRESAGSLYRLLISFRTDATELFGGELKDSLPWDNIDNCVEIIKCFI
120

Wyeth
LTEYILWVSHRWTHRETAGPLYRLLISFRTDATELEGGELKDSLPWDNIDNCVEIIKCFI
120

Lister
LTEYILWVSHRWTHRESAGSLYRLLISFRTDATELFGGELKDSLPWD---NCVEIIKCFI
86

****************:** *************************** **********

Cop
RNDSMKTAEELRAIIGLCTQSAIVSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTAK
180

Wyeth
RNDSMKTAEELRAIIGLCTQSAIVSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTAK
180

Lister
RNDSMKTAEELRAIIGLCTQSAIVSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTAK
146

************************************************************

Cop
Y
181

Wyeth
Y
181

Lister
Y
147

*

B23R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MIAFIIFREIGIISTRIAMDYCGRECTILCRLLDEDVTYKKIKLEIETCHNLSKHIDRRG
60

Wyeth
MIAFIIFREIGIISTRIAMDCT----CILCRLLDEDVTYKKIKLEIETCHNLSKHIDRRG
56

******************** *********************************

Cop
NNALHCYVSNKCDTDIKIVRLLLSRGVERLCRNNEGLTPLGAYSKHRYVKSQIVHLLISS
120

Wyeth
NNALHCYVFNKCDTDIKIVRLLLSRGVERLCRNNEGLTPLGVYSKHRYVKSQIVHLLISS
116

******** ********************************.******************

Cop
YSNSSNELKSNINDFDLSSDNIDLRLLKYLIVDKRIRPSKNTNYAINGLGLVDIYVTTPN
180

Wyeth
YSNSSNELKSNINDFDLSSDNIDLRLLKYLIVDKRIRPSKRTNYAINSLGLVDIYVTTPN
176

***********************************************.************

Cop
PRPEVLLWLLKSECYSTGYVFRTCMYNSDMCKNSLHYYISSHRESQSLSKDVIKCLINNN
240

Wyeth
PRPEVLLWLLKSECYSTGYVFRTCMYNSDMCKNSLHYYISSHRESQSLSKDVIKCLINNN
236

************************************************************

Cop
VSIHGRDEGGSLPIQYYWSFSTIDIEIVKLLLIKDVDTCRVYDVSPILEAYYLNKRFRVT
300

Wyeth
VSIHGRDEGGSLPIQYYWSFSTIDIEIVKLLLIKDVDTCRVYDVSPILEAYYLNKRFRVT
296

************************************************************

Cop
PYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYNHYIIDNILKRFRQQDESIVQAMLIN
360

Wyeth
PYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYNHYIIDNILKRFRQQDESIVQAMLIN
356

************************************************************

Cop
YLHYGDMVVRCMLDNGQQLSSARLLC
386

Wyeth
YLHYGDMVVRCMLDNGQQLSSARLLC
382

**************************

B24R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDDIDMIDNENKTLLYYAVDVNNIQFAKR
60

Wyeth
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDNIDMIDNENKTLLYYAVDVNNIQFAKR
60

*********************************:**************************

Cop
LLEYGASVTTSRSIINTAIQKSSYQRENKTRIVDLLLSYHPTLETMIDAFNRDIRYLYPE
120

Wyeth
LLEYGASVTTSRSIINTAIQKSSYRRENKTKLVDLLLSYHPTLETMIDAFNRDIRYLYPE
120

************************:*****::****************************

Cop
PLFACIRYALILDDDFPSKVSMISPVIIRN------------------------------
150

Wyeth
PLFACIRYALILDDDFPSKVKYDISGRHKELKRYRVDINRMKNAYISGVSMFDILFKRSK
180

********************. ::

Cop
------------------

Wyeth
RHRLRYAKNPTSNGTKKN
198

B25R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MBRINITKKIYCSVFLFLFLFLSYISNYEKVNDEMYEMGEMDEIVSIVRDSMWYIPNVFM
60

WR
----------------------------------------MDEIVRIVRDSMWYIPNVFM
20

Wyeth
MBRINITKKIYCSVFLF--LFLSYISNYEKVNDEMYEMGEMDEIVSIVRDSMWYIPNVFM
58

***** **************

Cop
DDGKNEGHVSVNNVCHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
120

WR
DDGKNEGHVSVNNVCHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
80

Wyeth
DDGKNEGHVSVNNVCMMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
118

************************************************************

Cop
VLKILLHHGMRNFDSKDEKGHHYLIHSLSIDNKIFDILTDTIDDFSKSSDLLLCYLRYKF
180

WR
VLKILLHHGMRNFDSKDEKGHHYQSITRSLIY----------------------------
112

Wyeth
VLKILLHHGMRNFDSKD---DHYQSITRSLIY----------------------------
147

***************** .** : *:

Cop
NGSLNYYVLYKGSDPNCADEDELTSLHYYCKHISTFYKSNYYKLSHTKMRAEKRFIYAII
240

WR
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
DYGANINAVTHLPSTVYQT
259

WR
-------------------

Wyeth
-------------------

B26R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILIVHPSWNDCATGHILIMLLNWHEQK
60

WR
-------------------MLFYLEEPIRGYVIILIVHPSWNDCATGHILIMLLNWHEQK
41

Wyeth
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILIVHPSWNDCATGHILIMLLNWHEQK
60

Lister
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILIVHPSWNDCATGHILIMLLNWHEQK
60

. ********************************

Cop
EEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQKDEYIYRLSKL-----------------
103

WR
EEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
101

Wyeth
EEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
120

Lister
EEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
120

************************************ :

Cop
--------

WR
KDGIRLGV
109

Wyeth
KDGIRLGV
128

Lister
KDGIRLGV
128

B27R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDIIKHRLKVSLPMIKSLFYKMSEFS
60

WR
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDIIHHRLKVSLPMIKSLFYKMSLPT
60

Wyeth
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDIIHHRLK--VPMIKSLFYKMSEFS
58

Lister
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDIIHHRLKVSLPMIKSLFYKMSLPT
60

******************************************* :*********** :

Cop
PYDDYYVKKILAYCLLRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSIIVT
113

WR
TITT-------------------------------------------------
64

Wyeth
PYDDYYVKKILAYCLLRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSIIVT
111

Lister
TITT-------------------------------------------------
64

B28R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCKDNEYKRHNLCPGTYASALCDSKTNTQC
60

WR
MKSVLYSYILELSCIIINGRDIAPHAPSDGKCKDNEYKRHNLCPGTYASRLCDSKTNTQC
60

Wyeth
-----------------------MHHPMESVKTTN--TNAIICV---REHTLPDYANTQC
32

* * :. . * .. :* .: . :****

Cop
TPCGSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNALPDIIVFSKDHPDARHVFPKQN
120

WR
TPCGSGTFTSRNNHLPACLSCNGRRDRVTRLTIESVNALPDIIVFSKDHPDARHVFPKQN
120

Wyeth
TPCGSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNALPDIIVFSKDHPDARHVFPKQN
92

***************************** ******************************

Cop
VE
122

WR
VE
122

Wyeth
V-
93

*

C23L/B29R

CLUSTAL O(1.2.4) multiple sequence alignment

Cop
--------------MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTKQDQTPTNDKI
46

WR
--------------MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTKQDQTPTNDKI
46

Tian
--------------MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTKQDQTPTNDKI
46

Wyeth
--------------MHVPASLQQ---SSSSCTEEENKHHMGIDVIIKVTKQDQTPTNDKI
43

Lister
MKQYIVLACMCLAAAAMPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTKQDQTPTNDKI
60

:****** **********************************

Cop
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSIS
106

WR
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSIS
106

Tian
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSIS
106

Wyeth
CQSVTEITESESDPDPEVESEDDSTSVEDVDIPTTYYSIIGGGLRMNFGFTKCPQIKSIS
103

Lister
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSIS
120

******************************* ****************************

Cop
ESADGNTVNARLSSVSPGQGKDSPAITREEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
166

WR
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
166

Tian
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
166

Wyeth
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
163

Lister
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
180

***************************:********************************

Cop
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGSASE
226

WR
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGSASE
226

Tian
LGSNISHKKVSYEDIIGSTIVDTKCVENLEFSVRIGDMCKESSELEVKDGFKYVDGSASE
226

Wyeth
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGSASE
223

Lister
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGSASE
240

************************************************************

Cop
GATDDTSLIDSTKLKACV
244

WR
GATDDTSLIDSTKLKACV
244

Tian
GATDDTSLIDSTKLKACV
244

Wyeth
GATDDTSLIDSTKLKACV
241

Lister
GATDDTSLIDSTKLKACV
258

******************

Assays for Measuring Virus Characteristics

Assays known in the art to measure the tumor spreading and virulence of a virus include but are not limited to measuring plaque size, syncytia formation, and/or comet assays (EEVs). Assays known in the art to measure the immunostimulatory activity of a virus include but are not limited to NK activation (measured in % CD69 expression), NK degranulation (measured in fold increase of CD107a), and/or T-cell priming assays. Assays known in the art to measure the selectivity of a virus include, but are not limited to, tail pox lesions, biodistribution, and/or body mass measurements.

Examples of Proteins Encoded by Orthopoxvirus Genes

Exemplary proteins encoded by orthopoxvirus genes described in this disclosure are reproduced below in Tables 31-40. As used below, the term “location” refers to the location of the gene with respect to the deleted nucleic acids in exemplary orthopoxvirus vectors described herein. For various genes, amino acid sequence information and protein accession ID numbers are provided.

TABLE 31

Examples of proteins encoded by Copenhagen Vaccinia genes deleted in CopMD5p vector

SEQ ID

Protein

NO.
Gene
Accession ID
Amino Acid Sequence
Location

SEQ ID
C2L
AAA47999.1
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO: 23
(26% 5′)

EAIILNGINYHAFESLLDYIRWKKINITINNVEMILVA
Deletion

AIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGI

KKLYNASMSEIINNITAVTSDPEFGKLSKDELTTILS

HENVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETICKWKTVTNMSS

LKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYVITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEKQ

FLQ

SEQ ID
C1L
AAA48000.1
MVKNNKISNSCRMIMSTNPNNILMRHLKNLTDDEF
Inside

NO: 24

KCIIHRSSDFLYLSDSDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
N1L
AAA48001.1
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO: 25

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
N2L
AAA48002.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMD
Inside

NO: 26

CINRHINMCIQRTYSSSIIAILNRFLTMNKDELNNTQ
Deletion

CHIIKEFMTYEQMAIDHYGEYVNAILYQIRKRPNQH

HTIDLFKKIKRTPYDTFKVDPVEFVKKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFINYVETKYF

SEQ ID
M1L
AAA48003.1
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLS
Inside

NO: 27

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKSVDTPLYYLSGTDDE

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYE

TMVDYLLFNHFSVDSVVNGHTCMSECVRLNNPVIL

SKLMLHNPTSETMYLTMKAIEKDKLDKSIIIPFIAYF

VLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFD

DYF

SEQ ID
M2L
AAA48004.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO: 28

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNICLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
HR/K1L
AAA48005.1
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO: 29

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGMDDSQFDDKGNTALYYAVDSG

NMQTVKLFVKKNWRLMFYGKTGWKTSFYHAVML

NDVSIVSYFLSEIPSTFDLAILLSCIHTTIKNGHVDMM

ILLLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQAL

FKYDINIYSVNLENVLLDDAEITKMIIEKHVEYKSDS

YTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

SEQ ID
SPI-
AAA48006.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO: 30
3/K2L

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPLYYQQYHRFGLYRLNFRRDAVNKINSIVERRSG

MSNVVDSNMLDNNTLWAIINTIYFKGTWQYPFDIT

KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDE

EYDMVRLPYKDANISMYLAIGDNMTHFIDSITAAK

LDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMM

APSMFNPDNASFKHMTRDPLYIYKMFQNAKIDVDE

QGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITG

FILFMGKVESP

SEQ ID
K ORF A
AAA48007.1
MGHIITYCQVHTNISILIRKAHHIIFFVIDCDCISLQFS
Inside

NO: 31

NYVHHGNRFRTVLISKTSIACFSDIKRILPCTFKIYSI
Deletion

NDCP

SEQ ID
K ORF B
AAA48008.1
MGTVFVPYLLVKLALRVLVISNGYCHVPLKYIVLMI
Inside

NO: 32

AHRVLLSSILESTTLDIPDLRSTIELILLTASRLICFNLY
Deletion

RPNL

SEQ ID
K ORF B
AAA48009.1
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPH
Inside

NO: 33

SEAILAESVICMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
K4L
AAA48010.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO: 34

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPIIYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNIDIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
K5L
AAA48011.1
MGATISILASYDNPNLFTAMILMSPLVNADAVSRLN
Inside

NO: 35

LLAAKLMGTITPNAPVGKLCPESVSRDMDKVYKYQ
Deletion

YDPLINHEKIKAGFASQVLKATNKVRKIISKINTPRL

SYSREQTMRLVMFQVHIISCNMQIVIE

SEQ ID
K6L
AAA48012.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO: 36

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
K7R
AAA48013.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO: 37

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
F1L
AAA48014.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO: 38

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

ICDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
DUT/F2L
AAA48015.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO: 39

SAYDYTIPPGERQLIKTDISMSMPKICYGRIAPRSGLS
Deletion

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTNRGDQGFSTGLR

SEQ ID
F3L
AAA48016.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO: 40
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFITYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYATGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

PRDNPELIIVDNKLLLIGGFYRGSYIDTIEVYNHHTY

SWNIWDGK

TABLE 32

Examples of proteins encoded by Western Reserve Vaccinia genes equivalent

to those deleted in CopMD5p vector

SEQ ID

Protein

NO.
Gene
Accession ID
AA Sequence
Location

SEQ ID
VACWR026
AAO89305.1
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO: 41
(26% 5′)

EAIILNGINYHAFESLLDYIRWKKINITINNVEMILVA
Deletion

AIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGI

KKLYNASMSEIINNITAVTSDPEFGKLSKDELTTILS

HENVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSS

LKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYVITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEKQ

FLQ

SEQ ID
VACWR027
AAO89306.1
MVKNNKIQKNKISNSCRMIMSTDPNNILMRHLKNL
Inside

NO: 42

TDDEFKCIIHRSSDFLYLSDSDYTSITKETLVSEIVEE
Deletion

YPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAI

LDKMTEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSY

RTRAINKYSKELGLATEYFNKYGHLMFYTLPIPYNR

FFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKF

KKELMSK

SEQ ID
VACWR028
AAO89307.1
MRTLLIRYILWRNDNDQTYYNDNFKKLMLLDELVD
Inside

NO: 43

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
VACWR029
AAO89308.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMD
Inside

NO: 44

CINRHINMCIQRTYSSSIIAILDRFLMMNKDELNNTQ
Deletion

CHIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQH

HTIDLFKRIKRTRYDTFKVDPVEFVKKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFINYVETKYF

SEQ ID
VACWR030
AAO89309.1
MIFVIESKLLQIYRNRNRNINFYTTNIDNIMSAEYYLS
Inside

NO: 45

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKTPLYYLSGTDDE

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYE

TMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVIL

SKLMLHNPTSETMYLTMKAIEKDRLDKSIIIPFIAYF

VLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFD

DYF

SEQ ID
VACWR031
AAO89310.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO: 46

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYSMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
VACWR032
AAO89311.1
MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALY
Inside

NO: 47

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNICDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

SEQ ID
SPI-3
AAO89312.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO: 48

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPSYYQQYHRFGLYRLNFRRDAVNKINSIVERRSG

MSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDITK

TRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEE

YDMVRLPYKDANISMYLAIGDNMTHFTDSITAAKL

DYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAP

SMFNPDNASFKHMTRDPLYIYKMFQNAKIDVDEQG

TVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI

LFMGKVESP

SEQ ID
VACWR034
AAO89313.1
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPH
Inside

NO: 49

FEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
VACWR035
AAO89314.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO: 50

TTKTLDISSFYWSLSDEVGTNFGTIILNKIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPIIYSKAGNI

LFWPYIEDELRRAAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCRLLK

NMKQCINDIYCDEIQPEKEIPEYSLE

SEQ ID
VACWR036
AAO89315.1
MQHANCNREIKIYEGAKHHLHKETDEVKKSVMKEI
Outside

NO: 51

ETWIFNRVK
Deletion

SEQ ID
VACWR037
AAO89316.1
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMIL
Inside

NO: 52

MSPLVNADAVSKLNLLAAKLMGTITLNAPVGKLCP
Deletion

ESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKA

TNKVRKIISKINTPRLSYSREQTIRLAMF

SEQ ID
VACWR038
AAO89317.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO: 53

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
VACWR039
AAO89318.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO: 54

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
VACWR040
AAO89319.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO: 55

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVETIKMFTLLSHT

ICDDYFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
DUT
AAO89320.1
MFNMNINSPVRFVKETNRAKSPTROQSPYAAGYDLY
Inside

NO: 56

SAYDYTIPPGERQLIKTDISMSMPKFCYGRIAPRSGL
Deletion

SLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTG

DRIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGLR

SEQ ID
VACWR042
AAO89321.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO: 57
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTWVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

PRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYS

WNIWDGK

TABLE 33

Examples of proteins encoded by Tian Tan Vaccinia genes

equivalent to those deleted in CopMD5p vector

SEQ ID

Protein

NO
Gene
Accession ID
AA Sequence
Location

SEQ ID
TC2L
AAF33878.1
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO: 58
(26% 5′)

EAIILNGINYHAFESLLDYIRWKKINITINNVEMILVA
Deletion

AIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKQYGI

KKLYNASMSEIINNITAVTSDPEFGKLSKDELTTILS

HEDVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSS

LKSEVSTCVNNGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYVITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEKQ

FLQ

SEQ ID
TC1L
AAF33879.1
MVKNNKISNSCRMIMSTDPNNILMRHLKNLTDDEF
Inside

NO: 59

KCIIHRSSDFLYLSDSDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
TN1L
AAF33880.1
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO: 60

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
TN2L
AAF33881.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPYIMD
Inside

NO: 61

CINRHINMCIQRTYSSSIIAILDRFLMMNKDELNNTQ
Deletion

CHIIKNL

SEQ ID
TM1L
AAF33882.1
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLS
Inside

NO: 62

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKTPLYYLSGTDDE

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYE

TMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVIL

SKLMLHNLTSETMYLTMKAIEKDRLDKSIIIPFIAYF

VLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFD

DYF

SEQ ID
TM2L
AAF33883.1
MSSSTRLPVLVLAAELTIGVNYDINSTIIGECHMSES
Inside

NO: 63

YIDRNANIVLTGYGLEINMTIMDTDQRFVAAAEGV
Deletion

GKDNKLSVLLFTTQRLDKVHHNISVTITCMEMNCG

TTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPT

KINPHYLHPKDKYLYHNSEYGMRGSYGVTFIDELN

QCLLDIKELSYDICYRE

SEQ ID
TK1L
AAF33884.1
MLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHV
Inside

NO: 64

EYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNC
Deletion

VKKN

SEQ ID
TK2L
AAF33885.1
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO: 65

YAIADNNVRLVCTLLNSGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTVDKHQ

SEQ ID
TK3L
AAF33886.1
MIALLILSLACSASAYRLQGFTNAGIVAYKNIQDDNI
Inside

NO: 66

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPSYYQQYHRFGLYRLNFRRDAVNKINSIVERRSG

MSNVVDSNMLDNNTLWAIINTIYFKGTWQYPFDIT

KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDE

EYDMVRLPYKDANISMYLAIGDNMTHFTDSITAAK

DYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAP

SMFNPDNASFKHMTRDPLYIYKMFQNAKIDVDEQG

TVAEASTIMVATARSSPEELEFNTPFVFIIRHDITGFIL

FMGKVESP

SEQ ID
ORFR
AAF33887.1
MGHIITYCQVHTNISILIRKAYHIIFFVIDCDCISLQFS

NO: 67

NYVHHGNRFRTVLISKTSIACFSDIKRILPCTFKIYSI

NDCP

SEQ ID
TK4L
AAF33888.1
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPH
Inside

NO: 68

SEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
TK6L
AAF33889.1
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMIL
Inside

NO: 69

MSPLVNADAVSKLNLLAAKLMGTITLNAPVGKLCP
Deletion

ESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKA

TNKVRKIISKINTPRLSYSREQTIRLAMF

SEQ ID
TK8R
AAF33890.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO: 70

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
TF1L
AAF33891.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDR
Inside

NO: 71

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

ICDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
TF2L
AAF33892.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO: 72

SAYDYTIPPGERQLIKTDISMSMPKICYGRIAPRSGLS
Deletion

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTDRGDQGFGSTGLR

SEQ ID
TF3L
AAF33893.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO: 73
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

PRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYS

WNIWDGK

TK5L

ORFR

TK7L

TABLE 34

Examples of proteins encoded by Wyeth Vaccinia genes

equivalent to those deleted in CopMD5p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
VAC_DPP20_035
AEY74729.1
MESVTFSINGEIIQVNKEIITASPYNFFKRIQEHHINDE
Inside

NO: 74
(26% 5′)

VIILNGINYHAFESLLDYMRWKKINITINNVEMILVA
Deletion

AVIIDVTPVVDLCVKTMIHNINSTNCIRMFNFSKRYG

IKKLYNASMSEIINNITAVTSDPEFGKLSKDELTTILS

HEDVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSS

LKSEVSTCVNNGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYAITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETHKSLPSFLSNCEKQFLQ

SEQ ID
VAC_DPP10_036
AEY74730.1
MVKNNKISNSCRMIMSTNPNNILMRHLKNLTDDEF
Inside

NO: 75

KCIIHRSSDFLYLSDRDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
N1L
AEY74731.1
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO: 76

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
VAC_DPP11_038
AEY74732.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIIDC
Inside

NO: 77

INRHINMCIQRTYSSSIIAILDRFLTMNKDELNNTQC
Deletion

HIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQHH

TIDLFKKIKRTRYDTFKVDPVEFVKKVIGFVSILNKY

KPVYSYVLYENVLYDEFKCFIDYVETKYF

SEQ ID
VAC_DPP11_039
AEY74733.1
MLHNPTSETMYLTMNAIKKDKLDKSIIIPFIAYFVLM
Not

NO: 78

HPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
Present

SEQ ID
VAC_DPP12_040
AEY74734.1
MSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWM
Inside

NO: 79

MKLGISPSKPDHDGNTPLHIVCSKTVKYVDIIDLLLP
Deletion

STDVNKQNKFGDSPLTLLIKTLSLAHINKLLSTSNV

ITDQTVNICIFYDRDDVLEIINDKGKQYDFKMAVEV

GSIKCVKYLLDNDIICEDAMYYAVLSEYKTMVDYL

LFNHFSVDSVVNGHTCMSECVKLNNRHFIEADVT

SEQ ID
VAC_DPP12_041
AEY74735.1
MIFVLESKLLQIYRNRNRNINFYTTMDNIMSAEYYLS
Not

NO: 80

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Present

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKTPLYYLSGTDDE

VIERINLLVQYGAKINN

SEQ ID
VAC_DPP12_042
AEY74736.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO: 81

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
VAC_DPP10_043
AEY74737.1
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO: 82

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

SEQ ID
VAC_DPP20_044
AEY74738.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO: 83

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPSYYQQYHRLNFRRDAVNKINSIVERRSGMSNVV

DSNMLDNNTLWAIINTIYFKGIWQYPFDITKTRNAS

FTNKYGTKTVPMMNVVTKLQGNTITIDDKEYDMV

RLPYKDANISMYLAIGDNMTHFTDSITAAKLDYWS

FQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFN

PDNASFKHMTRDPLYIYKMFQNAKIDVDEQGTVAE

ASTIMVATARSSPEKLEFNTPFVFIIRHDITGFILFMG

KVESP

SEQ ID
VAC_DPP10_045
AEY74739.1
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPH
Inside

NO: 84

FEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
K4L
AEY74740.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO: 85

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPIIYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
VAC_DPP20_047
AEY74741.1
MGATISILASYDNPNLFTAMILMSPLVNADAVSKLN
Inside

NO: 86

LLAAKLMGTITPNAPVGKLCPESVSRDMDKVYKYQ
Deletion

YDPLINHEKIKAGFASQVLKATNKVRKIISKINTPPT

LILQGTNNEISDVLGAYYFMQHANCNREIKIYEGAK

HHLHKETDEVKKSVMKEIETWIFNRVK

SEQ ID
List034/
AEY74742.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO: 87
VAC_DPP20_048

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
K7R/
AEY74743.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO: 88
VAC_DPP20-49

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
LIVPclone14_046/
AEY74744.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO: 89
VAC_DPP20_047

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITKLGRRCMNPVKTIKMFTLLSH

TICDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIM

FATYKTLKYMIG

SEQ ID
F2L/
AEY74745.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO: 90
VAC_DPP20_051

SAYDYTIPPGERQLIKTDISMSMPKICYGRIAPRSGLS
Deletion

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGLR

SEQ ID
F3L
AEY74746.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO: 91
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

PRDNPELIIVDNKILLIGGFYRESYIDTIEVYNHHTYS

WNIWDGK

TABLE 35

Examples of proteins encoded by Lister Vaccinia genes equivalent to

those deleted in CopMD5p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
List023
ABD52473.1
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO: 92
(26% 5′)

EAIILNGINYHAFESLLDYMRWKKINITINNVEMILV
Deletion

AAIIIDVPPVVDLCVKTMIHNINFTNCIRMFNFSKRY

GIKKLYNASMSEIINNITAVTSDPEFGKLSKDELTTIL

SHEDVNVNHEDVTAMILLKWIHKNPNDVDIINILHP

KFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVI

KNSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLH

NCLYIIGGMINNRHVYSVSRVDLKTKKWKTVTNMS

SLKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHG

TSKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTY

EKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEY

DGDIYVITGITHETRNYLYKYIVKEDKWIELYMYFN

HVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFD

MSTRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEK

QFLQ

SEQ ID
C1L/List024
ABD52474.1
MVKNNKISNSCRMIMSTNPNNILMRHLKNLTDDEF
Inside

NO: 93

KCIIHRSSDFLYLSDSDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKM

TADIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
N1L/List025
ABD52475.1
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO: 94

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
List026
ABD52476.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMD
Inside

NO: 95

CINRHINMCIQRTYSSSIIAILDRFLTMNKDELNNTQ
Deletion

CHIIKEFMTYEQMAIDHYGEYVNAILYQIRKRPNQH

HTIDLFKKIKRTRYDTFKVDPVEFVKKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFIDYVETKYF

SEQ ID
List027
ABD52477.1
MIFVIESKLLQIYRNRNINFYTTMDNIMSAEYYLSLY
Inside

NO: 96

AKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCGIK
Deletion

GLDERFVEELLHRGYSPNETDDDGNYPLHIASKINN

NRIVAMLLTHGADPNACDKQHKTPLYYLSGTDDEV

IERINLLVQYGAKINNSVDEEGCGPLLACTDPSERVF

KKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTIS

WMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIIDL

LLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLST

SNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDFK

MAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYET

MVDYLLFNHFSVDSVVNGHTCMSECVRLNNPVILS

KLMLHNPTSETMYLTMKAIEKDRLDKSIIIPFIAYFV

LMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDD

YF

SEQ ID
List028
ABD52478.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO: 97

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
K1L/List029
ABD52479.1
MDLSRINTWKSKQLKSFLSSKDAFKADINGHSALY
Inside

NO: 98

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISKNKELKLMYVNCVKKN

SEQ ID
List030
ABD52480.1
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYT
Inside

NO: 99

DLTYQSFVDNTVCIKPSYYQQYHRFGLYRLNFRRD
Deletion

AVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTI

YFKGIWQYPFDITKTRNASFTNKYGTKTVPMMNVV

TKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDN

MTHFTDSITAAKLDYWSSQLGNKVYNLKLPKFSIEN

KRDIKSIAEMMAPSMFNPDNASFKHMTRDPLYIYK

MFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFN

TPFVFIIRHDITGFILFMGKVESP

SEQ ID
K3L/List031
ABD52483.1
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPH
Inside

NO: 100

SEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
K4L/List032
ABD52484.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO: 101

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPIIYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
List033
ABD52485.1
MGHSMGATISILASYDNPNLFTAMILMSPLVNADA
Outside

NO: 102

VSRLNLLAAKLMGTITPNAPVGKLCPESVSRDMDK
Deletion

VYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKI

NTPPTLILQGTNNKISDVLGAYYFMQHANCNREIKI

YEGAKHHLHKETDEVKKSVMKEIETWIFNRVK

SEQ ID
List034
ABD52486.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO: 103

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
K7R/List035
ABD52487.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO: 104

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
F1L/List036
ABD52489.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO: 105

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

ICDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
List037
ABD52490.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO: 106

SAYDYTIPPGERQLIKTDISMSMPKFCYGRIAPRSGL
Deletion

SLKGIDIGGGV1DEDYRGNIGVILINNGKCTFNVNTG

DRIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGLR

SEQ ID
List038
ABD52491.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO: 107
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSIKLILESDELNVPDEDYVV

DFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINN

VKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQII

DIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNY

ISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNPT

SVERWFHGDAAWVNMPSLLKPRCNPAVASINNVIY

VMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKS

CALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIYPR

DNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSW

NIWDGK

TABLE 36

Examples of proteins encoded by Copenhagen Vaccinia genes deleted in CopMD3p vector

SEQ ID

Protein

NO
Gene
Accession ID
Amino Acid Sequence
Location

SEQ ID
B14R
AAA49211.1
MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPT
Inside

NO: 108
(41% 3′)

EMVDVSMMSMYGELFNHASVKESFGNFSIIELPYV
Deletion

GDTSMMVILPDKIDGLESIEQNLTDTNFKKWCNSLD

AMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGSTG

DYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAATC

ALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRY

CSPTTNC

SEQ ID
B15R
AAA49212.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO: 109

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
B ORF E
AAA48213.1
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMV
Inside

NO: 110

FTSPVSSSICTKSDDGRNLSDGFLLIRYITTDDFCTIF
Deletion

DIIPRHIFYQLANVDEH

SEQ ID
B16R
AAA48214.1
MSILPVIFLPIFFYSSFVQTFNASECIDKGXYFASFME
Inside

NO: 111

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLN

LTIVSVSESNIDFISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIHPTM

QLPEGVVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGDGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
B ORF F
AAA48215.1
MVIIPGVRCLSLLFLRRRCPLHIISAFTLLAINALILGH
Inside

NO: 112

TISPVDLSFTICGYEIKSIFDSETDTIVKFNDIMSQ
Deletion

SEQ ID
B17L
AAA48216.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO: 113

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

CKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITP

VEAPLPGNVLVYTFPDINKRIPGYIHVNIEGCIDGMI

YINSSKFACVLKLHRSMYRIPPFPIDICSCCSQYTND

DIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYF

NNIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVN

HSNTFVNCLLEDNV

SEQ ID
B18R
AAA48217.1
MSRRLIYVLNINRKSTHKIQENEIYTYFSHCNIDHTS
Inside

NO: 114

TELDFVVKNYDLNRRQHVTGYTALHCYLYNNYFT
Deletion

NDVLKILLNHDVNVTMKTSSGRMPVYILLTRCCNIS

HDVVIDMIDKDKNHLSHRDYSNLLLEYIKSRYMLL

KEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLC

LAHVYKPGECRKPITIKKAKRIISLFIQHGANLNALD

NCGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKICN

NHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPI

DERRMIVFEFIKTYSTRPADSITYLMNRFKNINIYTR

YEGKTLLHVACEYNNTQVIDYLIRINGDINALTDNN

KHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLV

DQLPSLPIFDIKSFEKFISYCILLDDTFYDRHVKNRDS

KTYRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVL

DTTLYAVLRCHNSRKLRRYLTELKKYNNDKSFKIY

SNIMNERYLNVYYKDMYVSKVYDKLFPVFTDKNC

LLTLLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
B19R
AAA48218.1
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFN
Inside

NO: 115

KMRDTLPAKDSKWLNPACMFGGTMNDMATLGEPF
Deletion

SAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVS

NKRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDC

VQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGIL

YAKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPE

LEDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQ

DHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLI

EWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENV

TEEYIGNTYKCRGHNYYFEKTLTTTVVLE

SEQ ID
B20R
AAA48219.1
MDEDTRLSRYLYLTDREHINVDSIKQLCKISDPNAC
Inside

NO: 116

YRCGCTALHEYFYNYRSVNGKYKYRYNGYYQYYS
Deletion

SSDYENYNEYYYDDYDRTGMNSESDSESDNISIKTE

YENEYEFYDETQDQSTQHNDL

SEQ ID
B21R
AAA48220.1
MSLESFIITTFNNNSSTNIDNMCHLYVKVCPSSLLFR
Inside

NO: 117

LFVECCDINKLVEGTTPLHCYLMNEGFESSVLKNLL
Deletion

KEYVMNTFNVHDIHYTNI

SEQ ID
B22R
AAA48221.1
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCA
Inside

NO: 118

QFRPCHCHATKDSLNTVADVRHCLTEYILWVSHRW
Deletion

THRESAGSLYRLLISFRTDATELFGGELKDSLPWDNI

DNCVEIIKCFIRNDSMKTAEELRAIIGLCTQSAIVSGR

VFNDKYIDILLMLRKILNENDYLTLLDHIRTAKY

SEQ ID
B23R
AAA48222.1
MIAFIIFREIGIISTRIAMDYCGRECTILCRLLDEDVTY
Inside

NO: 119

KKIKLEIETCHNLSKHIDRRGNNALHCYVSNKCDTD
Deletion

IK1VRLLLSRGVERLCRNNEGLTPLGAYSKHRYVKS

QIVHLLISSYSNSSNELKSNINDFDLSSDNIDLRLLKY

LIVDKRIRPSKNTNYAINGLGLVDIYVTTPNPRPEVL

LWLLKSECYSTGYVFRTCMYNSDMCKNSLHYYISS

HRESQSLSKDVIKCLINNNVSIHGRDEGGSLPIQYY

WSFSTIDIEIVKLLLIKDVDTCRVYDVSPILEAYYLN

KRFRVTPYNVDMEIVNLLIERRHTLVDVMRSITSYD

SREYNHYIIDNILKRFRQQDESIVQAMLINYLHYGD

MVVRCMLDNGQQLSSARLLC

SEQ ID
B24R
AAA48223.1
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDDID
Inside

NO: 120

MIDNENKTLLYYAVDVNNIQFAKRLLEYGASVTTS
Deletion

RSIINTAIQKSSYQRENKTRIVDLLLSYHPTLETMIDA

FNRDIRYLYPEPLFACIRYALILDDDFPSKVSMISPVII

RN

SEQ ID
B ORF G
AAA48224.1
MRRCIHIKERKIHMTNIVDRNVTFILTVVHKYVRYV
Inside

NO: 121

PHTVANDAHNLVHLAHLIHFIIYFFIIRDVRKKKKKK
Deletion

KKNRTIYFFSNVYARHIK

SEQ ID
B25R
AAA48225.1
MSRINITKKIYCSVFLFLFLFLSYISNYEKVNDEMYE
Inside

NO: 122

MGEMDEIVSIVRDSMWYIPNVFMDDGKNEGHVSV
Deletion

NNVCHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNS

PLHCYTMNTRFNPSVLKILLHHGMRNFDSKDEKGH

HYLIHSLSIDNKIFDILTDTIDDFSKSSDLLLCYLRYK

FNGSLNYYVLYKGSDPNCADEDELTSLHYYCKHIST

FYKSNYYKLSHTKMRAEKRFIYAIIDYGANINAVTH

LPSTVYQT

SEQ ID
B26R
AAA48226.1
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILI
Inside

NO: 123

VHPSWNDCATGHILIMLLNWHEQKEEGQHLLYLFI
Deletion

KHNQGYTLNILRYLLDRFDIQKDEYIYRLSKL

SEQ ID
B27R
AAA48227.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO: 124

IHHRLKVSLPMIKSLFYKMSEFSPYDDYYVKKILAY
Deletion

CLLRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSII

VT

SEQ ID
B28R
AAA48228.1
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCKDNEY
Inside

NO: 125

KRHNLCPGTYASRLCDSKTNTQCTPCGSGTFTSRNN
Deletion

HLPACLSCNGRRDRVTLLTIESVNALPDIIVFSKDHP

DARHVFPKQNVE

SEQ ID
C23L/B29R
AAA48229.1
MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTK
Inside

NO: 126
(44% 5′)

QDQTPTNDKKQSVTEITESESDPDPEVESEDDSTSV
Deletion

EDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSISESAD

GNTVNARLSSVSPGQGKDSPAITREEALAMIKDCEV

SIDIRCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGST

IVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVD

GSASEGATDDTSLIDSTKLKACV

TABLE 37

Examples of proteins encoded by Western Reserve Vaccinia genes

equivalent to those deleted in CopMD3p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
SPI-
AAO89474.1
MDIFREIASSMKGENVFISPASISSVLTILYYGANGST
Inside

NO: 127
2/B13R/

AEQLSKYVEKEENMDKVSAQNISFKSINKVYGRYS
Deletion

VACWR195

AVFKDSFLRKIGDKFQTVDFTDCRTIDAINKCVDIFT

(26% 3′)

EGKINPLLDEPLSPDTCLLAISAVYFKAKWLTPFEKE

FTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVKE

SFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTD

TNFKKWCNSLEATFIDVHIPKFKVTGSYNLVDTLVK

SGLTEVFGSTGDYSNMCNSDVSVDAMIHKTYIDVN

EEYTEAAAATCALVSDCASTITNEFCVDHPFIYVIRH

VDGKILFVGRYCSPTTNC

SEQ ID
VACWR196
AAO89475.1
MTANFSTHVFSPQHCGCDRLTSIDDVRQCLTEYIYW
Inside

NO: 128

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
VACWR197
AAO89476.1
MSILPVIFLSIFFYSSFVQTFNAPECIDKGQYFASFME
Inside

NO: 129

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
VACWR198
AAO89477.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO: 130

SAEKYMCRYTTLNHNCINVRRCALDSKLLHDIITNC
Deletion

KIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYP

VIFITHTSTRNLDKVSVKTYKGVKVKKLNRCADHAI

VINPFVKFKLTLPNKTSHAKVLVTFCKLKTDITPVEA

PLPGNVLVYTFPDINKRIPGYIHLNIEGCIDGMIYINS

SKFACVLKLHRSMYRIPPFPIDKSCCSQYINYDIEIPI

HDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNID

TAITQEHEYVKIALGIVCKLMINNMHSIVGVNHSNT

FVNCLLEDNV

SEQ ID
VACWR199
AAO89478.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO: 131

ELDFVVKNYDLNRRQPVTGYTALHCYLYNNYFTN
Deletion

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLLHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPITIKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFEICNN

HGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNIDIYTRYE

GKTLLHVACEYNNTHVIDYLIRINGDINALTDNNKH

ATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYNRHVRNRDSKT

YRYAFSKYMSFDKYDGIITKCHKETILLKLSTVLDT

TLYAVLRCHNSKKLRRYLTELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
B18R/
AAO89479.1
MTMKMMVHIYFVSLLLLLFHSYAIDIENEITEFFNK
Inside

NO: 132
VACWR200

MRDTLPAKDSKWLNPACMFGGTMNDIAALGEPFS
Deletion

AKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSN

KRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDCV

QGIVSHIRKPPSCIPKTYELGTHDKYGIDLYCGILY

AKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPEL

EDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQD

HRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIE

WENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVT

EEYIGNTYKCRGHNYYFEKTLTTTVVLE

SEQ ID
VACWR201
AAO89480.1
MHVIDVDVRLYMSTFIIIDQSTENTSIDTTVTINIIYL
Not

NO: 133

AIMKIIMNIIMMIMIELV
Present

SEQ ID
VACWR202
AAO89481.1
MNSESDNISIKTEYEFYDETQDQSTQLVGYDIKLKT
Not

NO: 134

NEDDFMANIDQWVSMII
Present

SEQ ID
VACWR203
AAO89482.1
MEMYPRHRYSKHSVFKGFSDKVRKNDLDMNVVKE
Inside

NO: 135

LLSNGASLTIKDSSNKDPITVYFRRTIMNLEMIDERK
Deletion

YIVHSYLKNYKNFDYPFFRKLVLTNKHCLNNYYNIS

DSKYGTPLHILASNKKLITPNYMKLLVYNGNDINAR

GEDTQMRTPLHKYLCKFVYHNIEYGIRYYNEKIIDA

FIELGADLTIPNDDGMIPVVYCIHSNAEYGYNNITNI

KIIRKLLNLSRRASHNLFRDRVMHDYISNTYIDLECL

DIIRSLDGFDINGYFEGRTPLHCAIQHNFTQIAKYLL

DRGADIVVPNTLIIHQYIQ

SEQ ID
VACWR204
AAO89483.1
MLNFSLCLYPVFILNKLVLRTQSIILHTINNASIKNR
Not

NOS 677

\\\\\\ and \\\\\\
Present

and 136

MEEDTNISNKVIRYNTVNNIWETLPNFWTGTINPGV

VSHKDDIYVVCDIKDEKNVKTCIFRYNTNTYNGWE

LVTTTESRLSALHTILYNNTIMMLHCYESYMLQDTF

NVYTREWNHMCHQHSNSYIMYNILPIY

SEQ ID
SPI-1/
AAO89484.1
MDIFKELILKHTDENVLISPVSILSTLSILNHGAAGST
Outside

NO: 137
VACWR205

AEQLSKYIENMNENTPDDNNDMDVDIPYCATLATA
Deletion

NKIYGSDSIEFHASFLQKIKDDFQTVNFNNANQTKE

LINEWVKTMTNGKINSLLTSPLSINTRMTVVSAVHF

KAMWKYPFSKHLTYTDKFYISKNIVTSVDMMVSTE

NNLQYVHINELFGGFSIIDIPYEGNSSMVIILPDDIEGI

YNIEKNITDEKFKKWCGMLSTKSIDLYMPKFKVEM

TEPYNLVPILENLGLTNIFGYYADFSKMCNETITVEK

FLHTTFIDVNEEYTEASAVTGVFMTNFSMVYRTKV

YINHPFMYMIKDNTGRILFIGKYCYPQ

SEQ ID
C13L/
AAO89485.1
MMIYGLIACLIFVTSSIASPLYIPVIPPISEDKSFNSVE
Outside

NO: 138
VACWR206

VLVSLFRDDQKDYTVTSQFNNYTIDTKDWTIGVLST
Deletion

PDGLDIPLTNITYWSRFTIGRALFKSESEDIFQKKMSI

LGVSIECKKSSTLLTFLTVRKMTRVFNKFPDMAYYR

GDCLKAVYVTMTYKNTKTGETDYTYLSNGGLPAY

YRNGVDG

SEQ ID
VACWR207
AAO89486.1
MKLFTQNDRYFGLLDSCTHIFCITCINIWHKTRRETG
Outside

NO: 139

ASDNCPICRTRFRNITMSKFYKLVN
Deletion

SEQ ID
p28/
AAO89487.1
MEFDPAKINTSSIDHVTILQYIDEPNDIRLTVCIIRNIN
Outside

NO: 140
VACWR208

NITYYINITKINTHLANQFRAWKKRIAGRDYMTNLS
Deletion

RDTGIQQSKLTETIRNCQKNRNIYGLYIHYNLVINVV

IDWITDVIVQSILRGLVNWYIANNTYTPNTPNNTTTI

SELDIIKILDKYEDVYRVSKEKECGICYEVVYSKR

SEQ ID
C10L/
AAO89488.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO: 141
VACWR209

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDSTIT
Deletion

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPICGGKIKLYHPTGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSMTTIHYNDDVDIDIHTDK

NGKELCYCYITIDDHYLVDVETIGVIVNRSGKCLLV

NNHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFS

LTNDDNRNIAWDTDKLDDDTDIWTPVTEDDYKFLS

RLVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRF

YFNMPK

SEQ ID
VGF-1/
AAO89489.1
MSMKYLMLLFAAMIIRSFADSGNAIETTSPEITNATT
Outside

NO: 142
VACWR210

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

GYTGIRCQHVVLVDYQRSENPNTTTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTKLPIQDMVVP

SEQ ID
VACWR211
AAO89490.1
MDEIVRIVRDSMWYIPNVFMDDGKNEGHVSVNNV
Inside

NO: 143

CHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHC
Deletion

YTMNTRFNPSVLKILLHHGMRNFDSKDEKGHHYQS

ITRSLIY

SEQ ID
C20L/
AAO89491.1
MLFYLEEPIRGYVIILIVHPSWNDCATGHILIMLLNW
Inside

NO: 144
VACWR212

HEQKEEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQ
Deletion

KDEYYNTAFQNCNNNVASYIGYDINLPTKDGIRLGV

SEQ ID
VACWR213
AAO89492.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO: 145

IHHRLKVSLPMIKSLFYKMSLPTTITT
Deletion

SEQ ID
VACWR214
AAO89493.1
MYDDLIEQCHLSMERKSKLVDKALNKLESTIGQSRL
Outside

NO: 146

SYLPPEIMRNII
Deletion

SEQ ID
B28R/
AAO89494.1
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCKDNEY
Inside

NO: 147
VACWR215

KRHNLCPGTYASRLCDSKTNTQCTPCGSGTFTSRNN
Deletion

HLPACLSCNGRRDRVTRLTIESVNALPDIIVFSKDHP

DARHVFPKQNVE

SEQ ID
VACWR216
AAO89495.1
MDSLRPVVVVNWIQINFHIDIVKGITGYGFAFICGRD
Outside

NO: 148

GVRICSETTRRTDDVSGYSVSYSTFCLGNTCLASG
Deletion

SEQ ID
VACWR217
AAO89496.1
MWKLICIQLTTTTGLSESISTSELTITMNHKDCNPVF
Outside

NO: 149

REEYFSVLNKVATSGFFTGERCAL
Deletion

SEQ ID
B29R/
AAO89497.1
MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTK
Inside

NO: 150
VACWR218

QDQTPTNDKICQSVTEITESESDPDPEVESEDDSTSV
Deletion

(44% 5′)

EDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSISESAD

GNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEV

SIDIRCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGST

IVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVD

GSASEGATDDTSLIDSTKLKACV

TABLE 38

Examples of proteins encoded by Tian Tan Vaccinia genes

equivalent to those deleted in CopMD3p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
TF3L
AAF34083.1
MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPT
Inside

NO: 151
(41% 3′)

EMVDVSMMSMYGKAFNHASVKESFGNFSIIELPYV
Deletion

GDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFM

DAMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGST

GDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAA

TCALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVG

RYCSPTTNC

SEQ ID
TB15R
AAF34084.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO: 152

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
ORFL
AAF34085.1
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMV

NO: 153

FTSPVSSSICTKSDDGRNLSDGFLLIRYITTDDFCTIF

DIIPRHIFYQLANVDEH

SEQ ID
TB16R
AAF34086.1
MELENEPVILPCPQINTLSSGYNILDILWEKRGADND
Inside

NO: 154

RIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMS
Deletion

LNLTIVSVLESNIDLISYPQIVNERSTGEMVCPNINAF

IASNVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRK

NDAGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPS

TMQLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWIS

NGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRL

NINPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
ORFL
AAF34087.1
MVIIPGVRCLSLLFLRRRCPLHIISAFTLLAINALILGH

NO: 155

TISPVDLSFTICGYEIRSIFDSKTDTIVKFNDIMSQ

SEQ ID
TB17L
AAF34088.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO: 156

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

CKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQ

IEAPLSGNVLVYTFPNINKRIPGYIHVNIEGCIDGMIY

INSSKFACVLKLHRSMYRIPPFPIDKSCCSQYTNGDI

EIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFN

NIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVNH

SNTFVNCLLEDNV

SEQ ID
TB18R
AAF34089.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO: 157

ELDFVVKNYDLNRRHPVTGYTALHCYLYNNYFTN
Deletion

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLLHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPITIKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKICNN

HGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNINIYTRYE

GKTLLHVACEYNNTQVIDYLIRINGDINALTDNNKH

ATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYDRHVKNRNSKT

YRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDT

TLYAVLRCHNSRKLRRYLTELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
TB19R
AAF34090.1
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFN
Inside

NO: 158

KMRDTLPAKDSKWLNPACMFGGTMNDIAALGEPF
Deletion

SAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVS

NKRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDC

VQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGIL

YAKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPE

LEDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQ

DHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLI

EWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENV

TEEYIGNTYKCRGHNYYFEKTLTTTVVLE

SEQ ID
ORFR
AAF34091.1
MHVIDVDVRLYMSTFIIIDQSTENTSIDTTVTINIIYL

NO: 159

AIMKIIMNIIMMIMIELV

SEQ ID
TB21R
AAF34092.1
LKNVECVDIDSTITFMKYDPNDDNKRTCSNWVPLT

NO: 160

NNYMEYCLVIYLETPICGGKIKLYHPTGNIKSDKDI

MFAKTLDFKSTKVLTGRKTIAVLDISVSYNRSMTTI

HYNDDVDIDIHTDKNGKELCYCYITIDDHYLVDVET

IGVIVNRSGKCLLVNNHLGIGIVKDKRISDSFGDVC

MDTIFDFSEARELFSLTNDDNRNIAWDTDKLDDDT

DIWTPVTENDYKFLSRLVLYAKSQSDTVFDYYVLT

GDTEPPTVFIFKVTRFYFNMPK

SEQ ID
TB22L
AAF34093.1
MYCRCSHGYTGIRCQHVVLVDYQRSEKPNTTTSYIP

NO: 161

SPGIMLVLVGIIIITCCLLSVYRFTRRTKLPLQDMVVP

SEQ ID
TB23R
AAF34094.1
MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTK
Inside

NO: 162
(44% 5′)

QDQTPTNDKICQSVTEITESESDPDPEVESEDDSTSV
Deletion

EDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSISESAD

GNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEV

SIDIRCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGST

IVDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVD

GSASEGATDDTSLIDSTKLKACV

TB20R

ORFL

TABLE 39

Examples of proteins encoded by Wyeth Vaccinia genes equivalent to

those deleted in CopMD3p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
VAC_DPP20_207
AEY74905.1
MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPT
Inside

NO: 163

EMVDVSMMSMYGKAFNHASVKESFGNFSIIELPYV
Deletion

GDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFM

DAMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGST

GDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAA

TCALVSDCASTVTNEFCADHPFIYVIRHVDGKILFV

GRYCSPTTNC

SEQ ID
VAC_DPP10_208
AEY74906.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO: 164

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KHMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
VAC_DPP12_209
AEY74907.1
MSILPVIFLSIFFYSSFVQTFNASECIDKGQYFASFME
Inside

NO: 165

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDTDVFWISNG

MYYEEDDGDGDGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
VAC_DPP20_210
AEY4908.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO: 166

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

CKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQ

IEAPLSGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIY

INSSKFACVLKLHRSMYRIPPFPIDKSCCSQYTNDDI

EIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFN

NIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVNH

SNTFVNCLLEDNV

SEQ ID
VAC_DPP20_211
AEY74909.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO: 167

ELDFVVKNYDLNRRQPVTGYTALHCYLYNNYFTN
Deletion

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLSHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPITIKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKICNN

HGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNINIYTRYE

GKTLLHVACEYNNTHVIDYLIRINGDINALTDNNKH

AIQLIIDNKENSPYTIDCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYNRHVRNRNSKT

YRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDT

TLYAVLRCHNSKKLRRYLNELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
VAC_DPP20_212
AEY74910.1
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFN
Inside

NO: 168

KMRDTLPAKDSKWLNPACMFGGTMNDIATLGEPFS
Deletion

AKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSN

KRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDCV

QGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILY

AKHYNNITWYKDNKEINIDDIKYSQTGKKLIIHNPEL

EDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQD

HRFKLKRNCGYASN

SEQ ID
VAC_DPP10_217
AEY74911.1
MRQIKINGTDMLTVMYMLNKPTKKRYVNNPIFTD
Not

NO: 169

WANKQYKFYNQIIYNANKLIEQSKKIDDMIEEVSID
Present

DNRLSTLPLEIRHLIFSYAFL

SEQ ID
VAC_DPP10_218
AEY74912.1
MSSKGGSGGMWSVFIHGHDGSNKGSKTYTSGGGG
Outside

NO: 170

MWGGGSSSGVNGGVKSGTGKI
Deletion

SEQ ID
VAC_DPP10_219
AEY74913.1
MFDYLENEEVALDELKQMLRDRDPNDTRNQFKNN
Outside

NO: 171

ALHAYLFNEHCNNVEVVKLLLDSGTNPLRKNWRQ
Deletion

LPH

SEQ ID
VAC_DPP10_220
AEY74914.1
MLKLKDIAMALLEATGFSNINDFNIFSYMKSKNVD
Outside

NO: 172

VDLIKVLVEHGFDLSVKCENHRSVIENYVMTMILFI
Deletion

ENGCSVLYEDEY

SEQ ID
VAC_DPP10_221
AEY74915.1
MKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNK
Outside

NO: 173

DVDLVKLFLENGKPHGIMCSIVPLWRNDKETIFLILK
Deletion

TMNSDVLQHILIEYMTFGDIPLVEYGTVVNKEAIHG

YFRNINIDSYTMKYLLKKEGRCHQLSRLDTYVNPT

MDVIISTLIHTKRVFVTCLMLAQFLVL

SEQ ID
VAC_DPP10_222
AEY74916.1
MPSIISIGHLCKSNYGCYNFYTYTYKKGLCDMSYAC
Outside

NO: 174

PILSTINICLPYLKDINMIDKRGETLLHKAVRYNKQS
Deletion

LVSLLLESGSDVNIRSNNGYTCIAIAINESKNIELLKM

LLCHKPTLDYVIDSLREISNIVDNDYAIKQCIKYAMII

DDCTSSKIPEFISQRYNDYIDLCN

SEQ ID
VAC_DPP10_223
AEY49171.1
MKKIMVGGNTMFSLIFTDHGAKIIHRYANNPELREY
Outside

NO: 175

YELKQNKIYVEAYDIISNAIVKHDRIHKTIESVDDNT
Deletion

YISNLPYTIKYKIFEQQ

SEQ ID
VAC_DPP10_224
AEY74918.1
MRILFLIAFMYGCVHSYVNAVETKCSNLDIVTSSGE
Outside

NO: 176

FHCSGCVEHMPNFSYMYWLAKDMRSDEDAKFIEH
Deletion

LGEGIKEDETVRTIDGRIVTLQKVLHVTDTNKFAHY

RFTCVLTTIDGVSKKNIWLK

SEQ ID
VAC_DPP10_225
AEY74919.1
MKLFTQNDRYFGLLDSCNHIFCITCINIWFIKTRRET
Outside

NO: 177

GASDNCPICRTRFRNITMSKFYKLVN
Deletion

SEQ ID
VAC_DPP10_226
AEY74920.1
MHYPKYYINITKINPHLANQFRAWKKRIAGRDYMT
Outside

NO: 178

NLSKDTGIQQSKLYVTVKKIETYMVYIYTTI
Deletion

SEQ ID
VAC_DPP20_227
AEY74921.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO: 179

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDNTIT
Deletion

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPICGGKIKLYHPTGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSITTIHYNDDVDIDIHTDKN

GKELCYCYITIDDHYLVDVETIGVIVNRSGKCLLVN

NHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFSL

TNDDNRNIAWDTDKLDDDTDIWTPVTENDYKFLSR

LVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRFY

FNMFK

SEQ ID
VAC_DPP20_228
AEY74922.1
MLINYLMLLFAAMIIRSFADSGNAIETTLPEITNATT
Outside

NO: 180

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

GYTGIRCQHVVLVDYQRSEKPNITTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTNKLPLQDMVVP

SEQ ID
VAC_DPP20_229
AEY74923.1
MDIFKELIVKHPDENVLISPVSILSTLSILNHGAAGST
Outside

NO: 181

AEQLSKYIENMNENTPDDKKDDNNDMDVDIPYCAT
Deletion

LATANKIYGSDSIEFHASFLQKIKDDFQTVNFNNAN

QTKELINEWVKTMTNGKINSLLTSPLSINTRMTVVS

AVHFKAMWKYPFSKHLTYTDKFYISKNIVTSVDMM

VGTENNLQYVHINELFGGFSIIDIPYEGNSSMVIILPD

DIEGIYNIEKNITDEKFKKWCGMLSTKSIDLYMPKF

KVEMTEPYNLVPILENLGLTNIFGYYADFSKMCNET

ITVEKFLHTTFIDVNEEYTEASAVTGVFMTNFAMVY

RTKVYINHPFMYMIKDTTGRILFIGKYCYPQ

SEQ ID
C13L/
AEY74924.1
MMIYGLIACLIFVTSSIASPLYIPVIPPITEDKSFNSVE
Outside

NO: 182
VAC_DPP20_230

VLVSLFRDDQKDYTVISQFNNYTIDTKDWTIGVLST
Deletion

PDGLDIPLTNITYWSRFTIGRALFKSESEDIFQKKMSI

LGVSIECKKSSTLLTFLTVRKMTRVFNKFPDMAYYR

GDCLKAVYVTMTYKNTKTGETDYTYLSNGGLPAY

YRNGVDG

SEQ ID
VAC_DPP20_231
AEY74925.1
MNLQKLSLAIYLTATCSWCYETCIRKTALYHDIQLE
Outside

NO: 183

HVEDNKDSVASLPYK
Deletion

SEQ ID
VAC_DPP20_232
AEY74926.1
MSLESFIITTFNNSSTNIDNMCHLYVKVCPSSLLFR
Inside

NO: 184

LFVECCDINKLVEGTTPLHCYLMNEGFESSVLKNLL
Deletion

KEYVMTSITQIFNS

SEQ ID
VAC_DPP20_233
AEY74927.1
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCA
Inside

NO: 185

QFRPCHCHATKDSLNTVADVRHCLTEYILWVSHRW
Deletion

THRETAGPLYRLLISFRTDATELFGGELKDSLPWDNI

DNCVEIIKCFIRNDSMKTAEELRAIIGLCTQSAIVSGR

VFNDKYIDILLMLRKILNENDYLTLLDHIRTAKY

SEQ ID
VAC_DPP20_234
AEY74928.1
MIAFIIFREIGIISTRIAMDCTCILCRLLDEDVTYKKIK
Inside

NO: 186

LEIETCHNLSKHIDRRGNNALHCYVFNKCDTDIKIV
Deletion

RLLLSRGVERLCRNNEGLTPLGVYSKHRYVKSQIVH

LLISSYSNSSNELKSNINDFDLSSDNIDLRLLKYLIVD

KRIRPSKNTNYAINSLGLVDIYVTTPNPRPEVLLWLL

KSECYSTGYVFRTCMYNSDMCKNSLHYYISSHRES

QSLSKDVIKCLINNNVSIHGRDEGGSLPIQYYWSFST

IDIEIVKLLLIKDVDTCRVYDVSPILEAYYLNKRFRV

TPYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYN

HYIIDNILKRFRQQDESIVQAMLINYLHYGDMVVRC

MLDNGQQLSSARLLC

SEQ ID
VAC_DPP20_235
AEY74929.1
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDNID
Inside

NO: 187

MIDNENKTLLYYAVDVNNIQFAKRLLEYGASVTTS
Deletion

RSIINTAIQKSSYRRENKTKLVDLLLSYHPTLETMID

AFNRDIRYLYPEPLFACIRYALILDDDFPSKVKYDIS

GRHKELKRYRVDINRMKNAYISGVSMFDILFKRSK

RHRLRYAKNPTSNGTKKN

SEQ ID
VAC_DPP20_236
AEY74930.1
MSRINITKKIYCSVFLFLFLSYISNYEKVNDEMYEMG
Inside

NO: 188

EMDEIVSIVRDSMWYIPNVFMDDGKNEGHVSVNNV
Deletion

CHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHC

YTMNTRFNPSVLKILLHHGMRNFDSKDDHYQSITRS

LIY

SEQ ID
VAC_DPP20_237
AEY74931.1
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILI
Inside

NO: 189

VHPSWNDCATGHILIMLLNWHEQKEEGQHLLYLFI
Deletion

KHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNN

NVASYIGYDINLPTKDGIRLGV

SEQ ID
VAC_DPP20_238
AEY74932.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO: 190

IHHRLKVPMIKSLFYKMSEFSPYDDYYVKKILAYCL
Deletion

LRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSIIVT

SEQ ID
VAC_DPP20_239
AEY74933.1
MHHPMESVKTTNTNAIICVREHTLPDYANTQCTPC
Inside

NO: 191

GSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNAL
Deletion

PDIIVFSKDHPDARHVFPKQNVE

SEQ ID
VAC_DPP20-241
AEY74934.1
MHVPASLQQSSSSCTEEENKHHMGIDVIIKVTKQDQ
Inside

NO: 192
(43% 5′)

TPTNDKKQSVTEITESESDPDPEVESEDDSTSVEDV
Deletion

DLPTTYYSIIGGGLRMNFGFTKCPQIKSISESADGNT

VNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDI

RCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGSTIVD

TKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGS

ASEGATDDTSLIDSTKLKACV

SEQ ID
VAC_DPP10_225
AEY74919.1
MKLFTQNDRYFGLLDSCNHIFCITCINIWHKTRRET
Outside

NO: 193

GASDNCPICRTRFRNITMSKFYKLVN
Deletion

SEQ ID
VAC_DPP10_226
AEY74920.1
MHYPKYYINITKINPHLANQFRAWKKRIAGRDYMT
Outside

NO: 194

NLSKDTGIQQSKLYVTVKKIETYMVYIYTTI
Deletion

SEQ ID
VAC_DPP20_207
AEY74921.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO: 195

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDNTIT
Deletion

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPICGGKIKLYHPTGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSITTIHYNDDVDIDIHTDKN

GKELCYCYITIDDHYLVDVETIGVIVNRSGKCLLVN

NHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFSL

TNDDNRNIAWDTDKLDDDTDIWTPVTENDYKFLSR

LVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRFY

FNMPK

SEQ ID
VAC_DPP20:228
AEY74922.1
MLINYLMLLFAAMIIRSFADSGNAIETTLPEITNATT
Outside

NO: 196

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

GYTGIRCQHVVLVDYQRSEKPNTTTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTNKLPLQDMVVP

SEQ ID
VAC_DPP20_239
AEY74933.1
MHHPMESVKTTNTNAIICVREHTLPDYANTQCTPC
Inside

NO: 197

GSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNAL
Deletion

PDIIVFSKDHPDARHVFPKQNVE

SEQ ID
VAC_DPP20-241
AEY74934.1
MHVPASLQQSSSSCTEEENKHHMGIDVIIKVTKQDQ
Inside

NO: 198
(43% 5′)

TPTNDKICQSVTEITESESDPDPEVESEDDSTSVEDV
Deletion

DLPTTYYSIIGGGLRMNFGFTKCPQIKSISESADGNT

VNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDI

RCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGSTIVD

TKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGS

ASEGATDDTSLIDSTKLKACV

TABLE 40

Examples of proteins encoded by Lister Vaccinia genes equivalent to

those deleted in CopMD3p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
B15R/
ABD52695.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO: 200
List191

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
List192
ABD52696.1
MSILPVIFLPIFFYSSFVQTFNAPECIDKGQYFASFME
Inside

NO: 201

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
B17L/
ABD52698.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO: 202
List193

SAEKYMCRYTTLNHNCINVRRCALDSKLLHDIITNC
Deletion

KIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYP

VIFITHTSTRNLDKVSVKTYKGVKVKKLNRCADHAI

VINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQIEA

PLSGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINS

SKFACVLKLHRSMYRIPPFPIDKSCCSQYTNDDIEIPI

HDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNID

TAITQEHEYVKIALGIVCKLMINNMHSIVGVNHSNT

FVNCLLEDNV

SEQ ID
crmE/
ABD52700.1
MTKVIIILGFLIINTNSLSMKCEQGVSYYNSQELKCC
Not

NO: 203
List195

KLCKPGTYSDHRCDKYSDTICGHCPSDTFTSIYNRSP
Present

WCHSCRGPCGTNRVEVTPCTPTTNRICHCDSNSYCL

LKASDGNCVTCAPKTKCGRGYGKKGEDEMGNTIC

KKCRKGTYSDIVSDSDQCKPMTR

SEQ ID
L6/
ABD52701.1
MAMPSLSACSSIEDDFNYGSSVASASVHIRMAFLRK
Not

NO: 204
List196

VYGILCLQFLLTTATTAVFLYFDCMRTFIQGSPVLIL
Present

ASMFGSIGLIFALTLHRHKHPLNLYLLCGFTLSESLT

LASVVTFYDVHVVMQAFMLTTAAFLALTTYTLQSK

RDFSKLGAGLFAALWILILSGLLGIFVQNETVKLVLS

AFGALVFCGFIIYDTHSLIHKLSPEEYVLASINLYLDII

NLFLHLLQLLEVSNKK

SEQ ID
List197
ABD52704.1
MASPCAKFRPCHCHATKDSLNTVADVRHCLTEYIL
Inside

NO: 205

WVSHRWTHRESAGSLYRLLISFRTDATELFGGELKD
Deletion

SLPWDNCVEIIKCFIRNDSMKTAEELRAIIGLCTQSAI

VSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTA

KY

SEQ ID
List199C
ABD52706.I
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILI
Inside

NO: 206

VHPSWNDCATGHILIMLLNWHEQKEEGQHLLYLFI
Deletion

KHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNN

NVASYIGYDINLPTKDGIRLGV

SEQ ID
List199D
ABL63830.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO: 207

IHHRLKVSLPMIKSLFYKMSLPTTITT
Deletion

SEQ ID
C23L/
ABL63827.1
MKQYIVLACMCLAAAAMPASLQQSSSSSSSCTEEE
Inside

NO: 208
List201

NKHHMGIDVIIKVTKQDQTPTNDKICQSVTEITESES
Deletion

(47% 5′)

DPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNF

GFTKCPQIKSISESADGNTVNARLSSVSPGQGKDSPA

ITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPVLGS

NISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCK

ESSELEVKDGFKYVDGSASEGATDDTSLIDSTKLKA

CV

List198A

List198B

List199A

List199B

List200

List194

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventor regards as her invention.

Example 1—Creation of the CopMD5p3p “SKV-B8R+” Recombinant Orthopoxvirus

The open reading frames (ORFs) from 59 poxvirus strains were clustered into orthologs and aligned at the amino acid level (see FIG. 1 for phylogenetic analysis). Bayesian analysis was performed to determine relatedness of all strains. Poxviruses are very diverse in gene content and host range. There are several naturally occurring Vaccinia wild-type strains, which are different from one another.

Five Vaccinia wild type strains (Copenhagen, TianTan, Lister, Wyeth, and Western Reserve) were mixed at equal plaque forming unit counts and sequenced with NGS (Input pool). The resulting mixture was passaged three times in different cancer cell lines (HeLa, 786-O, HT29, MCF7). The final population was sequenced with NGS illumina sequencing. Reads (short DNA fragments) were assigned to various strains based on sequence identity and used to calculate the percent of each strain in the final population. The relative abundance of the different viral strains was then quantified. As shown in FIG. 2, the Copenhagen strain was the most abundant vaccinia strain after three passages in any of the four cancer cell lines indicating that this strain was able to outgrow other strains and therefore replicates faster.

Different Vaccinia wild type strains were also used to infect at low PFU (1×10⁴) various patient tumor cores. Each strain infected on average 4 replicates each containing three 2×2 mm tumor cores. Replication was assessed through virus titering and is expressed as plaque forming units (PFU) as shown in FIG. 3. The Copenhagen strain grows to higher titers than other strains and therefore replicates faster in patient ex-vivo samples. Patient ex-vivo cores are a good mimic of a patient's 3D tumor.

Vaccinia wild-type strains were then subjected to a plaque assay on U2-OS cells with a 3% CMC overlay. Two days past infection, 20-30 plaques for each strain were measured for their size. Plaque size measurements for Copenhagen, Western Reserve, Wyeth, Lister, and Tian Tan are shown in FIG. 4. Plaque formation is affected by the ability of the virus to replicate, spread, and kill. The larger plaque sizes observed for the Copenhagen strain suggest that this strain is superior in these abilities, which are important for the development of an oncolytic virus.

Then, the number of TTAA sites across 1 kb regions in Vaccinia Copenhagen genome were counted (see FIG. 5A). Ilumina NGS sequencing was combined and used to identify Transposon Insertion Sites (Tn-Seq). The input library was passaged three times in either HeLa or U2-OS cells after which frequencies of transposon knockouts were determined. The frequency of a knockout directly corresponds to the amount of reads supporting the event (see FIG. 5B and FIG. 6).

Finally, all 59 poxvirus genomes from FIG. 1 were used to find ORFs and clustered into orthologous groups. Groups containing Copenhagen genes were plotted based on location of the gene in the Copenhagen genome (x-axis) and size of the group (y-axis). When all 59 species share the same gene the conservation is considered to be 100%. The TTAA motif is required for a transposon insertion and this motif is ubiquitous along the genome, meaning transposons can insert anywhere in the genome. However, it was noted that transposons insert preferentially in areas of low poxvirus gene conservation. While sequencing transposon knockouts, major deletions were identified and labelled as CopMD5p and CopMD3p (see FIG. 5C and FIG. 6). Genes that are present in the middle of the genome and that have an elevated gene conservation (FIG. 5C) are important for viral replication. This is because knocking these genes out with transposon insertions causes a decrease in fitness (less frequency after passaging). Genes that are part of the major deletions CopMD5p and CopMD3p were found to be less important for viral replication as their deletion does not impact fitness.

Illumina NGS deep sequencing revealed presence of major deletions during the plaque purification process. CopMD5p and CopMD3p represent clones, which were plaque purified and found to harbor major genomic deletions. These 2 clones were used to co-infect a monolayer of HeLa cells at a high MOI (MOI 10) to induce recombination. Random plaque picking and PCR revealed presence of a double deleted CopMD5p3p which contained both genome deletions (see FIG. 8). These 2 deletions were combined and purified to give a replicating virus, referred to herein as “CopMD5p3p”, that exhibits deletions in the C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, and B20R genes, as well as single deletions in each of the ITR genes B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R. As used herein, “CopWT” refers to wild-type Copenhagen vaccinia virus, “CopMD5p” refers to a Copenhagen vaccinia virus harboring deletions in representative 5′ genes (C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L), and “CopMD3p” refers to a Copenhagen vaccinia virus harboring deletions in representative 3′ genes (B14R, B15R, B16R, B17L, B18R, B19R, and B20R) as well as single deletions in each of the ITR genes B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R.

The 59 poxvirus genomes were then assessed for the presence of these 31 genes deleted in the CopMD5p3p. Homology searches were used to query poxviruses from other clades with amino acid sequences of Table 2 genes from the Copenhagen genome. As shown in FIG. 36, the percentage of these 32 genes present in various poxvirus strains decreases with increasing divergence from the Copenhagen strain (each dot on the plot represents one poxvirus genome). However, a majority of the members of the orthopox family, comprise at least 85% of the genes which are deleted in the CopMD5p3p recombinant vector.

Example 2—Cancer Cell Death

Cancer cells were infected with CopMD5p3p at a range of MOIs (1 to 0.01) in 24-well plates in 4 replicates. Two days post infection with virus, plates were stained with crystal violet. Crystal violet stain was dissolved into SDS and read by spectrophotometry. Data is represented as percent of non-infected cells (see FIG. 9). This data shows that the majority of cancer cell lines die faster when exposed to the CopMD5p3p virus.

The ability of wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions to induce an anti-tumor immune response and to propagate in various cancer cell lines is also shown in FIGS. 26, 27, and 29-35.

Example 3—Growth in Cancer Cells

Four cancer cell lines were infected with CopMD5p3p at a low MOI (0.001) in 24-well plates in triplicates, and at different time points, the virus was collected and tittered. Time 0 h represents input. The growth curves of HeLa, 786-O, HT-29, and MCF7 are shown in FIG. 10. This data shows that the modified CopMD5p3p virus is not impaired in its ability to grow in vitro. This means that the virus is replication competent, even in presence of interferon response. The ability to replicate in mammalian cell lines provides another important advantage. As such, viruses may be manufactured with enhanced speed and efficiency.

Example 4—Growth in Patient Tumor Samples

Patient tumor samples were obtained immediately after surgery and cut into 2 mm×2 mm cores. Three cores were infected with a small amount of virus (1×10⁴PFU), either wild-type Copenhagen or CopMD5p3p. After 72 h virus output was assessed by plaque assay and final Viral Titer expressed as PFU (see FIG. 11). This data shows that the modified CopMD5p3p virus can replicate in fresh patient tumor samples. Replication in patient tumor samples is a good model of replication in a patient 3D tumor.

Example 5—Syncytia in U2-OS Cells

Monolayers of U2-OS cells were infected with either Copenhagen wild-type or CopMD5p3p virus. After 2 h, the media was changed for overlay media as done for a plaque assay. At 48 h post infection, pictures were taken with EVOS to assess plaque phenotype (see FIG. 12). Cell fusion, also known as syncytia, is thought to help the virus spread, since uninfected cells merge with infected cells. Additionally, it has been shown that fused cells are immunogenic and in the case of cancer cells can help initiate an anti-tumor immune response. See, e.g., http://cancerres.aacrjournals.org/content/62/22/6566.long.

Example 6—Syncytia in 786-O Cells

Monolayers of 786-O cells were infected with either Copenhagen wild-type or CopMD5p3p virus. After 24 h pictures were taken with EVOS at 10× magnification (see FIG. 13). This is additional evidence for the occurrence of syncytia. In FIG. 12, the phenotype of a plaque is shown. In the current experiment, monolayers of cells were infected without overlay. Most cells infected by the CopMD5p3p virus have fused.

Example 7—Tumor Control and Weight Loss in Mouse Model

Nude CD-1 (Crl:CD1-Foxn1 nu) mice were seeded with HT-29 human colon cancer xenograft (5e6 cells). Once subcutaneous tumours have established an approximate 5 mm×5 mm size, mice were treated three times (dashed lines) 24 h apart with 1×10⁷PFU of either vaccinia virus intravenously. Mice were measured approximately every other day for tumor size and weight loss (see FIG. 14). This experiment shows that CopMD5p3p is a much safer virus because it does not cause any weight loss or other signs of sickness in immunocompromised nude mice. This experiment also shows CopMD5p3p is able to control tumor growth similarly to the parental Copenhagen wild-type virus.

Example 8—Pox Lesion Formation

Nude CD-1 mice were treated once with 1×10⁷PFU of either vaccinia virus intravenously, six mice per group. Two weeks post treatment, mice were sacrificed and pictures of tails were taken. Pox lesions on tails were counted manually on every mouse tail. Representative pictures shown in FIG. 15. This experiment shows that CopMD5p3p is a much safer virus because it does not cause any pox lesions in immunocompromised nude mice. This is important since prior Oncolytic Vaccinia clinical data has shown patients developing pox lesions upon treatment. Knockout of thymidine kinase (TK) is a popular way of increasing the safety of an OV (oncolytic virus), currently present in a Phase III Oncolytic Vaccinia and in FDA approved Oncolytic T-Vec. The data shows that deleting TK does not play a crucial role in this assay, where mice develop pox lesions when challenged with TK deleted viruses, but do not develop pox lesions with CopMD5p3p which has an intact TK.

Example 9—IVIS Bio-Distribution of Vaccinia after Systemic Administration

Vaccinia viruses wild-type Wyeth, wild-type Copenhagen, and CopMD5p3p were engineered to express Firefly Luciferase (Flue) and YFP through transfection of infected cells with a pSEM1 plasmid replacing TK with Fluc and YFP. Viruses were plaque purified and expanded. All viruses are TK knockouts and encode functional Fluc in their TK locus.

Nude CD-1 mice were then seeded with HT-29 human colon cancer xenograft. Once subcutaneous tumors have established an approximate 5 mm×5 mm size, mice were treated once with 1e7 PFU of either vaccinia Fluc encoding virus intravenously, four mice per group. Four days post treatment, mice were injected i.p. (intraperitoneal) with luciferin and imaged with IVIS for presence of virus (see FIG. 16). This experiment shows that CopMD5p3p is a much safer virus because it is more specific to the tumor. Other viruses show off target replication in the tail, muscle, paws and intra-nasal cavity. CopMD5p3p is only localized in the tumor. As shown in previous FIGS. 15 and 16, there is less detectable CopMD5p3p in the tail compared to the other strains. FIG. 17 shows that CopMD5p3p also has lower titers in other organs when compared to other oncolytic Vaccinia. Since the CopMD5p3p replicates at the same level as the other viruses in the tumor but less in off-target tissues, CopMD5p3p fits the profile of an oncolytic virus better.

An additional example of the biodistribution of various vaccinia viral vectors, including the wild-type Copenhagen vaccinia virus and several modified Copenhagen vaccinia virions, is shown in FIG. 28.

Example 10—Immunogenicity of Vaccinia in Human PBMCs

PBMCs were isolated from blood of healthy human donors (n=2). PBMCs were incubated with either Vaccinia for 24 h and checked for early activation markers using Flow Cytometry (see FIG. 18). This experiment shows that CopMD5p3p is more immunogenic and more readily detectable by immune cells. We believe that this is a desirable trait, since OVs replicating in tumor tissue need to activate immune cells for a successful anti-tumor immune response.

Example 11—Immunogenicity of Vaccinia in Mouse Splenocytes

Immune competent Balb/C mice were injected with 1×10 7 Vaccinia PFU Vaccinia virus intravenously. After one or two days, mice were sacrificed, spleens were harvested and analyzed for immune activation using Flow Cytometry (see FIG. 19). This experiment shows that CopMD5p3p is more immunogenic and more readily detectable by mouse immune cells. This data complements nicely the previous FIG. 18, since most of the in vivo experiments are done in mice.

Example 12—Immunogenicity of Vaccinia in Human Cells

Human cancer cells 786-O were infected at an MOI of 0.01 with either virus. The next day, cells were harvested and nuclei and cytoplasm were separated by cell fractionation. Protein was extracted from each fraction and blotted for NF-kB subunits p65 and p50 (see FIG. 20). NF-kB immune transcription factor initiated an immune response once it's subunit p65 and p50 are translocated to the nucleus. Some viruses are immunosuppressive and block this translocation, preventing an immune response. Suppressing NF-kB function is counter-intuitive to the goal of using oncolytic viruses in combination with immunotherapeutic approaches. Thus, CopMD5p3p is a more advantageous virus as it behaves similarly to MG-1.

Example 13—Synergy with Immune Checkpoint Inhibitor Anti-CTLA4 in Aggressive Melanoma Model

Immune competent C57BL/6 mice were seeded (5e5 cells) subcutaneously with B16-F10 melanoma tumors. Treatment began once subcutaneous tumors have established an approximate 5 mm×5 mm size. Mice treated with CopMD5p3p virus received three 1×10⁷PFU doses into the tumor (intra-tumor) one day apart. Mice treated with anti-CTLA4 received five 100 μg doses of antibody i.p. one day apart. Survival were recorded every other day once treatment started (see FIG. 21). In this experiment, we tested if the oncolytic effect of our CopMD5p3p virus can synergize with blockade of a well-known immune checkpoint CTLA-4 in a very aggressive melanoma murine model. Surprisingly, the median survival of mice treated with virus and checkpoint was higher than any other group. This suggests that CopMD5p3p has some stimulating properties that synergize with checkpoint blockade immunotherapy.

Example 14—Synergy with Immune Checkpoint Inhibitor Anti-CTLA4

Example 15—Synergy with Immune Checkpoint Inhibitor Anti-PD1

Example 16—Synergy with Immune Checkpoint Inhibitor Anti-PD1 and Anti-CDLA4

Immune competent Balb/C mice were seeded (5×10⁵cells) subcutaneously with CT26-LacZ tumors. Treatment began once subcutaneous tumors have established an approximate 5 mm×5 mm size. Mice treated with Vaccinia virus received three (24 h apart, first three dashed lines) 1×10⁷PFU doses into the tumor (intra-tumor). Mice treated with Anti-CTLA4 received five (24 h apart, first five dashed lines) 100 μg doses of antibody i.p. Mice treated with Anti-PD1 received five (24 h apart, last five dashed lines) 100 μg doses of antibody i.p. 24 h after the last dose of Vaccinia virus. Tumor size and survival were recorded every other day once treatment started (see FIG. 24). In this experiment we tested whether a lower dose (25 μg instead of 100 μg) of checkpoint inhibitor antibody could work if we blocked both checkpoints simultaneously. The CopMD5p3p still managed to achieve cures in this murine model with a lower dose (50 μg total) of both inhibitors of checkpoints. Since checkpoint inhibitors have dose dependent toxicity, it is advantageous that very small doses of checkpoint blockers can still achieve an observable phenotype. As in other experiments, the CopMD5p3p virus manages to cure established tumors, and this effect is not observed with wild-type virus lacking the corresponding deletions of CopMD5p3p.

Example 17—Administration for the Treatment of a Subject

Using the methods described herein, a clinician of skill in the art can administer to a subject (e.g., a patient) a pharmaceutical composition containing a recombinant orthopoxvirus vector described herein to treat cancer or tumor cells. The cancer may be, for example, leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, or throat cancer, among others.

For instance, a clinician of skill in the art may assess that a patient is suffering from cancer or tumors and may administer to the patient a therapeutically effective amount (e.g., an amount sufficient to decrease the size of the tumor) of a pharmaceutical composition containing the recombinant orthopoxvirus vector disclosed herein. The pharmaceutical composition may be administered to the subject in one or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more) per a specified time interval (e.g., weekly, daily, or hourly). The patient may be evaluated between doses to monitor the effectiveness of the therapy and to increase or decrease the dosage based on the patient's response. The pharmaceutical composition may be administered to the patient orally, parenterally (e.g., topically), intravenously, intramuscularly, subcutaneously, or intranasally. The treatment may involve a single dosing of the pharmaceutical composition. The treatment may involve continued dosing of the pharmaceutical composition (e.g., days, weeks, months, or years). The treatment may further involve the use of another therapeutic agent (e.g., an immune checkpoint inhibitor, such as an anti-PD-1 or anti-CTLA-4 antibody or antigen-binding fragment thereof, IL-12, FLT3L).

Example 18—Targeted Deletions of CopMD5p and CopMD3p

The following protocol for producing modified vaccinia viral vectors utilizes techniques described, e.g., in Rintoul et al. PLoS One. 6(9): e24643 (2011), the disclosure of which is incorporated herein by reference.

Briefly, CopMD5p (Copenhagen vaccinia virus harboring deletions in 5′ genes: C2L, C1L, N1L, N2L, M1L, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L) and CopMd3p (Copenhagen vaccinia virus harboring deletions in 3′ genes: (B14R, B15R, B16R, B17L, B18R, B19R, and B20R as well as single deletions in each of the ITR genes B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29R targeting recombinant constructs were synthesized by g-Block technology (IDT, Coralville Iowa). U2OS cells were infected with wildtype vaccinia virus (Wyeth, Western Reserve, Tian Tan, Lister) at an MOI of 0.01 in serum free DMEM for 1.5 hours. Viral supernatant was aspirated and U2OS cells were transfected with PCR amplified CopMD5p or CopMd3p targeting g-Blocks by Lipofectamine 2000 (Invitrogen) in OptiMEM (Gibco). DMEM supplemented with 10% FBS was added to cells 30 minutes after transfection and left overnight. The following day, transfection media was aspirated and fresh DMEM 10% FBS media was added to cells. 48 hours after infection transfection, U2OS cells were harvested and lysed by a single freeze thaw cycle. Serially diluted lysates were plated onto a confluent monolayer of U2OS cells and eGFP positive (CopMD5p targeted) or mCherry positive (CopMd3p targeted) plaques were isolated and purified through 5 rounds of plaque purifications.

Double major deleted vaccinia viruses were generated by co-infection of CopMD5p and CopMd3p deleted vaccinia viruses at an MOI of 5 for each virus in U2OS cells. Cells were harvested the next day and lysed by one round of freeze thaw. Lysates were serially diluted and plated onto a confluent monolayer of U2OS cells and selected for double positive plaques (eGFP+mCherry). Plaques were purified by 5 rounds of plaque purification.

An exemplary scheme for the production of modified orthopoxvirus vectors (e.g., modified vaccinia viral vectors, such as modified Copenhagen vaccinia viral vectors) of the disclosure is shown in FIG. 25.

Example 19—SKV-GFP (CopMD5p3p-B8R−) has Similar Efficacy in Tumour Control Compared to SKV (CopMD5p3p-B8R+)

The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to gamma interferon receptor (IFN-γ). In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of gamma interferon including human and rat gamma interferon; it does not, however, bind significantly to murine IFN-γ. Here we describe the construction and characterization of recombinant VVs lacking the B8R gene. Homologous recombination between the targeting construct and the B8R locus resulted in the replacement of 75% of the B8R gene with the eGFP transgenes flanked by two loxP sites (SKV-GFP).

B8R− viruses showed similar efficacy to B8R+ viruses. FIG. 39. Survival of mice treated with either SKV or SKV-GFP was assessed. 5×10⁶CT26-LacZ cells were seeded subcutaneously on day 0. On day 14, 16 and 18 tumours were treated at a dose of 10⁷pfu with an intratumoural injection of either SKV or SKV-GFP. No significant decrease in efficacy was seen when the viruses injected had a deletion of the B8R locus.

Example 20—Infection of Normal Versus Cancer Cell Lines of SKV (CopMD5p3p-B8R+) Virus

Primary health cell viability was compared to that of cancer cells. Confluent normal or cancer cells were infected at a range of MOI (pfu/cell) for 48 hrs, after which viability was quantified. As indicated in FIG. 37, SKV-B8R+ virus preferentially infects cancer cells.

Example 21—SKV (CopMD5p3p-B8R+) does not Impair Interferon Signaling

Interferon signaling was assessed by determining the number of genes in the interferon pathway that are upregulated (induced expression) or downregulated (repressed expression) in a variety of normal cell lines and one cancer cell line (786-O). FIG. 38 Confluent monolayers of 1 million cells were infected at an MOI of 3 (3×10⁶PFU) for 18 h with either SKV (CopMD5p3p-B8R+) or the parental Copenhagen virus strain having the TK gene disabled. RNA was sequenced using RNA-seq and gene expression of interferon genes was determined after read mapping a expression normalization. While the SKV (CopMD5p3p-B8R+) virus mostly induces genes in the interferon pathway the parental Copenhagen represses genes. This suggests SKV (CopMD5p3p-B8R+) is able to induce Type I Interferon signaling which is critical in viral clearance of normal cells.

Example 22—B8R Negative Vaccinia Virus Engineered to Express Flt3L, IL-12 TM and Anti-hCTLA-4

Modified vaccinia viruses containing both the CopMD5p3p and B8R deletions, as described above, were further engineered to express the immunotherapeutic transgenes. An SKV-123 virus 3 (CopMD5p3p-B8R+-IL12TM-FLT3-antiCLTA4) expressing three transgenes was evaluated in terms of transgene expression kinetics. Confluent monolayers of 786-O human adenocarcinoma cell lines were infected with SKV-123 virus at an MOI of 3 (3×10⁶pfu). RNA was sequenced using RNA-seq and gene expression of inserted transgenes were determined after read mapping after expression normalization. Transgene expression peaked at 3-4 hours after cell infection. See FIG. 40.

Example 23 SKV Expressing Murine IL-12 p35 Membrane Bound (SKV3) has Greater Efficacy in Controlling Murine Tumors

The survival of mice treated with either SKV (CopMD5p3p-B8R+) or SKV-3 (CopMD5p3p-B8R+-IL12TM) virus (expressing murine membrane bound p35 IL-12) was assessed. 5×10⁶CT26-LacZ cells were seeded sub cutaneously on day 0. On day 14, 16 and 18 tumours were treated at a dose of 1e7 pfu with an intratumoural injection of either SKV or SKV-3. Although SKV virus extend survival of mice bearing CT26 colon tumours. SKV-3 expression of IL-12 is able to induce remissions that lead to durable cures. See FIG. 41.

Example 24—Major Double Deletions in Engineered in Various Vaccinia Strains Enhance Cancer Cell Killing In Vitro

Hela cells were infected at an MOI of 0.1 with the following strains of engineered vaccinia viruses: (1) parental wildtype virus (wt); (2) 5 prime major deleted (5p), (3) 3 prime major deleted (3p), and (4) recombined 5 prime and 3 prime major double deleted (5p3p). Cell viability was quantified by alamar blue assay 72 hours post infection. Both 5p and 5p3p major double deleted vaccinia strains are more cytotoxic in HelLa cells when compared to their parental wildtype and 3p major deleted strains. See FIG. 42. FIG. 43 depicts a summary of the major deleted Vaccinia strains, and the effect of 5p, 3p and 5p3p deletions on syncytia, cytotoxicity and replication. CD-1 nude mice were treated with 1×10⁷pfu via intravenously tail vein injection and measured at the indicated timepoints. 5p3p vaccinia strains did not induce weight loss compared to wildtype strains. FIG. 44. Mice were also examined for pox lesions 6 days post-injection. 5p3p vaccinia strains do not induce pox lesions compared to wildtype strains. FIG. 45.

Some Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Some embodiments are within the claims.

	Number	Date	Country
	62784371	Dec 2018	US
	62614349	Jan 2018	US

	Number	Date	Country
Parent	16959632	Jul 2020	US
Child	18461241		US

MODIFIED ORTHOPOXVIRUS VECTORS

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