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, N1 L, N2L, M1 L, M2L, K1 L, K2L, K3L, K4L, K5L, K6L, K7R, F1 L, 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, 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-CTLA4 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, ML, M2L, K1L, K2L, K3L, K4L, K5L, K6L, K7R, F1L, F2L, F3L, B14R, B15R, B16R, B17L, B18R, B19R, 820R. 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 88R 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, F1 L, 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 N L.

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 or 3 genes each independently selected from the group consisting of B15R, B17L, and B14R. 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, 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 mom 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 anyone 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 anyone 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 anyone 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 F3L. 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, B17L, and B14R.

In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 1 gene selected from the group consisting of B15R, B17L, and B14R. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 2 genes selected from the group consisting of B15R, B17L, and B14R. In some embodiments, the recombinant orthopoxvirus genome has a deletion of at least 3 genes selected from the group consisting of B15R, B17L, 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, 1B28R, and B29R. In some embodiments, the deletion includes each of B21R, B22R, B23R, B24R, B25R, B26R, B27R, B28R, and B29L In anyone 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 vaccinia virus is a strain selected from the group consisting of Copenhagen, Western Reserve, Wyeth, Lister, EM63, ACAM2000, 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, 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, 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 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, sézary 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-alpha, IL-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 Fe 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 codon, 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, 1983). 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-lg, (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, Calif., 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_Dof 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_Dof 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_Dof 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 “VH” 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 C1L 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, “ML” 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 “K1 L” 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 pbospholipase-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-γ). 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 titering.

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 786-0 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 immunotherapeutuic 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, 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, 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-CTLA4 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, 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 PICR 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

Bl4R
Pseudogene
Pseudogene
CopMD3p

Bl5R
Unknovvn
Unknown

Bl6R
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-7). In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of gamma inteterferon including human and rat gamma interferon; it does not, however, bind significantly to murine IFN-7. 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 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, transdennally 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, sézary 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(SEQ ID NO:
Wilkinson et al. Cancer Immunol.

210);
Immunother. 61(2):169-79 (2012).

SVSESDTIRSISIAS SEQ
Hural et al. J. Immunol. 169(1):557-

ID NO: 211;
65 (2002).

LLANGRMPTVLQCVN

(SEQ ID NO: 212); and

RMPTVLQCVNVSVVS

(SEQ ID NO: 213)

2

PBF

CTACRW1CKACQR(SEQ
Tsukahara et al. Cancer Res.

ID NO: 214)
64(15):5442-8 (2004).

3

PRAME

VLDGLDVLL SEQ ID
Kessler et al. J. Exp. Med.

NO: 215);
193(1):73-88 (2001).

SLYSFPEPEA(SEQ ID
Ikeda et al. Immunity 6(2):199-208

NO: 216);
(1997).

ALYVDSLFFL (SEQ ID

NO: 217);

SLLQHLIGL(SEQ ID NO:

218); and

LYVDSLFFL (SEQ ID NO:

219)

4

WTI

TSEKRPFMCAY (SEQ ID
Asemissen et al. Clin. Cancer Res.

NO: 220); CMTWNQMNL
12(24):7476-82 (2006)

(SEQ ID NO: 221);
Ohminami et al. Blood. 95(1):286-

LSHLQMHSRICH (SEQ ID
93 (2000).

NO: 222);
Guo et al. Blood. 106(4):1415-8

KRYFKLSHLQMHSRICH
2005).

(SEQ ID NO: 223); and
Lin et al. J. Immunother. 36(3):159-

KRYFKLSHLQMHSRKH
70 (2013).

(SEQ ID NO: 223)
Fujiki et al. J. Immunother.

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

5

HSDLI

CYMEAVAL (SEQ ID NO:
Wick et al. Clin. Cancer Res.

224)
20(5):1125-34 (2014).

6

Mesothelin

SLLFLLFSL (SEQ ID NO:
Hassan et al. Appl.

225)
Immunohistochem. Mol. Morphol.

VLPLTVAEV (SEQ ID
13(3):243-7 (2005).

NO: 226)
Thomas et al J Exp Med. 2004 Aug

ALQGGGPPY (SEQ ID
2; 200(3): 297-306.

NO: 227)

LYPKARLAF (SEQ ID

NO: 228)

AFLPWHRLF (SEQ ID

NO: 229)

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).

SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

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

NO: 232))
55 (2000).

SLLMWITQC SEQ ID
Valmori et al. Cancer Res.

NO: 230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzald et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILITR (SEQ ID
Ebert et al. Cancer Res. 69(3):1046-

NO: 237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID
Mandic et al. J Immunol.

NO: 242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

SLLMWITQCFLPVF (SEQ
Mizote et at. Vaccine. 28(32):5338-

ID NO: 244)
46 (2010).

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

L-ARRSLAQ SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

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

ID NO: 246)
Zeng et at. J Immunol. 165(2):1153-

PGVLIKEFTVSGNILTIR
9(2000).

L-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

ID NO: 248)
8 (2002).

QGAMLAAQERRVPRAA
Hasegawa et al. Clin Cancer Res.

E-VPR(SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIR

LT (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQ

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIR

L-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAE

L-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT(SEQ ID

NO: 255)

LLEFYLAMPFATPM

(SEQ ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

8

CEA

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

ID NO: 258)
82 (2010).

EIIYPNASLLIQN (SEQ ID
Bast et al. Am. J. Obstet. Gynecol.

NO: 259)
149(5):553-9 (1984).

YACFVSNLATGRNNS
Crosti et al. J Immunol.

(SEQ ID NO: 260)
176(8):5093-9 (2006).

LWWVNNQSLPVSP (SEQ
Kobayashi et al. Clin Cancer Res.

ID NO: 261)
8(10):3219-25 (2002).

LWWVNNQSLPVSP (SEQ
Campi et al. Cancer Res.

ID NO: 261)
63(23):8481-6 (2003).

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

ID NO: 261)
102 (1995).

EIIYPNASLLIQN (SEQ ID
Tsai et al. J Immunol. 158(4):1796-

NO: 259)
802 (1997).

NSIVKSITVSASG (SEQ
Kawakami et al. J Immunol.

ID NO: 262)
154(8):3961-8 (1995).

KTWGQYWQV (SEQ ID
Cox et al. Science. 264(5159):716-9

NO: 263)
(1994).

(A)MLGTHTMEV (SEQ
Kawakami et al. J Immunol.

ID NO: 264)
154(8):3961-8 (1995).

ITDQVPFSV (SEQ ID NO:
Kawakami et al. J Immunol.

265)
161(12):6985-92 (1998).

YLEPGPVTA (SEQ ID
Skipper et al. J Immunol.

NO: 266)
157(11):5027-33 (1996).

LLDGTATLRL (SEQ ID
Michaux et al. J Immunol.

NO: 267)
192(4):1962-71 (2014).

VLYRYGSFSV (SEQ ID

NO: 268)

SLADTNSLAV (SEQ ID

NO: 269)

RLMKQDFSV (SEQ ID

NO: 270)

RLPRIFCSC (SEQ ID NO:

271)

LIYRRRLMK (SEQ ID

NO: 272)

ALLAVGATK (SEQ ID

NO: 273)

IALNFPGSQK (SEQ ID

NO: 274)

RSYVPLAHR (SEQ ID

NO: 275)

9

p53

VVPCEPPEV (SEQ ID NO:
Hung et al. Immunol. Rev. 222:43-

276)
69 (2008).

10

Her2/Neu

HLYQGCQVV (SEQ ID
Nakatsuka et al. Mod. Pathol.

NO: 277)
19(6):804-814 (2006).

YLVPQQGFFC (SEQ ID
Pils et al. Br. J Cancer 96(3):485-91

NO: 278)
(2007).

PLQPEQLQV (SEQ ID
Scardino et al. Eur J Immunol.

NO: 279)
31(11):3261-70 (2001).

TLEEITGYL (SEQ ID NO:
Scardino et al. J Immunol.

280)
168(11):5900-6 (2002).

ALIHHNTHL (SEQ ID
Kawashima et al. Cancer Res.

NO: 281)
59(2):431-5 (1999).

PLTSIISAV (SEQ ID NO:
Okugawa et al. Eur J Immunol

282)
30(11):3338-46 (2000).

VLRENTSPK (SEQ ID

NO: 283)

TYLPTNASL (SEQ ID NO:

284)

11

EpCAM

RYQLDPKFI (SEQ ID NO:
Spizzo et al. Gynecol. Oncol.

285)
103(2):483-8 (2006).

Tajhna et al. Tissue Antigens.

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

12

CA125

ILFTINFTI (SEQ ID NO:
Bast et al. Cancer 116(12):2850-

286)
2853 (2010).

VLFTINFTI (SEQ ID NO:

287)

TLNFTITNL (SEQ ID NO:

288)

VLQGLLKPL (SEQ ID

NO: 289)

VLQGLLRPV (SEQ ID

NO: 290)

RLDPKSPGV (SEQ ID

NO: 291)

QLYWELSKL (SEQ ID

NO: 292)

KLTRGIVEL (SEQ ID NO:

293)

QLTNGITEL (SEQ ID NO:

294)

QLTHNITEL (SEQ ID NO:

295)

TLDRNSLYV (SEQ ID

NO: 296)

13

Folate

FLLSLALML (SEQ ID
Bagnoli et al. Gynecol. Oncol.

receptor a

NO: 297)
88:S140-4 (2003).

NLGPWIQQV (SEQ ID
Pampeno et al. (2016) High-ranking

NO: 298)
In Silico epitopes [determined by 3

algorithms: BISMAS, IEDB,

RANKPEP] unpublished

14

Sperm

ILDSSEEDK (SEQ ID NO:
Chiriva-Inernti et al. J.

protein 17

299)
Immunother. 31(8):693-703 (2008).

15

TADG-12

YLPKSWTIQV (SEQ ID
Bellone et al. Cancer 115(4):800-11

NO: 300)
(2009).

WIHEQMERDLKT SEQ
Underwood et al. BBA Mol. Basis of

ID NO: 301)
Disease. 1502(3):337-350 (2000).

16

MUC-I6

ILFTINFTI (SEQ ID NO:
Chekmasova et al. Clin. Cancer Res.

286)
16(14):3594-606 (2010).

VLFTINFTI (SEQ ID NO:

287)

TLNFTITNL (SEQ ID NO:

288)

VLQGLLKPL (SEQ ID

NO: 289)

VLQGLLRPV (SEQ ID

NO: 290)

RLDPKSPGV (SEQ ID

NO: 291)

QLYWELSKL (SEQ ID

NO: 292)

KLTRGIVEL (SEQ ID NO:

293)

QLTNGITEL (SEQ ID NO:

294)

QLTHNITEL (SEQ ID NO:

295)

TLDRNSLYV (SEQ ID

NO: 296)

17

LICAM

LLANAYIYV (SEQ ID
Hong et al. J. Immunother. 37(2):93-

NO: 302)
104(2014).

YLLCKAFGA (SEQ ID
Pampeno et al. (2016) High-ranking

NO: 303)
In Silico epitopes [determined by 3

KLSPYVHYT (SEQ ID
algorithms: BISMAS, IEDB,

NO: 304)
RANKPEP] unpublished

18

Mannan-MUC-1

PDTRPAPGSTAPPAHGV
Loveland et al. Clin. Cancer Res.

TSA (SEQ ID NO: 305)
12(3 Pt 1):869-77 (2006).

STAPPVHNV (SEQ ID
Godelaine et al. Cancer Immunol

NO: 306)
Immunother. 56(6):753-9 (2007).

LLLLTVLTV (SEQ ID NO:
Ma et al. Int J Cancer. 129(10):2427-

307)
34 (2011).

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

ID NO: 308)
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 (SEQ ID
Schiavetti et al. Cancer Res.

NO: 309)
62(19):5510-6 (2002).

20

KK-LC-1

RQKRILVNL (SEQ ID
Fukuyama et al. Cancer Res.

NO: 310)
66(9):4922-8 (2006).

21

KM-HN-1

NYNNFYRFL (SEQ ID
Fukuyama et al. Cancer Res.

NO: 311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID
Monji et al. Clin Cancer Res. 10(19

NO: 312)
Pt 1): 6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

22

LAGE-1

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID
Rimoldi et al. J Immunol.

NO: 230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAA
Slager et al. J Immunol.

EVP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

L (SEQ ID NO: 253)
30 (2000).

CLSRRPWKRSWSAGSCP
Slager et al. J Immuno!.

G-MPHL (SEQ ID NO:
170(3):1490-7 (2003).

316)
Wang et al. Immunity. 20(1):107-18

ILSRDAAPLPRPG (SEQ
(2004).

ID NO: 317)
Hasegawa et al. Clin Cancer Res.

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

(SEQ ID
NO: 257)

23

MAGE-A4

EVDPASNTY (SEQ ID
Kobayashi et al. Tissue Antigens.

NO: 318)
62(5):426-32 (2003).

GVYDGREHTV (SEQ ID
Duffour et al. Eur J Imrnunol.

NO: 319)
29(10):3329-37 (1999).

NYKRCFPVI (SEQ ID NO:
Miyahara et al. Clin Cancer Res.

320)
11(15):5581-9 (2005).

SESLICMIF (SEQ ID NO:
Ottaviani et al. Cancer Immuno

321)
Immunother. 55(7):867-72 (2006)

Zang et al. Tissue Antigens.

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

24

Spl 7

ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

25

SSX-4

INKTSGPKRGKHAWTHR
Ayyoub et al. Clin Immunol.

LRE (SEQ ID NO: 322)
114(1):70-8 (2005).

YFSKKEWEKMKSSEKIV
Valmori et al. Clin Cancer Res.

YVY (SEQ ID NO: 323)
12(2):398-404 (2006).

MKLNYEVMTKLGFKVT

LPPF (SEQ ID NO: 324)

KHAWTHRLRERKQLVV

YEEI (SEQ ID NO: 325)

LGFKVTLPPFMRSKRAA

DFH (SEQ ID NO: 326)

KSSEKIVYVYMKLNYEV

MTK (SEQ ID NO: 327)

KHAWTHRLRERKQLVV

YEEI (SEQ ID NO: 325)

26

TAG-1

SLGWLFLLL (SEQ ID
Adair et al. J Immunother. 31(1):7-

NO: 328)
17 (2008).

LSRLSNRLL (SEQ ID NO:

329)

27

TAG-2

LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

329)
17 (2008).

TABLE 4

Breast cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

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

NO: 330)
109(6):909-18 (2004).

2
mammaglobin-A
PLLENVISK (SEQ ID NO:
Jaramillo et al. Int. J. Cancer.

331)
102(5):499-506 (2002).

3
NY-BR-I
SLSKILDTV (SEQ ID NO:
Wang et al. Cancer Res. 66(13):6826-

332)
33 (2006).

4
EpCAM
RYQLDPICFI (SEQ ID NO:
Gastl et al. Lancet 356(9246):1981-2

285)
(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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

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

NO: 232))
55 (2000).

SLLMWITQC (SEQ ID
Valmori et al. Cancer Res.

NO: 230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer. 132(2):345-

NO: 234)
54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Inununol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzald et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID
Ebert et al. Cancer Res. 69(3):1046-

NO: 237)
54 (2009).

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

ID NO: 238)
54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S A.

NO: 241)
98(7):3964-9 (2001).

FATPMEAEL (SEQ ID
Mandic et al. J Immunol. 174(3):1751-

NO: 242)
9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S A.

NO: 243)
101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol. 172(8):5095-

232)
102 (2004).

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

ID NO: 244)
46 (2010).

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

L-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res. 60(17):4946-

EFYLAMPFATPM (SEQ
52 (2000).

ID NO: 246)
Zeng et al. J Iminunol. 165(2):1153-

PGVLLICEFTVSGNILTIR
9(2000).

L-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74(2009)

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

ID NO: 248)
(2002).

QGAMLAAQERRVPRAA
Hasegawa et al. Clin Cancer Res.

E-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLICEFTVSGNILTIR

LT (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLICEFTVSGNILTIR

L-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAE

L-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM

(SEQ ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

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

NO: 333)
75 (1995).

7
HERV-K-
MLAVISCAV (SEQ ID
Schiavetti et al. Cancer Res.

MEL
NO: 309)
62(19):5510-6 (2002).

8
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

9
KM-HN-I
NYNNFYRFL (SEQ ID
Fukuyama et al. Cancer Res.

NO: 311)
66(9):4922-8 (2006).

EYSICECLKEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18 Pt

NO: 312)
1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

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

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID
Rimoldi et al. J Immunol.

NO: 230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S A.

ID NO: 244)
98(7):3964-9 (2001).

QGAMLAAQERRVPRAA
Slager et al. J hnmunol. 172(8):5095-

EVP-R(SEQ ID NO: 249)
102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

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

G-MPHL (SEQ ID NO: 316)
7(2003).

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

ID NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

11
MAGE-AI
I EADPTGHS (SEQ ID
Traversari et al. J Exp Med.

NO: 334)
176(5):1453-7 (1992).

KVLEYVIKV (SEQ ID
Ottaviani et al. Cancer Immunol

NO: 335)
Immunother. 54(12):1214-20 (2005).

SLFRAVITK (SEQ ID NO:
Pascolo et al. Cancer Res.

336)
61(10):4072-7 (2001).

EVYDGREHSA (SEQ ID
Chaux et al. J Immunol. 163(5):2928-

NO: 337)
6 (1999).

RVRFFFPSL (SEQ ID NO:
Luiten et al. Tissue Anitgens.

338)
55(2):49-52 (2000).

EADPTGHSY (SEQ ID
Luiten et al. Tissue Antigens.

NO: 334)
56(1):77-81 (2000).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

KEADPTGHSY (SEQ ID
Stroobant et al. Eur J Immunol.

NO: 340)
42(6):1417-28 (2012).

DPARYEFLW (SEQ ID
Corbiere et al. Tissue Antigens.

NO: 341)
63(5):453-7 (2004).

ITKKVADLVGF (SEQ ID
Goodyear et al. Cancer Immunol

NO: 342)
Immunother. 60(12):1751-61 (2011).

SAFPTTINF (SEQ ID NO:
van der Bruggen et al. Eur J Immunol.

343)
24(9):2134-40 (1994).

SAYGEPRKL (SEQ ID
Wang et al. Cancer Immunol

NO: 344)
Immunother. 56(6):807-18 (2007).

RVRFFFPSL (SEQ ID NO:
Chaux et al. J Exp Med. 189(5):767-78

338)
(1999).

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

ID NO: 345)
1910-6 (2001).

PRALAETSYVKVLEY

(SEQ ID NO: 346)

FLLLKYRAREPVTKAE

(SEQ ID NO: 347)

EYVIKVSARVRF (SEQ ID

NO: 348)

12
MAGE-A2
YLQLVFGIEV (SEQ ID
Kawashima et al. Hum Immunol.

NO: 349)
59(1):1-14 (1998).

EYLQLVFGI (SEQ ID NO:
Tahara et al. Clin Cancer Res.

350)
5(8):2236-41 (1999).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID
Breckpot et al. J Immunol.

NO: 351)
172(4):2232-7 (2004).

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

(SEQ ID NO: 352)
(1999).

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

TSA (SEQ ID NO: 305)
62 (1993).

14
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

15
SSX-2
ICASEKHYV (SEQ ID NO:
Ayyoub et al. J Immunol.

353)
168(4):1717-22 (2002).

EKIQICAFDDIAKYFSK
Ayyoub et al. J Immunol.

(SEQ ID NO: 354)
172(11):7206-11 (2004).

FGRLQGISPKI (SEQ ID
Neumann et al. Cancer Immunol

NO: 355)
Immunother. 60(9):1333-46 (2011).

WEKMICASEKIFYVYMK
Ayyoub et al. Clin Immunol.

RK (SEQ ID NO: 356)
114(1):70-8 (2005).

KIFYVYMICRICYEAMT
Neumann et al. Int J Cancer.

(SEQ ID NO: 357)
112(4):661-8 (2004).

KIFYVYMKRKYEAM
Ayyoub et al. J Clin Invest.

(SEQ ID NO: 358)
113(8):1225-33 (2004).

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

NO: 328)
(2008).

LSRLSNRLL (SEQ ID NO:

329)

17
TAG-2
LSRLSNRLL SEQ ID NO:
Adair et al. J Immunother. 31(1):7-17

329)
(2008).

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

(SEQ ID NO: 359)
27 (2006).

19
Her2/Neu
HLYQGCQVV (SEQ ID
Nakatsuka et al. Mod. Pathol.

NO: 277)
19(6):804-814 (2006).

YLVPQQGFFC (SEQ ID
Pils et al. Br. J. Cancer 96(3):485-91

NO: 278)
(2007).

PLQPEQLQV (SEQ ID
Scardino et al. Eur J Immunol.

NO: 279)
31(11):3261-70 (2001).

TLEEITGYL (SEQ ID NO:
Scardino et al. J Immunol.

280)
168(11):5900-6 (2002).

ALIHHNTHL (SEQ ID NO:
Kawashima et al. Cancer Res.

281)
59(2):431-5 (1999).

PLTSHSAV (SEQ ID NO:
Okugawa et al. Eur J Immunol.

282)
30(11):3338-46 (2000).

VLRENTSPK (SEQ ID NO:

283)

TYLPTNASL (SEQ ID NO:

284)

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
p.53
VVPCEPPEV (SEQ ID NO:
Hung et al. Immunol. Rev. 222:43-69

276)
(2008).

http://cancerimmunity.org/peptide/mut

ations/

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 (SEQ ID NO:
Tomita et al. Cancer Sci.

360)
102(4):697-705 (2011).

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

NO: 361)
(2007).

3
PRAME
VLDGLDVLL (SEQ ID
Kessler et al. J Exp Med.

NO: 215), SLYSFPEPEA
193(1):73-88 (2001).

(SEQ ID NO: 216),
Ikeda et al. Immunity 6(2):199-

ALYVDSLFFL (SEQ ID
208 (1997).

NO: 217), SLLQHLIGL

(SEQ ID NO: 218),

LYVDSLFFL (SEQ ID NO:

219)

4
RU2AS
LPRWPPPQL (SEQ ID NO:
Van Den Eynde et al. J. Exp.

362)
Med. 190(12):1793-800 (1999).

5
Telomerase
ILAKFLHWL (SEQ ID
Vonderheide et al. Immunity

NO: 363);
10(6):673-9 (1999).

RLVDDFLLV (SEQ ID
Miney et al. Proc. Natl. Acad.

NO: 364);
Sci. U.S.A. 97(9):4796-801

RPGLLGASVLGLDDI
(2000).

(SEQ ID NO: 365); and
Schroers et al. Cancer Res.

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

(SEQ ID NO: 366)
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 (SEQ ID NO:
Di Modugno etal. Int. J.

330)
Cancer. 109(6):909-18

(2004).

2
PBF
CTACRWICKACQR (SEQ ID
Tsukahara et al. Cancer Res.

NO: 214)
64(15):5442-8 (2004).

3
K-ras
VVVGAVGVG (SEQ ID NO:
Gjertsen et al. Int. J. Cancer.

367)
72(5):784-90 (1997).

4
Mesothelin
SLLFLLFSL (SEQ ID NO:
Le et al. Clin. Cancer Res.

225)
18(3):858-68 (2012).

VLPLTVAEV (SEQ ID NO:
Hassan et al. Appl.

226)
Immunohistochem. Mol.

ALQGGGPPY (SEQ ID NO:
Morphol. 13(3):243-7 (2005).

227)
Thomas et al J Exp Med.

LYPKARLAF (SEQ ID NO:
2004 Aug 2; 200(3): 297-306.

228)

AFLPWHRLF (SEQ ID NO:

229)

5
mucink
PDTRPAPGSTAPPAHGVTSA
Jerome et al. J Immunol.

(SEQ ID NO: 305)
151(3):1654-62 (1993).

TABLE 7

Liver cancer

Tumor-

associated
Reported immunogenic

No
antigen
epitopes
Sources

1
G250/
HLSTAFAR (SEQ ID NO:
Vissers et al. Cancer Res. 59(21):5554-9

MN/
368);
(1999).

CAIX
KIFGSLAFL (SEQ ID NO:
Fisk et al. J Exp Med. 181(6):2109-17

369);
(1995).

IISAVVGIL (SEQ ID NO:
Brossart et al. Cancer Res. 58(4):732-6

370);
(1998).

ALCRWGLLL (SEQ ID NO:
Kawashima et al. Hum Immunol.

371);
59(1):1-14 (1998).

ILHNGAYSL (SEQ ID NO:
Rongcun et al. J Immunol. 163(2):1037-

372);
44 (1999).

RLLQETELV (SEQ ID NO:

373);

VVKGVVFGI (SEQ ID NO:

374); and YMIMVKCWMI

(SEQ ID NO: 375)

2
Hepsin
SLLSGDWVL (SEQ ID NO:
Guo et al. Scand J Immunol. 78(3):248-

376);
57 (2013).

GLQLGVQAV (SEQ ID

NO: 377); and

PLTEYIQPV (SEQ ID NO:

378)

3
Intestinal
SPRWWPTCL (SEQ ID NO:
Ronsin et al. J Immunol. 163(1):483-90

carboxyl
379)
(1999).

esterase

4
alpha-
GVALQTMKQ (SEQ ID
Butterfield et al. Cancer Res.

foetoprote
NO: 380);
59(13):3134-42 (1999).

in
FMNICFIYEI (SEQ ID NO:
Pichard et al. J Immunother. 31(3):246-

381); and QLAVSVILRV
53 (2008)

(SEQ ID NO: 382)
Alisa et al. Clin. Cancer Res.

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

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

ID NO: 383)
193(10):1189-98 (2001).

6
PBF
CTACRWIUCACQR (SEQ
Tsukahara et al. Cancer Res.

ID NO: 214)
64(15):5442-8 (2004).

7
SMA
NYARTEDFF (SEQ ID NO:
Horiguchi et al. Clin Cancer Res.

384)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res. 60(16):4499-

230)
506 (2000).

MLMAQEALAFL SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer. 132(2):345-

NO: 234)
54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzaki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

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

ID NO: 238)
54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12 (2002).

NO: 240)
Zeng et al. Proc Natl Acad Sci U S A.

MPFATPMEA (SEQ ID NO:
98(7):3964-9 (2001).

241)
Mandic et al. J Immunol. 174(3):1751-

FATPMEAEL (SEQ ID NO:
9(2005).

242)
Chen et al. Proc Natl Acad Sci U S A.

FATPMEAELAR (SEQ ID
101(25):9363-8 (2004).

NO: 243)
Ayyoub et al. Clin Cancer Res.

LAMPFATPM (SEQ ED NO:
16(18):4607-15 (2010).

231)
Slager et al. J Immunol. 172(8):5095-

ARGPESRLL (SEQ ID NO:
102 (2004).

232)
Mizote et al. Vaccine. 28(32):5338-

SLLMWITQCFLPVF (SEQ
46 (2010).

ID NO: 244)
Jager et al. J Exp Med. 191(4):625-

LLEFYLAMPFATPMEAEL
30 (2000).

-ARRSLAQ (SEQ ID NO:
Zarour et al. Cancer Res. 60(17):4946-

245)
52 (2000).

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

NO: 246)
9 (2000).

PGVLLKEFTVSGNILTIRL-
Bioley et al. Clin Cancer Res.

TAADHR (SEQ ID NO: 247)
15(13):4467-74 (2009).

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

NO: 248)
(2009).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLICEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLICEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILURL (SEQ

ID NO: 254)

PGVLLICEFTVSGNILTIRL-

TAADHR (SEQ ID NO: 247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ
ID NO: 257)

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

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang etal. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol Immunother.

314)
55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S A.

ID NO: 244)
98(7):3964-9 (2001).

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

VP-R (SEQ ID NO: 249)
102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

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

G-MPHL (SEQ ID NO: 316)
7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

10
HERV-K-
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

MEL
309)
62(19):5510-6 (2002).

11
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

12
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

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

NO: 312)
1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

13
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Intemati et al. Int J Cancer.

299)
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 (SEQ ID NO:
Hung et al. Immunol. Rev. 222:43-69

276)
(2008).

http://cancerimmunity.org/peptide/mutat

ions/

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 (hMe
TMNGSKSPV (SEQ I D
Di Modupo et al. Int. J Cancer.

na)
NO: 330)
109(6):909-18 (2004).

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

carboxyl
NO: 379)
90 (1999).

esterase

3
CASP-5
FLIIWQNTM (SEQ ID NO:
Schwitalle et al. Cancer Immun. 4: 14

385)
(2004).

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

(SEQ ID NO: 386)
63(20):6735-43 (2003).

5
OGT
SLYICFSPFPL (SEQ ID
Ripberger. J Clin Immunol. 23(5):415-

NO: 387)
23 (2003).

6
OS-9
KELEGILLL (SEQ ID NO:
Viperon et al. Cancer Immun. 2: 9

388)
(2002).

7
TGF-betaRII
RLSSCVPVA (SEQ ID NO:
Linnebacher et al. Int. J. Cancer.

389)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and
HLA-Cw6-Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

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

NO: 232))
55 (2000).

SLLMWITQC (SEQ ID
Valmori et al. Cancer Res.

NO: 230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer. 132(2):345-

NO: 234)
54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzaki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID
Ebert et al. Cancer Res. 69(3):1046-

NO: 237)
54 (2009).

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

ID NO: 238)
54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S A.

NO: 241)
98(7):3964-9 (2001).

FATPMEAEL (SEQ ID
Mandic et al. J Immunol. 174(3):1751-

NO: 242)
9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S A.

NO: 243)
101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol. 172(8):5095-

232)
102 (2004).

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

ID NO: 244)
46 (2010).

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

L-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res. 60(17):4946-

EFYLAMPFATPM (SEQ
52 (2000).

ID NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTLR
9(2000).

L-TAADHR SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

ID NO: 248)
(2002).

QGAMLAAQERRVPRAA
Hasegawa et al. Clin Cancer Res.

E-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIR

LT (SEQ ID NO: 251)

VLLICEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIR

L-TAADRR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAE

L-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM

(SEQ ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

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

ID NO: 258)
(2001).

EHYPNASLLIQN (SEQ ID
Parkhurst et al. Mol. Ther. 19(3):620-6

NO: 259)
(2011).

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

(SEQ ID NO: 260)
82 (2010).

LWWVNNQSLPVSP (SEQ
Bast et at. Am. J. Obstet. Gynecol.

ID NO: 261)
149(5):553-9 (1984).

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

ID NO: 261)
(2006).

LWWVNNQSLPVSP (SEQ
Kobayashi et al. Clin Cancer Res.

ID NO: 261)
8(10):3219-25 (2002).

EHYPNASLLIQN (SEQ ID
Campi et al. Cancer Res. 63(23):8481-

NO: 259)
6 (2003).

NSIVKSITVSASG (SEQ
Bakker et at. Int J Cancer. 62(497-

ID NO: 262)
102 (1995).

KTWGQYWQV (SEQ ID
Tsai et at. J Immunol. 158(4):1796-

NO:263)
802 (1997).

(A)MLGTHTMEV (SEQ ID
Kawakami et al. J Immunol.

NO: 264)
154(8):3961-8 (1995).

ITDQVPFSV (SEQ ID NO:
Cox et al. Science. 264(5159):716-9

265)
(1994).

YLEPGPVTA (SEQ ID
Kawakami et al. J Immunol.

NO: 266)
154(8):3961-8 (1995).

LLDGTATLRL (SEQ ID
Kawakami et al. J Immunol.

NO: 267)
161(12):6985-92 (1998).

VLYRYGSFSV (SEQ ID
Skipper et at. J Immunol.

NO: 268)
157(11):5027-33 (1996).

SLADTNSLAV (SEQ ID
Michaux et al. J Immunol.

NO: 269)
192(4):1962-71 (2014).

RLMKQDFSV (SEQ ID

NO: 270)

RLPRIFCSC (SEQ ID NO:

271)

LIYRRRLMK (SEQ ID

NO: 272)

ALLAVGATK (SEQ ID

NO: 273)

IALNFPGSQK (SEQ ID

NO: 274)

RSYVPLAHR (SEQ ID

NO: 275)

10
HERV-K-
MLAVISCAV (SEQ ID
Schiavetti et al. Cancer Res.

MEL
NO: 309)
62(19):5510-6 (2002).

11
KK-LC-1
RQICRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

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

NO: 311)
66(9):4922-8 (2006).

EYSICECLICEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18 Pt

NO: 312)
1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

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

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID
Rimoldi et al. J Immunol.

NO: 230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et at. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S A.

ID NO: 244)
98(7):3964-9 (2001).

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

EVP-R (SEQ ID NO: 249)
102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

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

G-MPHL (SEQ ID NO:
7(2003).

316)
Wang et al. Immunity. 20(1):107-18

ILSRDAAPLPRPG (SEQ
(2004).

ID NO: 317)
Hasegawa etal. Clin Cancer Res.

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

(SEQ
ID NO: 257)

14
MAGE-A2
YLQLVFGIEV (SEQ ID
Kawashima et al. Hum Immunol.

NO: 349)
59(1):1-14 (1998).

EYLQLVFGI (SEQ ID NO:
Tahara et al. Clin Cancer Res.

350)
5(8):2236-41 (1999).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID
Breckpot et al. J Immunol.

NO: 351)
172(4):2232-7 (2004).

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

(SEQ ID NO: 352)
(1999).

15
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

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

NO: 328)
(2008).

LSRLSNRLL (SEQ ID NO:

329)

17
TAG-2
LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-17

329)
(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 (SEQ ID NO:
Hung et al. Immunol. Rev. 222:43-69

276)
(2008).

http://cancerimmunity.org/peptide/mut

ations/

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 (SEQ ID NO:
El Hage et al. Proc. Natl.

390)
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 SEQ
Scie. U.S.A. 103(39):14453-8

ID NO: 230)), HLA-Cw3-
(2006).

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

FATPM (SEQ ID NO: 231)) and
September 26,2000 vol. 97

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

(ARGPESRLL (SEQ ID NO:
Jager et al. J Exp Med.

232))
187(2):265-70 (1998).

SLLMWITQC (SEQ ID NO:
Chen et al. J Immunol.

230)
165(2):948-55 (2000).

MLMAQEALAFL (SEQ ID
Valmori et al. Cancer Res.

NO: 233)
60(16):4499-506 (2000).

YLAMPFATPME (SEQ ID NO:
Aarnoudse et al. Int J Cancer.

234)
82(3):442-8 (1999).

ASGPGGGAPR (SEQ ID NO:
Eikawa et al. Int J Cancer.

235)
132(2):345-54 (2013).

LAAQERRVPR (SEQ ID NO:
Wang et al. J Immunol.

236)
161(7):3598-606 (1998).

TVSGNILTIR (SEQ ID NO:
Matsuzaki et al. Cancer

237)
Immunol Immunother.

APRGPHGGAASGL (SEQ ID
57(8)1185-95 (2008).

NO: 238)
Ebert et al. Cancer Res.

MPFATPMEAEL (SEQ ID NO:
69(3):1046-54 (2009).

239)
Eikawa et al. Int J Cancer.

KEFTVSGNILTI (SEQ ID NO:
132):345-54 (2013).

240)
Knights et al. Cancer

MPFATPMEA (SEQ ID NO:
Immunol Immunother.

241)
58(3):325-38 (2009).

FATPMEAEL (SEQ ID NO:
Jager et al. Cancer Immun.

242)
2:12 (2002).

FATPMEAELAR(SEQ ID NO:
Zeng et al. Proc Natl Acad Sci

243)
U S A. 98(7):3964-9 (2001).

LAMPFATPM (SEQ ID NO:
Mandic et al. J Immunol.

231)
174(3):1751-9 (2005).

ARGPESRLL (SEQ ID NO:
Chen et al. Proc Natl Acad

232)
Sci U S A. 101(25):9363-

SLLMWITQCFLPVF (SEQ ID
8(2004).

NO: 244)
Ayyoub et al. Clin Cancer

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

ARRSLAQ (SEQ ID NO: 245)
Slager et al. J Immunol.

EFYLAMPFATPM (SEQ ID
172(8):5095-102 (2004).

NO: 246)
Mizote et al. Vaccine.

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

TAADIAR (SEQ ID NO: 247)
Jager et al. J Exp Med.

RLLEFYLAMPFA (SEQ ID
191(4):625-30 (2000).

NO: 248)
Zarour et al. Cancer Res.

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

QGAMLAAQERRVPRAAE-
Zeng et al. J Immunol.

VPR (SEQ ID NO: 249)
165(2):1153-9 (2000).

PFATPMEAELARR (SEQ ID
Bioley et al. Clin Cancer Res.

NO: 250)
15(13):4467-74 (2009).

PGVLLKEFTVSGNILTIRLT
Zarour et al. Cancer Res.

(SEQ ID NO: 251)
62(1):213-8 (2002).

VLLICEFTVSG (SEQ ID NO:
Hasegawa et al. Clin Cancer

252)
Res. 12(6):1921-7 (2006).

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ ID

NO: 254)

PGVLLICEFTVSGNILTIRL-

TAADHR (SEQ ID NO: 247)

LLEFYLAMPFATPMEAEL-

ARRSLAQ (SEQ ID NO: 245)

KEFTVSGNILT (SEQ ID NO:

255)

LLEFYLAMPFATPM (SEQ ID

NO: 256)

AGATGGRGPRGAGA (SEQ

ID NO: 257)

3
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

4
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

5
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID NO:
Monji et al. Clin Cancer Res.

312)
10(18 Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO: 13)

6
AGE-1
MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID NO:
Wang et al. J Immunol

236)
161(7):3598-606 (1998).

ELVRRILSR (SEQ ID NO: 314)
Sun et al. Cancer Immunol

APRGVRMAV (SEQ ID NO:
Immunother. 55(6):644-52

315)
(2006).

SLLMWITQCFLPVF (SEQ ID
Slager et al. Cancer Gene

NO: 244)
Ther. 11(3):227-36 (2004).

QGAMLAAQERRVPRAAEVP-
Zeng et al. Proc Nat! Acad Sci

R (SEQ ID NO: 249)
U S A. 98(7):3964-9 (2001).

AADHRQLQLSISSCLQQL
Slager et al. J Immunol.

(SEQ ID NO: 253)
172(8):5095-102 (2004).

CLSRRPWICRSWSAGSCPG-
Jager et al. J Exp Med.

MPHL (SEQ ID NO: 316)
191(4):625-30 (2000).

ILSRDAAPLPRPG (SEQ ID
Slager et al. J Immunol.

NO: 317)
170(3):1490-7 (2003).

AGATGGRGPRGAGA (SEQ
Wang et al. Immunity.

ID NO: 257)
20(1):107-18 (2004).

Hasegawa et al. Clin Cancer

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

7
Spl 7
ILDSSEEDK (SEQ ID NO: 299)
Chiriva-Intemati 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 (SEQ ID NO:
Munir et al. Cancer Res. 73(6):1764-76

391)
(2013).

2
mdm-2
VLFYLGQY (SEQ ID NO:
Asai et al. Cancer Immun. 2: 3 (2002).

392)

3
alpha-
FIASNGVKLV (SEQ ID
Echchakir et al. Cancer Res.

actinin-4
NO: 393)
61(10):4078-83 (2001).

4
Elongation
ETVSEQSNV (SEQ ID NO:
Hogan et al. Cancer Res. 58(22):5144-

factor 2
394)
50 (1998).

(squamous

cell

carcinoma of

the lung)

5
ME1(non-
FLDEFMEGV (SEQ ID NO:
Karanikas et al. Cancer Res.

small cell
395)
61(9):3718-24 (2001).

lung

carcinoma)

6
NFYC
QQITKTEV (SEQ 1D NO:
Takenoyama et al. Int. J Cancer.

(squamous
396)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

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

NO: 232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer. 132(2):

NO: 234)
345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzalci et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

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

ID NO: 238)
345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S A.

NO: 241)
98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol. 174(3):1751-

242)
9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S A.

NO: 243)
101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol. 172(8):5095-

232)
102(2004).

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

ID NO: 244)
46 (2010).

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

L-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res. 60(17):4946-

EFYLAMPFATPM (SEQ
52 (2000).

ID NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
(2002).

QGAMLAAQERRVPRAA
Hasegawa et al. Clin Cancer Res.

E-VPR SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAE

L-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

8
GAGE-1,2,8
YRPRPRRY (SEQ ID NO:
Van den Eynde et al. J Exp Med.

397)
182(3):689-98 (1995).

9
HERV-K-
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

MEL
309)
62(19):5510-6 (2002).

10
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

11
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

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

NO: 312)
1):6047-57 (2004).

EYLSLSDKI SEQ ID NO:

313)

12
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang etal. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S A.

ID NO: 244)
98(7):3964-9 (2001).

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

EVP-R (SEQ ID NO: 249)
102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

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

G-MPHL (SEQ ID NO: 316)
7 (2003).

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

ID NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

13
MAGE-A2
YLQLVFGIEV (SEQ ID
Kawashima et al. Hum Immunol

NO: 349)
59(1):1-14 (1998).

EYLQLVFGI(SEQ ID NO:
Tahara et al. Clin Cancer Res.

350)
5(8):2236-41 (1999).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol

351)
172(4):2232-7 (2004).

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

ID NO: 352)
(1999).

14
MAGE-A6
MVKISGGPR (SEQ ID NO:
Zorn et al. Eur J Immunol. 29(2):602-7

(squamous
398)
(1999).

cell lung
EVDPIGHVY (SEQ ID NO:
Benlalam et al. J Immunol.

carcinoma)
399)
171(11):6283-9 (2003).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

ISGGPRISY (SEQ ID NO:
Vantomme et al. Cancer Immun.

400)
3:17 (2003).

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

ID NO: 352)
78 (1999).

15
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

16
TAG-1
SLGWLFLLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-17

328)
(2008).

LSRLSNRLL (SEQ ID NO:

329)

17
TAG-2
LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-17

329)
(2008).

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

(SEQ ID NO: 359)
(2006).

19
XAGE-
RQKKIRIQL (SEQ ID NO:
Ohue et al. Int J Cancer. 131(5):E649-

1b/GAGED2a
401)
58 (2012).

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

cell lung
(SEQ ID NO: 402)
40 (2007).

cancer)
CATWKVICKSCISQTPG

(SEQ ID NO: 403)

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 (SEQ ID
Nakatsuka et al. Mod. Pathol.

NO: 277)
19(6):804-814 (2006).

YLVPQQGFFC (SEQ ID
Pils et al. Br. J. Cancer 96(3):485-91

NO: 278)
(2007).

PLQPEQLQV (SEQ ID NO:
Scardino et al. Eur J Immunol.

279)
31(11):3261-70 (2001).

TLEEITGYL (SEQ ID NO:
Scardino et al. J Immunol.

280)
168(11):5900-6 (2002).

ALIHHNTHL (SEQ ID NO:
Kawashima et al. Cancer Res.

281)
59(2):431-5 (1999).

PLTSIISAV (SEQ ID NO:
Olcugawa et al. Eur J Immunol.

282)
30(11):3338-46 (2000).

VLRENTSPK (SEQ ID NO:

283)

TYLPTNASL (SEQ ID NO:

404)

23
MUC1

Reuschenbach et al. Cancer Immunol.

Immunother. 58:1535-1544 (2009)

24
p53
VVPCEPPEV (SEQ ID NO:
Hung et al. Immunol. Rev. 222:43-69

276)
(2008).

http://cancerimmunity.org/peptide/

mutations/

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 (SEQ ID NO:
Qian et al. Blood.

361)
110(5):1587-94 (2007).

2
ENAH (hMena)
TMNGSKSPV (SEQ ID NO:
Di Modugno et al. Int. J.

330)
Cancer. 109(6):909-18

(2004).

3
Kallikrein 4
FLGYLILGV (SEQ ID NO:
Wilkinson et al. Cancer

210); SVSESDTIRSISIAS (SEQ
Immunol Immunother.

ID NO: 211);
61(2):169-79 (2012).

LLANGRMPTVLQCVN (SEQ
Hural et al. J. Immunol.

ID NO: 212); and
169(1):557-65 (2002).

RMPTVLQCVNVSVVS (SEQ

ID NO: 213)

4
PSMA
NYARTEDFF (SEQ ID NO:
Horiguchi et al. Clin Cancer

384)
Res. 8(12):3885-92 (2002).

5
STEAP1
MIAVFLPIV (SEQ ID NO: 405)
Rodeberg et al. Clin. Cancer

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

ID NO: 406)
Kobayashi et al. Cancer

Res. 67(11):5498-504

(2007).

6
PAP
FLFLLFFWL (SEQ ID NO:
Olson et al. Cancer

407);
Immunol Immunother.

TLMSAMTNL (SEQ ID NO:
59(6):943-53 (2010).

408); and

ALDVYNGLL (SEQ ID NO:

409)

7
PSA (prostate
FLTPKKLQCV (SEQ ID NO:
Correale et al. J Natl.

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

ID NO: 411)
(1997).

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

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

ID NO: 230)), HLA-Cw3-
8 (2006).

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

FATPM (SEQ ID NO: 231)) and
September 26,2000 vol. 97

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

(ARGPESRLL (SEQ ID NO:
Jager et al. J Exp Med.

232))
187(2):265-70 (1998).

SLLMWITQC (SEQ ID NO:
Chen et al. J Immunol.

230)
165(2):948-55 (2000).

MLMAQEALAFL (SEQ ID
Valmori et al. Cancer Res.

NO: 233)
60(16):4499-506 (2000).

YLAMPFATPME (SEQ ID NO:
Aamoudse et al. Int J

234)
Cancer. 82(3):442-8 (1999).

ASGPGGGAPR (SEQ ID NO:
Eikawa et al. Int J Cancer.

235)
132(2):345-54 (2013).

LAAQERRVPR (SEQ ID NO:
Wang et al. J Immunol.

236)
161(7):3598-606(1998).

TVSGNILTIR (SEQ ID NO:
Matsuzaki et al. Cancer

237)
Immunol Immunother.

APRGPHGGAASGL (SEQ ID
57(8)1185-95 (2008).

NO: 238)
Ebert et al. Cancer Res.

MPFATPMEAEL (SEQ ID NO:
69(3):1046-54 (2009).

239)
Eilcawa et al. Int J Cancer.

KEFTVSGNILTI (SEQ ID NO:
132(2):345-54 (2013).

240)
Knights et al. Cancer

MPFATPMEA (SEQ ID NO:
Immunol Immunother.

241)
58(3):325-38 (2009).

FATPMEAEL (SEQ ID NO:
Jager et al. Cancer Immun.

242)
2:12 (2002).

FATPMEAELAR (SEQ ID NO:
Zeng et al. Proc Natl Acad

243)
Sci U S A. 98(7):3964-

LAMPFATPM (SEQ ID NO:
9(2001).

231)
Mandic et al. J Immunol.

ARGPESRLL (SEQ ID NO:
174(3):1751-9 (2005).

232)
Chen et al. Proc Natl Acad

SLLMWITQCFLPVF (SEQ ID
Sci U S A. 101(25):9363-

NO: 244)
8 (2004).

LLEFYLAMPFATPMEAEL-
Ayyoub et al. Clin Cancer

ARRSLAQ (SEQ ID NO: 245)
Res. 16(18):4607-15 (2010).

EFYLAMPFATPM (SEQ ID
Slager et al. J Immunol.

NO: 246)
172(8):5095-102 (2004).

PGVLLKEFTVSGNILTLRL-
Mizote et al. Vaccine.

TAADHR (SEQ ID NO: 247)
28(32):5338-46 (2010).

RLLEFYLAMPFA (SEQ ID
Jager et al. J Exp Med.

NO: 248)
191(4):625-30 (2000).

QGAMLAAQERRVPRAAE-
Zarour et al. Cancer Res.

VPR (SEQ ID NO: 249)
60(17):4946-52 (2000).

PFATPMEAELARR (SEQ ID
Zeng et al. J Immunol.

NO: 250)
165(2):1153-9 (2000).

PGVLLKEFTVSGNILTIRLT
Bioley et al. Clin Cancer

(SEQ ID NO: 251)
Res. 15(13):4467-74 (2009).

VLLKEFTVSG (SEQ ID NO:
Zarour et al. Cancer Res.

252)
62(1):213-8 (2002).

AADHRQLQLSISSCLQQL
Hasegawa et al. Clin Cancer

(SEQ ID NO: 253)
Res. 12(6):1921-7 (2006).

LKEFTVSGNILTIRL (SEQ
ID

NO: 254)

PGVLLICEFTVSGNILTERL-

TAADHR (SEQ ID NO: 247)

LLEFYLAMPFATPMEAEL-

ARRSLAQ (SEQ ID NO: 245)

KEFTVSGNILT (SEQ ID NO:

255)

LLEFYLAMPFATPM (SEQ ID

NO: 256)

AGATGGRGPRGAGA (SEQ

ID NO: 257)

9
BAGE-1(non-
AARAVFLAL (SEQ ID NO:
Boel et al. Immunity.

small cell lung
333)
2(2):167-75 (1995).

carcinoma)

10
GAGE-1,2,8
YRPRPRRY (SEQ ID NO: 397)
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 (SEQ ID NO:
De Backer et al. Cancer

(lung squamous
412)
Res. 59(13):3157-65 (1999).

cell carcinoma

and lung

adenocarcinoma)

12
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

13
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

14
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID NO:
Monji et al. Clin Cancer

312)
Res. 10(18 Pt 1):6047-57

EYLSLSDKI (SEQ ID NO: 313)
(2004).

15
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J

NO: 233)
Cancer. 82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID NO:
Wang et al. J Immunol.

236)
161(7):3598-606 (1998).

ELVRRILSR (SEQ ID NO: 314)
Sun et al. Cancer Immunol

APRGVRMAV (SEQ ID NO:
Immunother. 55(6):644-52

315)
(2006).

SLLMWITQCFLPVF (SEQ ID
Slager et al. Cancer Gene

NO: 244)
Ther. 11(3):227-36 (2004).

QGAMLAAQERRVPRAAEVP-
Zeng et al. Proc Natl Acad

R (SEQ ID NO: 249)
Sci U S A. 98(7):3964-

AADHRQLQLSISSCLQQL
9(2001).

(SEQ ID NO: 253)
Slager et al. J Immunol.

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

MPHL (SEQ ID NO: 316)
Jager et al. J Exp Med.

ILSRDAAPLPRPG (SEQ ID
191(4):625-30 (2000).

NO: 317)
Slager et al. J Immunol.

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

ID NO: 257)
Wang et al. Immunity.

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

Hasegawa et al. Clin Cancer

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

16
Sp17
ILDSSEEDK (SEQ ID NO: 299)
Chiriva-Intemati 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 (SEQ ID NO:
Hanada et al. Nature.

413)
427(6971):252-6 (2004).

2
Hepsin
SLLSGDWVL (SEQ ID NO:
Guo et al. Scand J Immunol.

376);
78(3):248-57 (2013).

GLQLGVQAV (SEQ ID NO:

377); and

PLTEYIQPV (SEQ ID NO:

378)

3
Intestinal
SPRWWPTCL (SEQ ID NO:
Ronsin et al. J Immunol

carboxyl
379)
163(1):483-90 (1999).

esterase

4
M-CSF
LPAVVGLSPGEQEY (SEQ
Probst-Kepper et al. J Exp

ID NO: 383)
Med. 193(10):1189-98 (2001).

5
RU2AS
LPRWPPPQL (SEQ ID NO:
Van Den Eynde et al. J. Exp.

362)
Med. 190(12):1793-800

(1999).

6
hsp70-2 renal
SLFEGIDIYT (SEQ ID NO:
Gaudin et al. J. Immunol.

cell carcinoma)
414)
162(3):1730-8 (1999).

7
Mannan-MUC-I
PDTRPAPGSTAPPAHGVTSA
Loveland et al. Clin. Cancer

(renal cell
(SEQ ID NO: 305)
Res. 12(3 Pt 1):869-77 (2006).

carcinoma)
STAPPVHNV (SEQ ID NO:
Loveland et al. Clin. Cancer

306)
Res. 12(3 Pt 1):869-77 (2006).

LLLLTVLTV (SEQ ID NO:
Godelaine et al. Cancer

307)
Immunol Immunother.

PGSTAPPAHGVT (SEQ ID
56(6):753-9 (2007).

NO: 308)
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 (SEQ ID NO:
Oehlrich et al. Int J Cancer.

cell carcinoma)
415)
117(2):256-64 (2005).

TABLE 13

Melanoma

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
Hepsin
SLLSGDWVL (SEQ ID NO:
Guo et at. Scand J Immunol.

376);
78(3):248-57 (2013).

GLQLGVQA (SEQ ID NO:

416); and

PLTEYIQPV (SEQ ID NO:

378)

2
ARTC1
YSVYFNLPADTIYTN
Wang et al J Immunol. 174(5):2661-

(SEQ ID NO: 417)
70 (2005).

3
B-RAF
EDLTVIUGDFGLATEKSR
Sharkey et at. Cancer Res.

WSGSHQFEQLS (SEQ ID
64(5):1595-9 (2004).

NO: 418)

4
beta-catenin
SYLDSGIHF (SEQ ID NO:
Robbins et al. J. Exp. Med.

419)
183(3):1185-92 (1996).

5
Cdc27
FSWAMDLDPKGA (SEQ
Wang et at. Science.

ID NO: 420)
284(5418):1351-4 (1999).

6
CDK4
ACDPHSGHFV (SEQ ID
Wolfel et at. Science.

NO: 421)
269(5228):1281-4 (1995).

7
CDK12
CILGKLFTK SEQ ID NO:
Robbins et at. Nat Med. 19(6):747-

422)
52. (2013).

8
CDKN2A
AVCPWTWLR (SEQ ID
Huang et al. J Immunol.

NO: 423)
172(10):6057-64 (2004).

9
CLPP
ILDKVLVHL SEQ ID NO:
Corbiere et al. Cancer Res.

424)
71(4):1253-62 (2011).

10
CSNK1A1
GLFGDIYLA (SEQ ID NO:
Robbins et at. Nat Med. 19(6):747-

425)
52 (2013).

11
FN1
MIFEKHGFRRTTPP (SEQ
Wang et al. J Exp Med.

ID NO: 426)
195(11):1397-406 (2003).

12
GAS7
SLADEAEVYL (SEQ ID
Robbins, et al. Nat Med. 19(6):747-

NO: 427)
52 (2013).

13
GPNMB
TLDWLLQTPK (SEQ ID
Lennerz et al. Proc. Natl. Acad. Sci.

NO: 428)
U.S.A. 102(44):16013-8 (2005).

14
HAUS3
ILNAMIAKI SEQ ID NO:
Robbins et al. Nat Med. 19(6):747-

429)
52 (2013).

15
LDLR-
WRRAPAPGA (SEQ ID
Wang et al. J Exp Med.

fucosyltransferase
NO: 430) and
189(10):1659-68 (1999).

PVTWRRAPA (SEQ ID

NO: 431)

16
MART2
FLEGNEVGKTY (SEQ ID
Kawakami et al. J Immunol.

NO: 432)
166(4):2871-7 (2001).

17
MATN
KTLTSVFQK (SEQ ID NO:
Robbins et al. Nat Med. 19(6):747-

433)
52 (2013).

18
MUM-1
EEKLIVVLF (SEQ ID NO:
Coulie et al. Proc. Natl. Acad. Sci.

434)
U.S.A. 92(17):7976-80 (1995).

19
MUM-2
SELFRSGLDSY (SEQ ID
Chiari et al. Cancer Res.

NO: 435)
and 59(22):5785-92 (1999).

FRSGLDSYV(SEQ ID NO:

436)

20
MUM-3
EAFIQPITR (SEQ ID NO:
Baurain et al. J. Immunol.

437)
164(11):6057-66 (2000).

21
neo-PAP
RVIKNSIRLTL (SEQ ID
Topalian et al. Cancer Res.

NO: 438)
62(19):5505-9 (2002).

22
Myosin class I
KINKNPKYK (SEQ ID NO:
Zorn, et al. Eur. J. Immunol.

439)
29(2):592-601 (1999).

23
PPP1R3B
YTDFHCQYV (SEQ ID
Robbins et al. Nat Med. 19(6):747-

NO: 440)
52 (2013).

Lu et al. J Immunol. 190(12):6034-

42 (2013).

24
PRDX5
LLLDDLLVSI (SEQ ID
Sensi et al. Cancer Res. 65(2):632-

NO: 441)
40 (2005).

25
PTPRK
PYYFAAELPPRNLPEP
Novellino et al. J. Immunol.

(SEQ ID NO: 442)
170(12):6363-70 (2003).

26
N-ras
ILDTAGREEY (SEQ ID
Linard et al. J. Immunol.

NO: 443)
168(9):4802-8 (2002).

27
RBAF600
RPHVPESAF (SEQ ID NO:
Lennerz et al. Proc. Natl. Acad. Sci.

444)
U.S.A. 102(44):16013-8 (2005).

28
SIRT2
KIFSEVTLK (SEQ ID NO:
Lennerz et al. Proc. Natl. Acad. Sci.

445)
U.S.A. 102(44):16013-8 (2005).

29
SNRPDI
SHETVIIEL (SEQ ID NO:
Lennerz et al. Proc. Natl. Acad. Sci.

446)
U.S.A. 102(44):16013-8 (2005).

30
Triosephosphate
GELIGILNAAKVPAD
Pieper et al. J Exp Med. 189(5):757-

isomerase
(SEQ ID NO: 447)
66 (1999).

31
OAI
LYSACFWWL (SEQ ID
Touloukian et al. J. Immunol.

NO: 448)
170(3):1579-85 (2003).

32
RAB38/NY-MEL-
VLHWDPETV (SEQ ID
Walton et al. J Immimol.

1
NO: 449)
177(11):8212-8 (2006).

33
TRP-I/gp75
MSLQRQFLR (SEQ ID NO:
Toulouldan et al. Cancer Res.

450);
62(18):5144-7 (2002).

ISPNSVFSQWRVVCDSLE
Robbins et al. J. Immunol.

DY (SEQ ID NO: 451);
(10):6036-47 (2002).

SLPYWNFATG (SEQ ID
Osen et al. PLoS One. 5(11):e14137

NO: 452); and
(2010).

SQWRVVCDSLEDYDT

(SEQ ID NO: 453)

34
TRP-2
SVYDFFVWL (SEQ ID
Parkhurst et al. Cancer Res.

NO: 454);
58(21):4895-901 (1998).

TLDSQVMSL (SEQ ID NO:
Noppen et al. hit. J. Cancer.

455);
87(2):241-6 (2000).

LLGPGRPYR (SEQ ID NO:
Wang et al. J. Exp. Med.

456);
1184(6):2207-16 (1996).

ANDPIFVVL (SEQ ID NO:
Wang et al. J. Immunol 160(2):890-

457);
7(1998).

QCTEVRADTRPWSGP
Castelli et al. J. Immunol.

(SEQ ID NO: 458); and
162(3):1739-48 (1999).

ALPYWNFATG (SEQ ID
Paschen et al. Clin. Cancer Res.

NO: 459)
(14):5241-7 (2005).

Robbins et al. J. Immunol.

169(10):6036-47 (2002).

35
tyrosinase
KCDICTDEY (SEQ ID NO:
Kittlesen et al. J. Immunol.

460);
160(5):2099-106 (1998).

SSDYVIPIGTY (SEQ ID
Kawakami et al. J. Immunol.

NO: 461);
(12):6985-92 (1998).

MLLAVLYCL (SEQ ID
Wolfel et al. Eur. J. Immunol.

NO: 462);
24(3):759-64 (1994).

CLLWSFQTSA (SEQ ID
Riley et al. I Immunother.

NO: 463);
24(3):212-20 (2001).

YMDGTMSQV (SEQ ID
Skipper et al. J. Exp. Med.

NO: 464);
183(2):527-34 (1996).

AFLPWHRLF (SEQ ID NO:
Kang et al. J. Immunol.

229);
155(3):1343-8 (1995).

IYMDGTADFSF (SEQ ID
Dalet et al. Proc. Natl. Acad. Sci.

NO: 465);
U.S.A. 108(29):E323-31 (2011)

QCSGNFMGF (SEQ ID
Lennerz et al. Proc. Natl. Acad. Sci.

NO: 466);
U.S.A. 102(44):16013-8 (2005).

TPRLPSSADVEF (SEQ ID
Benlalam et al. J. Immunol.

NO: 467);
171(11):6283-9 (2003).

LPSSADVEF (SEQ ID NO:
Morel et al. Int. J. Cancer.

468);
83(6):755-9 (1999).

LHHAFVDSIF (SEQ ID
Brichard et al. Eur. J. Immunol

NO: 469);
26(1):224-30 (1996).

SEIWRDIDF (SEQ ID NO:
Topalian et al. J. Exp. Med.

470);
(5):1965-71 (1996).

QNILLSNAPLGPQFP (SEQ
Kobayashi et al. Cancer Res.

ID NO: 471);
58(2):296-301 (1998).

SYLQDSDPDSFQD (SEQ

ID NO: 661); and

FLLHHAFVDSIFEQWLQR

HRP (SEQ ID NO: 472)

36
Melan-A/MART-1
YTTAEEAAGIGILTVILGV
Meng et al. J. Immunother. 23:525-

LLLIGCWYCRR (SEQ ID
534(2011)

NO: 473)

37
g59100/Pmell7
ALNFPGSQK (SEQ ID NO:
El Hage et al. Proc. Natl. Acad. Sci.

474)
U.S.A. 105(29):10119-24 (2008).

ALNFPGSQK (SEQ ID NO:
Kawashima et al. Hum Immunol.

474)
59(1):1-14 (1998).

VYFFLPDHL (SEQ ID NO:
Robbins et al. J Immunol.

475)
159(1):303-8 (1997).

RTKQLYPEW (SEQ ID
Sensi et al. Tissue Antigens.

NO: 476)
59(4):273-9 (2002).

HTMEVTVYHR (SEQ ID
Lennerz et al. Proc Natl Acad Sci U

NO: 477)
S A. 102(44):16013-8 (2005).

SSPGCQPPA (SEQ ID NO:
Benlalam et al. I Immunol.

478)
171(11):6283-9 (2003).

VPLDCVLYRY (SEQ ID
Vigneron et al. Tissue Antigens.

NO: 479)
65(2):156-62 (2005).

LPHSSSHWL (SEQ ID NO:
Castelli et al. J Immunol.

480)
162(3):1739-48 (1999).

SNDGPTLI (SEQ ID NO:
Touloulcian et al. J Immunol.

481)
164(7):3535-42 (2000).

GRAMLGTHTMEVTVY
Parkhurst et al. J Immunother.

(SEQ ID NO: 482)
27(2):79-91 (2004).

WNRQLYPEWTEAQRLD
Lapointe et at. J Immunol.

(SEQ ID NO: 483)
167(8):4758-64 (2001).

TTEWVEITARELPIPEPE
Kobayashi et al. Cancer Res.

(SEQ ID NO: 484)
61(12):4773-8 (2001).

TGRAMLGTHTMEVTVY

H(SEQ ID NO: 485)

GRAMLGTHTMEVTVY

(SEQ ID NO: 482)

38
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).

SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager etal. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. hit J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzald et at. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID
NO: Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ED NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVILICEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

39
BAGE-1
AARAVFLAL (SEQ ID NO:
Boel et al. Immunity. 2(2):167-

333)
75 (1995).

40
GAGE-1,2,8
YRPRPRRY (SEQ ID NO:
Van den Eynde et al. J Exp Med.

397)
182(3):689-98 (1995).

41
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer etal. Cancer Res.

(cutaneous
NO: 412)
59(13):3157-65 (1999).

melanoma)

42
GnTVf
VLPDVFIRC(V) (SEQ ID
Guilloux et al. J Exp Med.

NO: 486)
183(3):1173-83 (1996).

43
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

44
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

45
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSICECLICEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

46
LAGE-1
MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Stager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPW1CRSWSAGSCP
Slager et al. J Immunol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

47
LY6K
RYCNLEGPPI (SEQ ID
Suda et al. Cancer Sci. 98(11):1803-

NO: 487)
8 (2007).

KWTEPYCVIAAVKIFPRF
Tomita et al. Oncoimmunology.

FMV-AKQ (SEQ ID NO:
3:e28100 (2014).

488)

KCCICIRYCNLEGPPINSSV

F (SEQ ID NO: 489)

48
MAGE-A1
EADPTGHSY (SEQ ID NO:
Traversari et al. J Exp Med.

334)
176(5):1453-7 (1992).

KVLEYVHCV (SEQ ID NO:
Ottaviani et al. Cancer Immunol

335)
Immunother. 54(12):1214-20 (2005).

SLFRAVITK (SEQ ID NO:
Pascolo et al. Cancer Res.

336)
61(10):4072-7 (2001).

EVYDGREHSA (SEQ ID
Chaux et al. J Immunol.

NO: 337)
163(5):2928-36 (1999).

RVRFFFPSL (SEQ ID NO:
Luiten et al. Tissue Antigens.

338)
55(2):149-52 (2000).

EADPTGHSY (SEQ ID NO:
Luiten et al. Tissue Antigens.

334)
56(1):77-81 (2000).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

KEADPTGHSY (SEQ ID
Stroobant et al. Eur J Immunol.

NO: 340)
42(6):1417-28 (2012).

DPARYEFLW (SEQ ID
Corbiere et al. Tissue Antigens.

NO: 341)
63(5):453-7 (2004).

ITKKVADLVGF (SEQ ID
Goodyear et al. Cancer Immunol

NO: 342)
Immunother. 60(12):1751-61 (2011).

SAFPTITNF (SEQ ID NO:
van der Bruggen et al. Eur J

343)
Immunol24(9):2134-40 (1994).

SAYGEPRKL (SEQ ID NO:
Wang et al. Cancer Immunol

344)
Immunother. 56(6):807-18 (2007).

RVRFFFPSL (SEQ ID NO:
Chaux et al. J Exp Med. 189(5):767-

338)
78 (1999).

TSCILESLFRAVITK (SEQ
Chaux et al. Eur J Immunol.

ID NO: 345)
31(6):1910-6 (2001).

PRALAETSYVKVLEY

(SEQ ID NO: 346)

FLLIKYRAREPVTKAE

(SEQ ID NO: 347)

EYVIKVSARVRF (SEQ ID

NO: 348)

49
MAGE-A6
MVKISGGPR (SEQ ID NO:
Zorn et al. Eur J Immunol.

398)
29(2):602-7 (1999).

EVDPIGHVY (SEQ ID NO:
Benlalam et al. J Immunol.

399)
171(11):6283-9 (2003).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

ISGGPRISY (SEQ ID NO:
Vantomme et al. Cancer Immun.

400)
3:17 (2003).

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

ID NO: 352)
78 (1999).

50
MAGE-A10
GLYDGMEHL (SEQ ID
Huang et al. J Immunol.

NO: 490)
162(11):6849-54 (1999).

DPARYEFLW (SEQ ID
Chaux et al. J Immunol.

NO: 341)
163(5):2928-36 (1999).

51
MAGE-Al2
FLWGPRALV (SEQ ID
van der Bruggen et al. Eur J

NO: 491)
Immunol. 24(12):3038-43 (1994).

VRIGHLYIL (SEQ ID NO:
Heidecker et al. J Immunol.

492)
164(11):6041-5 (2000).

EGDCAPEEK (SEQ ID NO:
Panelli et al. J Immunol.

351)
164(8):4382-92 (2000).

REPFTICAEMLGSVIR
Breckpot et al. J Immunol.

(SEQ ID NO: 493)
172(4):2232-7 (2004).

AELVHFLLLKYRAR (SEQ
Wang et al. Cancer Immunol

ID NO: 494)
Immunother. 56(6):807-18 (2007).

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

78(1999).

52
MAGE-C2
LLFGLALIEV (SEQ ID
Ma et al. Int .1. Cancer. 109(5):698-

NO: 495)
702 (2004).

ALKDVEERV (SEQ ID
Godelaine et al. Cancer Immunol

NO: 496)
Immunother. 56(6):753-9 (2007).

SESIKKKVL (SEQ ID NO:
Ma et al. Int J Cancer. 129(10):2427-

497)
34 (2011).

ASSTLYLVF (SEQ ID NO:
Wen et al. Cancer Sci. 102(8):1455-

498)
61 (2011).

SSTLYLVFSPSSFST (SEQ

ID NO: 499)

53
NA88-A
QGQHFLQKV (SEQ ID
Moreau-Aubry et al. J Exp Med.

NO: 500)
191(9):1617-24 (2000).

54
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

55
SSX-2
KASEKIFYV (SEQ ID NO:
Ayyoub et al. J Immunol.

353)
168(4):1717-22 (2002).

EKIQKAFDDIAKYFSK
Ayyoub et al. J Immunol.

(SEQ ID NO: 354)
172(11):7206-11 (2004).

FGRLQGISPKI SEQ ID
Neumann et al. Cancer Immunol

NO: 355)
Immunother. 60(9):1333-46 (2011).

WEKMKASEKIFYVYMKR
Ayyoub et al. Clin Immunol.

K (SEQ ID NO: 356)
114(1):70-8 (2005).

KIFYVYMKRKYEAMT
Neumann et al. Int J Cancer.

(SEQ ID NO: 357)
112(4):661-8 (2004).

KIFYVYMKRKYEAM
Ayyoub et al. J Clin Invest.

(SEQ ID NO: 358)
113(8):1225-33 (2004).

56
SSX-4
INKTSGPKRGKHAWTHR
Ayyoub et al. J Immunol.

LRE (SEQ ID NO: 322)
174(8):5092-9 (2005).

YFSKKEWEKMKSSEKIV
Valmori et al. Clin Cancer Res.

YVY (SEQ ID NO: 323)
12(2):398-404 (2006).

MKLNYEVMTKLGFKVTL

PPF (SEQ ID NO: 324)

KHAWTHRLRERKQLVV

YEEI (SEQ ID NO: 325)

LGFKVTLPPFMRSKRAA

DFH (SEQ ID NO: 326)

KSSEKIVYVYMICLNYEV

MTK (SEQ ID NO: 327)

KHAWTHRLRERKQLVV

YEEI (SEQ ID NO: 325)

57
TRAG-3
CEFHACWPAFTVLGE
Janjic et al. J Immunol. 177(4):2717-

(SEQ ID NO: 359)
27 (2006).

58
TRP2-INT2g
EVISCKLIKR (SEQ ID NO:
Lupetti et al. J Exp Med.

501)
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 (SEQ ID
Mandruzzato et al. J. Exp. Med.

NO: 502)
186(5):785-93 (1997).

2
p53
VVPCEPPEV (SEQ ID
Ito et al. Int. J. Cancer.

NO: 276)
120(12):2618-24 (2007).

3
SAGE
LYATVIHDI (SEQ ID
Miyahara et al. Clin Cancer Res.

NO: 503)
11(15):5581-9 (2005).

TABLE 15

Chronic myeloid leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
BCR-ABL
SSKALQRPV(SEQ ID NO:
Yotnda et al. J. Clin. Invest.

504);
101(10):2290-6 (1998).

GFKQSSKAL(SEQ ID NO:
Bosch et al. Blood. 88(9):3522-7

505);
(1996).

ATGFKQSSICALQRPVAS
Makita et al. Leukemia.

(SEQ ID NO: 506); and
16(12):2400-7 (2002).

ATGFKQSSICALQRPVAS

(SEQ ID NO:
506)

2
dek-can
TMKQICKKEIRRLHQY
Makita et al. Leukemia.

(SEQ ID NO: 507)
16(12):2400-7 (2002).

3
EFTUD2
KILDAVVAQK (SEQ ID
Lennerz et al. Proc. Natl. Acad.

NO: 508)
Sci. U.S.A. 102(44):16013-8

(2005).

4
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

TABLE 16

Acute lymphoblastic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
ETV6-AML1
RIAECILGM (SEQ ID NO:
Yotnda et al. J. Clin. Invest.

509) and
(2):455-62 (1998).

IGRIAECILGMNPSR
Yun et al. Tissue Antigens.

(SEQ ID NO: 510)
54(2):153-61 (1999).

2
GAGE-3,4,5,6,7
YYWPRPRRY(SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

TABLE 17

Acute myelogenous leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
FLT3-ITD
YVDFREYEYY (SEQ ID
Graf et al. Blood. 109(7):2985-8

NO: 511)
(2007).

2
Cyclin-Al
FLDRFLSCM (SEQ ID
Ochsenreither et al. Blood.

NO: 512) and
119(23):5492-501 (2012).

SLIAAAAFCLA (SEQ ID

NO: 513)

3
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

TABLE 18

Chronic lymphocytic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
FNDC3B
VVMSWAPPV (SEQ ID
Rajasagi et al. Blood. 124(3):453-

NO: 514)
62 (2014).

2
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

TABLE 19

Promyelocytic leukemia

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
pml-RARalpha
NSNHVASGAGEAMETQS
Gambacorti-Passerini et al. Blood.

SSSEEIV (SEQ ID NO: 515)
81(5):1369-75 (1993).

2
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

TABLE 20

Multiple myeloma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
MAGE-C1
ILFGISLREV (SEQ ID NO:
Anderson et al. Cancer Immunol

516)
Immunother. 60(7):985-97 (2011).

KVVEFLAML (SEQ ID
Nuber et al. Proc Natl Acad Sci U S

NO: 517)
A. 107(34):15187-92 (2010).

SSALLSIFQSSPE (SEQ ID

NO: 518)

SFSYTLLSL (SEQ ID NO:

519)

VSSFFSYTL (SEQ ID NO:

520)

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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzaki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR SEQ

ID NO: 250)

PGVLLICEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLICEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

3
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (199)

SLMMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPWKRSWSAGSCP
Slager et al. J Immunol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

ILSRDAAPLPRPG (SEQ ID
Wang etal. Immunity. 20(1):107-18

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

SEQ ID NO: 257)
12(6):1921-7 (2006).

4
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

5
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

6
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

7
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

TABLE 21

B-cell lymphoma

Tumor-
Reported

associated
immunogenic

No.
antigen
epitopes
Sources

1
KPLFRRMSSLELVIA
Vauchy
et al. Int J Cancer.

D393-CD20
(SEQ ID
137(1):116-26 (2015).

NO: 521)

TABLE 22

Bladder carcinoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
BAGE-1
AARAVFLAL (SEQ ID NO:
Boel et al. Immunity. 2(2):167-

333)
75 (1995).

2
GAGE-1,2,8
YRPRPRRY (SEQ ID NO:
Van den Eynde et al. J Exp Med.

397)
182(3):689-98 (1995).

3
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

4
MAGE-A4
EVDPASNTY (SEQ ID NO:
Kobayashi et al. Tissue Antigens.

(transitional cell
318)
62(5):426-32 (2003).

carcinoma of
GVYDGREHTV (SEQ ID
Duffour et al. Eur J Immunol.

urinary bladder)
NO: 319)
29(10):3329-37 (1999).

NYKRCFPVI (SEQ ID NO:
Miyahara et al. Clin Cancer Res.

320)
11(15):5581-9 (2005).

SESLICMIF (SEQ ID NO:
Ottaviani et al. Cancer Immunol

321)
Immunother. 55(7):867-72 (2006).

Zhang et al. Tissue Antigens.

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

5
MAGE-A6
MVKISGGPR (SEQ ID NO:
Zorn et al. Eur J Immunol.

398)
29(2):602-7 (1999).

EVDPIGHVY (SEQ ID NO:
Benlalam et al. J Immunol.

399)
171(11):6283-9 (2003).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

ISGGPRISY (SEQ ID NO:
Vantomme et al. Cancer Immun.

400)
3:17 (2003).

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

ID NO: 352)
78 (1999).

6
SAGE
LYATVIHDI (SEQ ID NO:
Miyahara et al. Clin Cancer Res.

503)
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).

(SEQ ID NO: 230)), HLA-

Cw3-restricted p92-100
Gnjatic et al. PNAS

(LAMP-FATPM (SEQ ID
September 26, 2000 vol. 97 no. 20 p.

NO: 231)) and HLA-Cw6-
10919

restricted p80-88
Jager et al. J Exp Med. 187(2):265-

(ARGPESRLL (SEQ ID NO:
70 (1998).

232))
Chen et al. J Immunol. 165(2):948-

SLLMWITQC (SEQ ID NO:
55 (2000).

230)
Valmori et al. Cancer Res.

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

MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matusuzki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

8
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

AADHRQLQLSISSCLQQL
Jager etal. J Exp Med. 191(4):625-

(SEQ ID NO: 253)
30 (2000).

CLSRRPWICRSWSAGSCP
Slager et al. J Immunol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

9
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

10
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

No.
Tumor-
Reported immunogenic
Sources

associated
epitopes

antigen

11
KM-11N-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLICEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

12
SP17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
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 (SEQ ID NO:
Boel et al. Immunity. 2(2):167-

neck squamous cell
333)
75 (1995).

carcinoma)

2
GAGE-1,2,8
YRPRPRRY (SEQ ID NO:
Van den Eynde et al. I Exp Med.

397)
182(3):689-98 (1995).

3
GAGE-3,4,5,6,7
YYWPRPRRY (SEQ ID
De Backer et al. Cancer Res.

NO: 412)
59(13):3157-65 (1999).

4
LY6K
RYCNLEGPPI (SEQ ID
Suda et al. Cancer Sci. 98(11):1803-

NO: 487)
8 (2007).

KWTEPYCVIAAVKIFPRF
Tomita et al. Oncoimmunology.

FMV-AKQ (SEQ ID NO:
3:e28100 (2014).

488)

KCCKIRYCNLEGPPINSSV

F (SEQ ID NO: 489)

5
MAGE-A3 (head
EVDPIGHLY (SEQ ID NO:
Gaugler et al. J Exp Med.

and neck squamous
662)
179(3):921-30 (1994).

cell carcinoma)
FLWGPRALV (SEQ ID
van der Bruggen et al. Eur J

NO: 491)
Immunol. 24(12):3038-43 (1994).

KVAELVHFL (SEQ ID NO:
Kawashima et al. Hum Immunol.

663)
59(1):1-14 (1998).

TFPDLESEF (SEQ ID NO:
Oiso et al. Int J Cancer. 81(3):387-

664)
94 (1999).

VAELVHFLL (SEQ ID NO:
Miyagawa et al. Oncology. 70(1):54-

665)
62 (2006).

MEVDPIGHLY (SEQ ID
Bilsborough et al. Tissue Antigens.

NO: 666)
60(1):16-24 (2002).

EVDPIGHLY (SEQ ID NO:
Schultz et al. Tissue Antigens.

662)
57(2):103-9 (2001).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

AELVHFLLL (SEQ ID NO:
Schultz et at. J Exp Med.

667)
195(4):391-9 (2002).

MEVDPIGHLY (SEQ ID
Herman et al. Immunogenetics.

NO: 666)
43(6):377-83 (1996).

WQYFFPVIF (SEQ ID NO:
Russo et al. Proc Natl Acad Sci U S

668)
A. 97(5):2185-90 (2000).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

KKLLTQHFVQENYLEY
Schultz et al. Cancer Res.

(SEQ ID NO: 669)
60(22):6272-5 (2000).

RKVAELVHFLLLKYR
Cesson et al. Cancer Immunol

(SEQ ID NO: 670)
Immunother. 60(1):23-35 (2011).

KKLLTQHFVQENYLEY
Schultz et al. J Immunol.

(SEQ ID NO: 669)
172(2):1304-10 (2004).

ACYEFLWGPRALVETS
Zhang et al. J Immunol. 171(1):219-

(SEQ ID NO: 671)
25 (2003).

RKVAELVHFLLLKYR
Cesson et al. Cancer Immunol

(SEQ ID NO: 670)
Immunother. 60(1):23-35 (2010).

VIFSKASSSLQL (SEQ ID
Kobayashi et al. Cancer Res.

NO: 672)
61(12):4773-8 (2001).

VFGIELMEVDPIGHL
Cesson et al. Cancer Immunol

(SEQ ID NO: 673)
Immunother. 60(1):23-35 (2011).

GDNQIMPKAGLLIIV
Consogno et al. Blood. 101(3):1038-

(SEQ ID NO: 674)
44 (2003).

TSYVKVLIMMVKISG
Manici et al. J Exp Med. 189(5):871-

(SEQ ID NO: 675)
6 (1999).

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

(SEQ ID NO: 676)
78 (1999).

FLLLKYRAREPVTKAE

(SEQ ID NO: 347)

6
MAGE-A6
MVIUSGGPR (SEQ ID NO:
Zorn et al. Eur J Immunol.

398)
29(2):602-7 (1999).

EVDPIGHVY (SEQ ID NO:
Benlalam et al. J Immunol.

399)
171(11):6283-9 (2003).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

ISGGPRISY (SEQ ID NO:
Vantomme et al. Cancer Immun.

400)
3:17 (2003).

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

ID NO: 352)
78 (1999).

7
SAGE
LYATVIHDI (SEQ ID NO:
Miyahara et al. Clin Cancer Res.

503)
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 (SEQ ID
De Backer et al. Cancer Res.

(Esophageal
NO: 412)
59(13):3157-65 (1999).

squamous cell

carcinoma and

esophageal

adenocarcinoma)

2
MAGE-A2
YLQLVFGIEV (SEQ ID
ICawashima et al. Hum Immunol.

NO: 349)
59(1):1-14 (1998).

EYLQLVFGI (SEQ ID NO:
Tahara et al. Clin Cancer Res.

350)
5(8):2236-41 (1999).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

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

ID NO: 352)
78 (1999).

3
MAGE-A6
MVKISGGPR (SEQ ID NO:
Zorn et al. Eur J Immunol.

398)
29(2):602-7 (1999).

EVDPIGHVY (SEQ ID NO:
Benlalam et al. J Immunol.

399)
171(11):6283-9 (2003).

REPVTKAEML (SEQ ID
Tanzarella et al. Cancer Res.

NO: 339)
59(11):2668-74 (1999).

EGDCAPEEK (SEQ ID NO:
Breckpot et al. J Immunol.

351)
172(4):2232-7 (2004).

ISGGPRISY (SEQ ID NO:
Vantommeet al. Cancer Immun.

400)
3:17 (2003).

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

ID NO: 352)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int. J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzaki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Nati Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

5
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPWKRSWSAGSCP
Slager et al. J Inununol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

6
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

7
KK-LC-1
RQICRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

8
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID
NO:

313)

9
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

TABLE 25

Brain cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

328)
17 (2008).

LSRLSNRLL (SEQ ID NO:

329)

2
TAG-2
LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

329)
17 (2008).

TABLE 26

Pharynx cancer

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

328)
17 (2008).

LSRLSNRLL (SEQ ID NO:

329)

2
TAG-2
LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

329)
17 (2008).

TABLE 27

Tumors of the tongue

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
TAG-1
SLGWLFLLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

328)
17 (2008).

LSRLSNRLL (SEQ ID NO:

329)

2
TAG-2
LSRLSNRLL (SEQ ID NO:
Adair et al. J Immunother. 31(1):7-

329)
17 (2008).

TABLE 28

Synovial cell sarcoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol.

NO: 236)
161(7):3598-606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPWICRSWSAGSCP
Slager et al. J Immunol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
12(6):1921-7 (2006).

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

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

(SEQ ID NO: 230)), HLA-
Gmjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
p. 10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol.

NO: 235)
161(7):3598-606 (1998).

LAAQERRVPR (SEQ ID
Matsuzaki et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager etal. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL (SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLICEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

3
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

4
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

5
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID

NO: 312)

EYLSLSDKI (SEQ ID NO:

313)

6
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

TABLE 29

Neuroblastoma

Tumor-

associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV (SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPWICRSWSAGSCP
Slager et al. J Inununol.

G-MPHL (SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM (SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzald et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Imununother. 58(3):325-38 (2009).

KEFTVSGNLLTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Natl Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et al. Clin Cancer Res.

247)
15(13):4467-74 (2009).

RLLEFYLAMPFA SEQ ID
Zarour et al. Cancer Res. 62(1):213

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR(SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

3
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti et al. Cancer Res.

309)
62(19):5510-6 (2002).

4
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

5
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF (SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

6
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Int J Cancer.

299)
107(5):863-5 (2003).

TABLE 30

Uterine cancer

Tumor-associated
Reported immunogenic

No.
antigen
epitopes
Sources

1
LAGE-1
MLMAQEALAFL (SEQ ID
Aarnoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

SLLMWITQC (SEQ ID NO:
Rimoldi et al. J Immunol.

230)
165(12):7253-61 (2000).

LAAQERRVPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 236)
606 (1998).

ELVRRILSR (SEQ ID NO:
Sun et al. Cancer Immunol

314)
Immunother. 55(6):644-52 (2006).

APRGVRMAV SEQ ID
Slager et al. Cancer Gene Ther.

NO: 315)
11(3):227-36 (2004).

SLLMWITQCFLPVF (SEQ
Zeng et al. Proc Natl Acad Sci U S

ID NO: 244)
A. 98(7):3964-9 (2001).

QGAMLAAQERRVPRAAE
Slager et al. J Immunol.

VP-R (SEQ ID NO: 249)
172(8):5095-102 (2004).

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

(SEQ ID NO: 253)
30 (2000).

CLSRRPWICRSWSAGSCP
Slager et al. J Immunol.

G-MPHL SEQ ID NO: 316)
170(3):1490-7 (2003).

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

NO: 317)
(2004).

AGATGGRGPRGAGA
Hasegawa et al. Clin Cancer Res.

(SEQ ID NO: 257)
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).

(SEQ ID NO: 230)), HLA-
Gnjatic et al. PNAS

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

(LAMP-FATPM SEQ ID
10919

NO: 231)) and HLA-Cw6-
Jager et al. J Exp Med. 187(2):265-

restricted p80-88
70 (1998).

(ARGPESRLL (SEQ ID NO:
Chen et al. J Immunol. 165(2):948-

232))
55 (2000).

SLLMWITQC (SEQ ID NO:
Valmori et al. Cancer Res.

230)
60(16):4499-506 (2000).

MLMAQEALAFL (SEQ ID
Aamoudse et al. Int J Cancer.

NO: 233)
82(3):442-8 (1999).

YLAMPFATPME (SEQ ID
Eikawa et al. Int J Cancer.

NO: 234)
132(2):345-54 (2013).

ASGPGGGAPR (SEQ ID
Wang et al. J Immunol. 161(7):3598-

NO: 235)
606 (1998).

LAAQERRVPR (SEQ ID
Matsuzald et al. Cancer Immunol

NO: 236)
Immunother. 57(8)1185-95 (2008).

TVSGNILTIR (SEQ ID NO:
Ebert et al. Cancer Res. 69(3):1046-

237)
54 (2009).

APRGPHGGAASGL (SEQ
Eikawa et al. Int J Cancer.

ID NO: 238)
132(2):345-54 (2013).

MPFATPMEAEL (SEQ ID
Knights et al. Cancer Immunol

NO: 239)
Immunother. 58(3):325-38 (2009).

KEFTVSGNILTI (SEQ ID
Jager et al. Cancer Immun. 2:12

NO: 240)
(2002).

MPFATPMEA (SEQ ID
Zeng et al. Proc Nat! Acad Sci U S

NO: 241)
A. 98(7):3964-9 (2001).

FATPMEAEL SEQ ID NO:
Mandic et al. J Immunol.

242)
174(3):1751-9 (2005).

FATPMEAELAR (SEQ ID
Chen et al. Proc Natl Acad Sci U S

NO: 243)
A. 101(25):9363-8 (2004).

LAMPFATPM (SEQ ID
Ayyoub et al. Clin Cancer Res.

NO: 231)
16(18):4607-15 (2010).

ARGPESRLL (SEQ ID NO:
Slager et al. J Immunol.

232)
172(8):5095-102 (2004).

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

ID NO: 244)
46 (2010).

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

-ARRSLAQ (SEQ ID NO:
30 (2000).

245)
Zarour et al. Cancer Res.

EFYLAMPFATPM (SEQ ID
60(17):4946-52 (2000).

NO: 246)
Zeng et al. J Immunol. 165(2):1153-

PGVLLKEFTVSGNILTIRL
9(2000).

-TAADHR (SEQ ID NO:
Bioley et at. Clin Cancer Res.

247)
15(13):4467-74 (2009).

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

NO: 248)
8 (2002).

QGAMLAAQERRVPRAAE
Hasegawa et al. Clin Cancer Res.

-VPR (SEQ ID NO: 249)
12(6):1921-7 (2006).

PFATPMEAELARR (SEQ

ID NO: 250)

PGVLLKEFTVSGNILTIRL

T (SEQ ID NO: 251)

VLLKEFTVSG (SEQ ID

NO: 252)

AADHRQLQLSISSCLQQL

(SEQ ID NO: 253)

LKEFTVSGNILTIRL (SEQ

ID NO: 254)

PGVLLKEFTVSGNILTIRL

-TAADHR (SEQ ID NO:

247)

LLEFYLAMPFATPMEAEL

-ARRSLAQ (SEQ ID NO:

245)

KEFTVSGNILT (SEQ ID

NO: 255)

LLEFYLAMPFATPM (SEQ

ID NO: 256)

AGATGGRGPRGAGA

(SEQ ID NO: 257)

3
HERV-K-MEL
MLAVISCAV (SEQ ID NO:
Schiavetti el al. Cancer Res.

309)
62(19):5510-6 (2002).

4
KK-LC-1
RQKRILVNL (SEQ ID NO:
Fukuyama et al. Cancer Res.

310)
66(9):4922-8 (2006).

5
KM-HN-1
NYNNFYRFL (SEQ ID NO:
Fukuyama et al. Cancer Res.

311)
66(9):4922-8 (2006).

EYSKECLKEF(SEQ ID
Monji et al. Clin Cancer Res. 10(18

NO: 312)
Pt 1):6047-57 (2004).

EYLSLSDKI (SEQ ID NO:

313)

6
Sp17
ILDSSEEDK (SEQ ID NO:
Chiriva-Internati et al. Intr 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 0(1.2.4) multiple sequence alignment (SEQ ID NOS 522-526, respectively, in

order of appearance)

cop
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIIINGINYHAFESLLDYIRW
60

WR
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESILDYIRW
60

Tian
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKDEAIILNGINYHAFESLLDYIRW
60

Wyeth
MESVTFSINGEIIQVNKEIITASPYNFFKRIQEHHINDEVIILNGINYHAFESLLDYMRW
60

Lister
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDRHLKDEAIILNGINYHAFESLLDYMRW
60

**** ***************************:**::**.*****************:**

cop
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMENFSKRYGIKKLYNA
120

WR
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMENFSKRYGIKKLYNA
120

Tian
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINSTNCIRMENFSKQYGIKKLYNA
120

Wyeth
KKINITINNVEMILVAAVIIDVTPVVDLCVKTMIHNINSTNCIRMFNESKRYGIKKLYNA
120

Lister
KKINITINNVEMILVAAIIIDVPPVVDLCVKTMIHNINFTNCIRMFNFSKRYGIKKLYNA
120

*****************:**** *************** ***********:*********

cop
SMSEIINNITAVTSDPEFGKLSKDELTTILSHENVNVNHEDVTAMILLKWIHKNPNDVDI
180

WR
SMSEIINNITAVTSDPEFGKLSKDELTTILSHENVNVNHEDVTAMILLKWIHKNPNDVDI
180

Tian
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

Wyeth
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

Lister
SMSEIINNITAVTSDPEFGKLSKDELTTILSHEDVNVNHEDVTAMILLKWIHKNPNDVDI
180

*********************************:**************************

cop
INILHPKFMTNTMRNAISLLGLTISKSTKPVTANGIKHNIVVIKNSDYISTITHYSPRTE
240

WR
INILHPKFMTNTMRNAISLLGLTISKSTKPVTANGIKHNIVVIKNSDYISTITHYSPRTE
240

Tian
INILHPKFMTNTMRNAISLLGLTISKSTKPVTANGIKHNIVVIKNSDYISTITHYSPRTE
240

Wyeth
INILHPKFMTNTMRNAISLLGLTISKSTKPVTANGIKHNIVVIKNSDYISTITHYSPRTE
240

Lister
INILHPKFMTNTMRNAISLLGITISKSTKPVTANGIKHNIVVIKNSDYISTITHYSPRTE
240

************************************************************

cop
YWTIVGNTDRQFYNANVIENCLYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

WR
YWTIVGNTDRQFYNANVLHNCLYIIGGMINNAHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Tian
YWTIVGNTDRQFYNANVIHNCLYIIGGMINNAHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Wyeth
YWTIVGNTDRQFYNANVIHNCLYIIGGMINNAHVYSVSRVDLETKKWKTVTNMSSLKSEV
300

Lister
YWTIVGNTDRQFYNANVIHNCLYIIGGMINNAHVYSVSRVDIKTKKWKTVTNMSSLKSEV
300

*******************************************:****************

cop
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

WR
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Tian
STCVNNGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Wyeth
STCVNNGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

Lister
STCVNDGKLYVIGGLEFSISTGVAEYLKHGTSKWIRLPNLITPRYSGASVFVNDDIYVMG
360

*****:******************************************************

cop
GVYTTYEKYVVLNDVECETKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

WR
GVYTTYEKYVVLNDVECETKNRWIKKSPMPRHESIVYAVEYDGDIYVITGITHETRNYLY
420

Tian
GVYTTYEKYVVLNDVECETKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

Wyeth
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYAITGITHETRNYLY
420

Lister
GVYTTYEKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYDGDIYVITGITHETRNYLY
420

**********************************************.*************

cop
KYIVREDKWIELYMYENHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLEDMSTRNIEY
480

WR
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDMSTRNIEY
480

Tian
KYIVKEDKWIELYMYFNHVGKMFVCSCGDYILIIADAKYETYPKSNTWNLFDMSTRNIEY
480

Wyeth
KYIVKEDKWIELYMYENHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLEDMSTRNIEY
480

Lister
KYIVKEDKWIELYMYENHVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLEDMSTRNIEY
480

************************************************************

cop
YDMETKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

WR
YDNFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

Tian
YDNFTKDETPKCNVTHKSLPSFLSNCEKQFLQ
512

Wyeth
YDMFTKDET------HKSLPSFLSNCEKQFLQ
506

Lister
YDMETKDETPKONVTHKSLPSFLSNCEKQFLQ
512

********* *****************

c1L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 527-531, respectively, in

order of appearance)

Cop
MVKNNKI-----SNSCRMIMSTNPNNILMRHLKULTDDEFKCIIHRSSDFLYLSDSDYTS
55

WR
MVKNNKIQKNKISNSCRMIMSTDPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
60

Tian
MVKNNKI-----SNSCRMINSTDPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
55

Wyeth
MVKNNKI-----SNSCRMINSTNPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDRDYTS
55

Lister
MVRNNKI-----SNSCRMIMSTNPNNILMRHLKNLTDDEFKCIIHRSSDFLYLSDSDYTS
55

******* ************************************************

Cop
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIETKLITKPAVRFAILDRMTEDIK
115

WR
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDINTEDIK
120

Tian
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIKTKLITKPAVRFAILDKMTEDIK
115

Wyeth
ITKETLVSEIVEEYFDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKMTEDIK
115

Lister
ITKETLVSEIVEEYPDDCNKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDINTADIK
115

*************************************:**********************

Cop
LTDLVRRYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

WR
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
180

Tian
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

Wyeth
LTDLVRHYFRYIEQDIPLGPLFRKIDSYRTRAINKYSKELGLATEYFNKYGHLMFYTLPI
175

Lister
LTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAINRYSKELGLATEYFNRYGHLMFYTLPI
175

************************************************************

Cop
PYNRFFCRNSIGFLAVISPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

WR
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
229

Tian
PYNRFFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

Wyeth
PYNRFFCRNSIGFLAVISPTIGHVKAFYKFIEYVSIDDRRKFKKELMSK
224

Lister
PYNRFFCRNSIGFLAVLSPTIGHVKAFYRFIEYVSIDDRRKFKKELMSK
224

*************************************************

N1L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 532-536, respectively, in

order of appearance)

Cop
MTYLLIRYILWANDNDQTYYNDNFKKLMILDELVDDGDVCTLIKNNRMTLSDGPLLDRLN
60

WR
MRTLLIRYILWANDNDQTYYNDNFKKLMILDELVDDGDVCTLIKNNRMTLSDGPLLDRLN
60

Tian
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

Wyeth
MRTLLIRYILWANDNDQTYYNDDFKKLMILDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

Lister
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVDDGDVCTLIKNMRMTLSDGPLLDRLN
60

**********************:*************************************

Cop
QPVNNIEDAKRMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

WR
QPVNNIEDARRMIAISARVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Tian
QPVNNIEDAERMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Wyeth
QPVNNIEDAKRMIAISARVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

Lister
QPVNNIEDAKRMSAISAKVARDIGERSEIRWEESFTILFRMIETYFDDLMIDLYGEK
117

*********************************************************

N2L

CLUSTAl 0(1.2.4) multiple sequence alignment (SEQ ID NOS 537-541, respectively, in

order of appearance)

Cop
MTSSAMDNNEPHVLEMVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILNRF
60

WR
MTSSANDNNEPKVLEMVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILDRF
60

Tian
MTSSAMDNNEFKVLEMVTDATILPEGSSMDPYINDCINRHINMCIQRTYSSSIIAILDRF
60

Wyeth
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIIDCINREINMCIQRTYSSSIIAILDRF
60

Lister
MTSSANDNNEPKVLENVYDATILPEGSSMDPNIMDCINRHINMCIQRTYSSSIIAILDRF
60

******************************* *:***********************:**

Cop
LTMNKDELNNTQCHIIREFMTYEQMAIDHYGEYVNAILYQIRKRPNQHHTIDLFKKIRRT
120

WR
LMMNKDELNNTQCHIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQHRTIDLFKRIKRT
120

Tian
LMMNKDELNNTQCHIIKNL-----------------------------------------
79

Wyeth
LTMNKDELNNTQCHIIKEFMTYEQMAIDHYGGYVNAILYQIRRRPNQHHTIDLFKKIKRT
120

Lister
LTMNRDELNNTQCHIIKEFMIYEQMAIDHYGEYVNAILYQIRERPNQHRTIDLFKKIKRT
120

* ***************::

Cop
PYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFINYVETRYF
175

WR
RYDTFKVDPVEEVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFINYVETKYF
175

Tian
-------------------------------------------------------

Wyeth
RYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVLYDEFKCFIDYVETKYF
175

Lister
RYDTFKVDPVEFVKKVIGFVSILNKYKPVYSYVLYENVINDEFKCFIDYVETRYF
175

M1L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 542-546, respectively, in

order of appearance)

Cop
MIEVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVERNMLQAIEPS
60

WR
MIETIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVERNMIQAIEPS
60

Tian
MIETIESKLLQIYRNRNIININFYTTMDNIMSAEYYLSLYAKYNKNLDVERNMIQAIEPS
60

Wyeth
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMIQAIEPS
60

Lister
MIFVIESKLLQIYRN--RNINFYTTMDNIMSAEYYLSLYAKYNSKNLDVFRNMLQAIEPS
58

*************** *******************************************

Cop
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMILTH
120

WR
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNIPLHIASKINNNRIVAMILTH
120

Tian
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNIPLHIASKINNNRIVAMLLTH
120

Wyeth
GNNYHILHAYCGIKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
120

Lister
GNNYHILHAYCGIKGIDERFVEELLHRGYSPNETDDDGNYPLHIASKINNNRIVAMLLTH
118

************************************************************

Cop
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLIACTDPSER
180

WR
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLIACTDPSER
180

Tian
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLIACTDPSER
180

Wyeth
GADPNACDKHNKTPLYYLSGTDDEVIERINLLVQYGAKINN-------------------
161

Lister
GADPNACDKQHKTPLYYLSGTDDEVIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER
178

*********::******************************

Cop
VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTISWMMKLGISPSKPDHDGNTPLHI
240

WR
VFKKIMSIGFEARIVDKEGKNHIHRHLMEDNPKASTISWMMKIGISPSKPDHDGNIPLHI
240

Tian
VEKKIMSIGFEARIVDKEGKNHIHRHLMSDNPKASTISWNMKLGISPSKPDHDGNTPLHI
240

Wyeth
------------------------------------------------------------

Lister
VFKKIMSIGFEARIVDKEGKNHIHRHLMSDNPKASTISWMMFIGISPSKPDHDGNIPLHI
238

Cop
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLILLIKILSPAHLINKLLSTSNVITDQTV
300

WR
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLILLIKILSPAHLINKLLSTSNVITDQTV
300

Tian
VCSKTVKNVDIIDLLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLSTSNVITDQTV
300

Wyeth
------------------------------------------------------------

Lister
VCSKTVFNVDIIDLLLPSTDVNKQNKFGDSPLILLIKILSPAHLINKLLSTSNVITDQTV
298

Cop
NICIFYDRDDVIEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVISE
360

WR
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
360

Tian
NICIFYDRDDVLEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
360

Wyeth
------------------------------------------------------------

Lister
NICIFYDRDDVIEIINDKGKQYDSTDFKMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSE
358

Cop
YETMVDYLLENHFSVDSVVNGHTCMSECVALNNPVILSKLMLHNPTSETMYLTMKAIEKD
420

WR
YETMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVILSKLMLHNPTSETMYLTMKAIEKD
420

Tian
YETMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVILSKLMLHNLTSETMYLTMKAIEKD
420

Wyeth
------------------------------------------------------------

Lister
YETMVDYLLENHFSVDSVVNGHTCMSECVALNNPVILSKLMIHNPTSETMYLTMKAIEKD
418

Cop
KLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVEDDYF
472

WR
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKRFVTDYVHEGVSIEVEDDYF
472

Tian
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKREVTDYVHEGVSYEVEDDYF
472

Wyeth
----------------------------------------------------

Lister
RLDKSIIIPFIAYFVLMHPDFCKNRRYFTSYKREVIDYVHEGVSYEVEDDYF
470

M2L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 547-551, respectively, in

order of appearance)

Cop
MVYKLVILFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

WR
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

Tian
------------------------MSSSTRLPVIVLAAELTIGVNYDINSTIIGECHMSE
36

Wyeth
MVYKIVILFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

Lister
MVYK/VILFCIASLGYSVEYKNTICPPRQDYRYWYFAAELTIGVNYDINSTIIGECHMSE
60

Cop
SYIDRNANIVLIGYGLEINMTIMDTDQREVAAAEGVGKONKLSVILFTTQRLDKVBHNIS
120

WR
SYIDRNNANIVTGYGLEINNTIMDTDOREVAAAEGVGEDNKLSVLLFTTQRLDKVEHNIS
120

Tian
SYIDRNANIVLTGYGLEINMTIMDTDQREVAAAEGVGKDNKLSVLLETTQRLDKVHBNIS
96

Wyeth
SYIDRNANIVLTGYGLEINMTIMDTDQREVAAAEGVGKDNKISVLLFTTQRLDKVHHNIS
120

Lister
SYIDRNANIVLTGYGLEINNTIMDTDQREVAAAEGVGKDNKLSVLLETTQRLDKVHENIS
120

------------------------------------------------------------

Cop
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDK
180

WR
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDATKINPHYLHAKDK
180

Tian
VTITCNEMNCGTTKYDSDLPESIEKSSSCDITINGSCVTCVNLETDATKINPHYLHPKDK
156

Wyeth
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDATKINPHYLHAKDK
180

Lister
VTITCMEMNCGTTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDATKINPHYLHAKDK
180

------------------------------------------------------------

Cop
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

WR
YLYHNSEYSMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

Tian
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
196

Wyeth
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

Lister
YLYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDICYRE
220

********_*******************************

K1L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 552-556, respectively,

in order of appearance)

Cop
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALYYAIADNNVALVCTLLNAGALKNLLE
60

WR
MDLSAINTWKSKQLKSFLSSKDAFKADVHGHSALYYAIADNNVRLVCTLLNAGALKNLLE
60

Tian
MDLSAINTWKSKQLKSFLSSKDTFKADVHGHSALYYAIADNNVALVCTLLNSGALKNLLE
60

Wyeth
MDLSRINTWKSKQLKSELSSKDTFKADVHGHSALYYAIADNNVALVCTLLNAGALKNLLE
60

Lister
MDLSRINTWKSKQLKSELSSKDAFKADINGHSALYYAIADNNVALVCTLLNAGALKNLLE
60

**********************:****::**********************:********

Cop
NEFPLHQAATLEDTKIVKILLFSGMDDSOFDDKGNTALYYAVDSGNMOTVKLFVKKNWRL
120

WR
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWAL
120

Tian
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLEVKKNWRL
120

Wyeth
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLEVKKNWRL
120

Lister
NEFPLHQAATLEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGNMQTVKLFVKKNWAL
120

************************:***********************************

Cop
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHTTIKNGHVDMMILLL
180

WR
MFYGKTGWETSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIRITIKNGHVDMMILLL
180

Tian
MFYGKTGWKTSFYHAVM11DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVD8MILLL
180

Wyeth
MFYGKTGWKTSFYHAVMLNDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

Lister
MFYGKTGWKTSFYHAVMINDVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMILLL
180

********************************************* **************

Cop
DYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSVNLENV1LDDAEITKMII
240

WR
DYNTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFKYDINIYSANLENVILDDAEIAKMII
240

Tian
DYMTVDKHQ---------------------------------------------------
189

Wyeth
DYMTSTNTNNSLLFIADIKLAIDNKDIEMLQALFKYDINIYSANLENVLLDDAEIAKMII
240

Lister
DYNTSTNTNNSLLFIPDIKLAIDNKDIEMIQALFKYDINIYSANLENVILDDAEIAKMII
240

**** : :

Cop
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKKN
284

WR
EKHVEYKSDSYTKDLDIVKNNKLDEITSKNKELRLMYVNCVKKN
284

Tian
--------------------------------------------

Wyeth
EKHVEYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNCVKEN
284

Lister
EKHVEYKSDSYTKDLDIVKNNKLDETISKNKELKLMYVNCVRKN
284

K2L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 557-561, respectively, in

order of appearance)

Cop
MIAILILSLTCSVSTYRLOGFTNAGIVAYKNIQDDNIVFSPFGYSFSMFMSLLPASGNTR
60

WR
MIALLILSLTCSVSTYRIQGFTNAGIVAYKNIODDNIVESPEGYSFSMEMSLLPASGNTR
60

Tian
MIALLILSLACSASAYRLOGFTNAGIVAYKNIODDNIVFSPFGYSFSMFMSLLPASGNTR
60

Wyeth
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNIVESPFGYSFSMEMSLLPASGNTR
60

Lister
------------------------------------------------------------

Cop
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPLYYQQYHR
120

WR
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Tian
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Wyeth
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
120

Lister
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCIKPSYYQQYHR
60

**************************************************** *******

Cop
FGLYRINFRADAVNKINSIVERRSGMBNVVDSNMIDNNTLWAIINTIYFKGTWQYPFDIT
180

WR
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMIDNNTLWAIINTIYFKGIWQYPFDIT
180

Tian
FGLYRLNFRADAVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTIYFKGTWQYFFDIT
180

Wyeth
-----LNFRADAVNKINSIVERRSGMSNVVDSNMIDNNTLWAIINTIYFKGIWQYFFDIT
175

Lister
FGLYRLNFRRDAVNKINSIVERRSGMSNVVDSNMIDNNTLWAIINTIYFKGIWQYPFDIT
120

********************************************** ********

Cop
KTRNASFTNKYGTKTVPMMINVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNET
240

WR
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNMT
240

Tian
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDNNT
240

Wyeth
KTRNASFTNXYGTKTVPMMINVTKLQGNTITIDDKEYDMVRLPYKDANISMYLAIGDNNT
235

Lister
KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEEYDMVALPYKDANISMYLAIGDNMT
180

**********************************:*************************

Cop
HFTDSITAAKLDYWSFQLGNEVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
300

WR
HFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMENPDNASFKHM
300

Tian
HFTDSITAA-KDYWSFQLONKVYNLKLPKFSIENXRDIKSIAEMMAPSMFNPDNASFKHM
299

Wyeth
HFTDSITAAKLDYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
295

Lister
HFTDSITAAKLDYWSSQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFNPDNASFKHM
240

********* **** ********************************************

Cop
TRDPLYIYKMEQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
360

WR
TRDPLYIYKMFQNAKIDVDEQGTVARASTIMVATARSSPEKLEFNTPFVFIIRHDITGFI
360

Tian
TRDPLYIYEMPQNAKIDVDEQGTVAEASTIMVATARSSPEELEFNTPFVFIIRHDITGFI
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 0(1.2.4) multiple sequence alignment (SEQ ID NOS 562-563, respectively, in

order of appearance)

Cop
MGHIITYCQVHTNISILIRKAHHIIFFVIDCDCISLQFSNYVHHGNRFRTVLISKTSIAC
60

Tian
MGHIITYCQVHTNISILIRKAYHIIFFVIDCDCISLQFSNYVHHGNRFRTVLISKTSIAC
60

************************************************************

Cop
FSDIKRILPCTFKIYSINDCP
81

Tian
FSDIKRILPCTFKIYSINDCP
81

*********************

K3L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 564-568, respectively, in

order of appearance)

Cop
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHSEAILAESVFMEMDRYVEYRDKLVG
60

WR
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHFEAILAESVKMEMDRYVEYRDKLVG
60

Tian
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPHSEAILAESVXMEMDRYVEYRDKLVG
60

Wyeth
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPHFEAILAESVKMHMDRYVEYRDXLVG
60

Lister
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPHSEAILAESVKMEMDRYVEYRDKLVG
60

*********************:************* ************************

Cop
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

WR
KTVXVKVIRVDYTKGYIDVNYKRMCRHQ
88

Tian
KTVKVKVIRVDYTKGYIDVNYKRMCRHQ
88

Wyeth
KTVEVKVIRVDYTKGYIDVNYKRMCRHQ
88

Lister
KTVKVXVIRVDYTKGYIDVNYKRMCRHQ
88

****************************

K4L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 569-572, respectively, in

order of appearance)

Cop
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

WR
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNITKTLDISSFYWSLSDEVGTNFG
60

Wyeth
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTIKTLDISSFYWSLSDEVGTNFG
60

Lister
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSNTTKTLDISSFYWSLSDEVGTNFG
60

************************************************************

Cop
TIILNEIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

WR
TIILNKIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

Wyeth
TIILNEIVQLPKRGVAVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

Lister
TIILNEIVQLPKRGVRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNILGGVLHTKFWI
120

*****:******************************************************

Cop
SDNTHIYLGSANMDWRSLTQKELGIAIFNNANLAADLTQIFEVYWYLGVNNLPYNWKNF
180

WR
SDNTHIYLGSANMDWRSLTQVKELGIAIFNNRLAADLTQIFEVYWYLGVNNLPYNWKNF
180

Wyeth
SDNTHIYLGSANMDWRSLTQKELGIAIFNNRNLAADLIQIFEVYWYLGVNNLPYNWKNF
180

Lister
SDNTHIYLGSANMDWRSLTQWELGIAIFNNRNLAADLIQIFEVYWYLGVNNLPYNWKNF
180

***********************************************************

Cop
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

WR
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCIMERTNDLTALLSCIRNASKFVYVSVMN
240

Wyeth
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

Lister
YPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMN
240

Cop
FIPIIYSKAGKILFWPYIEDELARSAIDRQVSVKLLISCWQRSSFIMANFLRSIAMIKSK
300

WR
FIPIIYSKAGNILFWPYIEDELRRAAIDRQVSVKLLISCWQRSSFIMANFLRSIAMIKSK
300

Wyeth
FIPIIYSKAGKILFWPYIEDEL8RSAIDRQVSVKLLISCWQRSSFIMENFLRSIAMIKSK
300

Lister
FIPIIYSKAGKILFWPYIEDELARSAIDRQVSVKLLISCWQRSSFIERNFLRSIAMIKSK
300

************************************************************

Cop
NIDIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

WR
NINIEVKLFIVPDADPPIPYSRVNEAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

Wyeth
NINIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

Lister
NINIEVKLFIVPDADPPIPYSRVNHAKYMVTDKTAYIGTSNWTGNYFTDTCGASINITPD
360

**:*********************************************************

Cop
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMKQCTNDIYCDEIQPEKEIPE
420

WR
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCALLKNMKQCTNDIYCDEIQPEKEIPE
420

Wyeth
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMKQCINDIYCDEIQPEKEIPE
420

Lister
DGLGLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLKNMEQCTNDIYCDEIQPEKEIPE
420

**********************************:*************************
420

Cop
YSLE
424

WR
YSLE
424

Wyeth
YSLE
424

Lister
YSLE
424

****

K5L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 573-577, respectively, in

order of appearance)

Cop
-------------MGATISILASYDNPNLFTAMILMSPLVNADAVSRLNLLAAKLMGTIT
47

WR
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKINGTIT
60

Tian
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKLMGTIT
60

Wyeth
-------------MGATISILASYDNPNLFTAMILMSPLVNADAVSKLNLLAAKLMGTIT
47

Lister
---------MGHSMGATISILASYDNPNLFTAMILMSPLVNADAVSRINLLAAELMGTIT
51

****************************************

Cop
PNAPVGKLCPESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLHATNKVRKIISKINTPR
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
TLILQGINNEISDVLGAYYFMHANCNREIKIYEGAKHHLHKETDEVEKSVMKEIETWIF
167

Lister
TLILQGTNNXISDVLGAYYFMHANCNREIKIYEGAEHELHKETDEVEKSVMKEIETWIF
171

Cop
----

WR
----

Tian
----

Wyeth
NRVK
171

Lister NRVK
175

K6L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 578-581, respectively,

appearance)

Cop
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKHSGRYDETAENISSLGILVISHDHIG
60

WR
MSANCMFNIDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

Wyeth
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

Lister
MSANCMFNLDNDYIIWKPITYPKALVFISHGAGKHSGRYDELAENISSLGILVFSHDHIG
60

************************************************************

Cop
HGRSNGEKMMIDDFGTARGNY
81

WR
HGRSNGEKMMIDDFGTARGNY
81

Wyeth
HGRSNGEKMMIDDFGTARGNY
81

Lister
HGRSNGEKMMIDDFGTARGNY
81

*********************

K7R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 582-586, respectively, in

order of appearance)

Cop
MATKIDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNEDITSCGRLYKELMKF
60

WR
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

Tian
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNKDITSCGRLYKELMKF
60

Wyeth
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVFNEDITSCGRLYKELMKF
60

Lister
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITWRNHVIVINKDITSCGRLYKELMKF
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
RFQSLGNITDLMTDDNINTLILFLEKKLN
149

Lister
RFQSLGNITDLMTDDNINTLILFLEKKLN
149

*****************************

F1L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 587-591, respectively, in

order of appearance)

Cop
MISMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYREDKSTNILDYLS
60

WR
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

Tian
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDRDYVYPIPENMVYRFDKSTNILDYLS
60

Wyeth
MISMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

Lister
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDHDYVYPLPENMVYRFDKSTNILDYLS
60

**********************************:*************************

Cop
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

WR
TERDHVMMAVRYYMSKQRLDDLYRQLPTKIRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Tian
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Wyeth
TERDRVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

Lister
TERDHVMMAVRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVSNDYNRDMNIMYDMAS
120

************************************************************

Cop
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRACMNPVKTIKMFTLLSHTICDD
180

WR
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRACMNPVETIKMFTLLSHTICDD
180

Tian
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRRUMNPVKTIKMFTLISHTICDD
180

Wyeth
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITKLGRACMNPVKTIKMFTLLSHTICDD
180

Lister
TKSFTVYDINNEVNTILMDNKGLGVRLATISFITELGRACMNPVKTIKMFTLLSHTICDD
180

**********************************:*********:***************

Cop
CFVDYITDISPPDNTIPNTSTREYLKLIGITALMFATYKTLKYMIG
226

WR
YFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

Tian
CFVDYITDISPPDNTIPNTSTREYLKLIGITALMFATYKILKYMIG
226

Wyeth
CFVDYITDISPPDNTIPNTSTREYLKLIGITAIMFATYKTLKYMIG
226

Lister
CFVDYITDISPPDNTIPNTSTREYLKIIGITAIMFATYKTLKYMIG
226

**********************************************

F2L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 592-596, respectively, in

order of appearance)

Cop
MFNMNINSPVREVEETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMIT
60

WR
MFNMNINSPVREVKETNRAKSPTRQSPYAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

Tian
MENMNINSPVRFVFETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

Wyeth
MENMNINSPVREVEETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMIT
60

Lister
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLYSAYDYTIPPGERQLIKTDISMSMPK
60

************************************************************

Cop
ICYGRIAPRSGISLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQIIYQRI
120

WR
FCYGRIAPRSGLSLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQLIYQRI
120

Tian
ICYGRIAPRSGISLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQIIYQRI
120

Wyeth
ICYGRIAPRSGISLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGDRIAQIIYQRI
120

Lister
FCYGRIAPRSGISLKGIDIGGGVIDEDYRGNIGVILINNGKCTENVNTGDRIAQIIYQRI
120

:***********************************************************

Cop
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

WR
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

Tian
YYPELEEVQSLDSTDRGDQGFGSTGLR
147

Wyeth
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

Lister
YYPELEEVQSLDSTNRGDQGFGSTGLR
147

**************:************

F3L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 597-601, respectively, in

order of appearance)

Cop
MPIEVNTVYCKNILALSMTKKEKTIIDAIGGNIIVNSTILKKISPYFRTHIRQKYTKNKD
60

WR
MPIETNTVYCKNILALSMTKKEKTIIDAIGGNIIVNSTILKKISPYFRTHLRQKYTKNKD
60

Tian
MPIEVNTVYCKNILALSMTKKEKTIIDAIGGNIIVNSTILKKISPYFRTHLRQKYTKNKD
60

Wyeth
MPIEVNTVYCKNILAISMTKKEKTIIDAIGGNIIVNSTILKELSPYFRTHLRQKYTKNKD
60

Lister
MPIFVNTVYCKNILAISMTKKERTIIDAIGGNSIVNSTILKKISPYFRTHLRQKYTKNKD
60

************************************************************

Cop
PVTRVCIDLDIHSLTSIVIYSYTGKVYIDSHNVVNLIRASILTSVEFIIYTCINFILRDF
120

WR
PVTWVCIDLDIHSLTSIVIYSYTGKVYIDSHNVVNLIRASILTSVEFIIYTCINFILRDF
120

Tian
PVTRVCIDLDIHSLTSIVIYSYTGKVYIDSHNVVNLIRASILTSVEFIIYTCINFILRDF
120

Wyeth
PVTRVCIDLDIHSLTSIVIYSYTGKVYIDSHNVVNLIRASILTSVEFIIYTCINFILRDF
120

Lister
PVTRVCIDIDIHSLTSIVIYSYTGKVYIDSHNVVNLIRASILTSVEFIIYTCINFILRDF
120

*** ********************************************************

Cop
RKEYCVECYMMGIEYGLSNLICHTKNFIAKHFLELEDDIIDNFDYLSMKLILESDELNVP
180

WR
RKEYCVECYMMGIEYGLSNLICHTKNFIAKHFLELEDDIIDNEDYLSMKLILESDELNVP
180

Tian
RKEYCVECYMMGIEYGLSNLICHTKNFIARHFLELEDDIIDNEDYLSMEIILESDELNVP
180

Wyeth
RKEYCVECYMMGIEYGLSNLICHTKNFIAEHFLELEDDIIDNEDYLSMKLILESDELNVP
180

Lister
RKEYCVECYMMGIEYGLSNLICHTKNFIAEHFLELEDDIIDNEDYLSIKLILESDELNVP
180

***********************************************:************

Cop
DEDYVVDEVIEWYIKRRNKLGNILLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

WR
DEDYVVDEVIEWYIKRANKIGNILLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

Tian
DEDYVVDEVIEWYIKRANKIGNILLLIKNVIRSNYLSPRGINNVKWILDCTKIETCDKQP
240

Wyeth
DEDYVVOFVIEWYIKRANKIGNILLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

Lister
DEDYVVDFVINNYIKRRNKLGNLLLLIKNVIRSNYLSPRGINNVKWILDCTKIFHCDKQP
240

************************************************************

Cop
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVILIGGWMNNEIHNNAIAVNYISNNWI
300

WR
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVILIGGWNNNEIHNNAIAVNYISNNWI
300

Tian
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWNVNEIHNNAIAVNYISNNWI
300

Wyeth
RKSYKYPFIEYPMFMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

Lister
RKSYKYPFIEYPMNMDQIIDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNYISNNWI
300

************************************************************

Cop
PIPPMNSPRLYATGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

WR
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Tian
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Wyeth
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

Lister
PIPPMNSPRLYASGIPANNKLYVVGGLPNPTSVERWFHGDAAWVNMPSLLKPRCNPAVAS
360

************:***********************************************

Cop
INNVIYVMGGHSETDTTTEYLLPNEDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

WR
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCAIVFGRRLFLVGRNAEFY
420

Tian
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRALFLVGRNAEFY
420

Wyeth
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

Lister
INNVIYVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKSCALVFGRRLFLVGRNAEFY
420

************************************************************

Cop
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRGSYIDTIEVYNHHTYSWNIWDGK
480

WR
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNINDGK
480

Tian
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNINDGK
480

Wyeth
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHHTYSWNIWDGK
480

Lister
CESSNTWTLIDDPIYPRDNPELIIVDNKLLLIGGFYRESYIDTIEVYNHETYSWNIWDGK
480

************************************* **********************

B14R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 602-605, respectively, in

order of appearance)

Cop
------------------------------------------------------------

WR
MDIFREIASSMEGENVFISPASISSVLTILYYGANGSTAEQLSKYVEKEENMDKVSAQNI
60

Tian
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
------------------------------------------------------------

WR
SFKSINKVYGRYSAVFKDSFLRKIGDKFQTVDFTDCRTIDAINKCVDIFTEGKINPLLDE
120

Tian
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
---MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGELFNHASVK
57

WR
PLSPDTCLLAISAVYFKAENLTPFEKEFTSDYPFYVSPTEMVDVSMNSMYGKAFNHASVK
180

Tian
---MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVY
57

Wyeth
---MNHCLLAISAVYFKAEWLTPFEKEFTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVK
57

: *********************************************: *******

Cop
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTOTNFKRWCNSLDAMFIDVHIPK
117

WR
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCNSLEATFIDVHIPK
240

Tian
ESFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFMDAMFIDVHIPK
117

Wyeth
ESIGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFMDAMFIDVHIPK
117

Cop
FKVTGSYNLVDTLVFSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAAT
177

WR
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNSDVSVDAMIEKTYIDVNEEYTEAAAAT
300

Tian
FINTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTFAAAAT
177

Wyeth
FKVTGSYNLVDTLVKSGLTEVFGSTGDYSNMCNLDVSVDAMIHKTYIDVNEEYTFAAAAT
177

************************************************************

Cop
CALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRYCSPTTNC
222

WR
CALVSDCASTITNEFCVDHPFIYVIREVDGKILFVGRYCSPTTNC
345

Tian
CALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRYCSPTTNC
222

Wyeth
CALVSDCASTVTNEFCADHPFIYVIRHVDGKILFVGRYCSPTTNC
222

**********:*****.****************************

B15R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 606-610, respectively, in

order of appearance)

Cop
MTANFSTHVESPQHCGCDRITSIDDVXQCLTEYITASSIAYRNRQCAGQLYSTLLSERDD
60

WR
MTANFSTHVESPQHCGCDRITSIDDVRQCLTEYIYWSSYAYANRQCAGQLYSTLLSFRDD
60

Tian
MTANFSTHVESPQHCGCDRLTSIDDVXQCLTEYIYWSSYAYANRQCAGQLYSTLLSERDD
60

Wyeth
MTANFSTHVESPQHCGCDRITSIDDVKQCLTEYITASSIAYRNRQCAGQLYSTLLSFRDD
60

Lister
MTANFSTHVESPQHCGCDRITSIDDVKQCLTEYITASSYAYRNRQCAGQLYSTLLSFRDD
60

Cop
AELVFIDIRELVKNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

WR
AELVFIDIRELVKNMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Tian
AELVFIDIRELVKNMEWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Wyeth
AELVFIDIRELVKNMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

Lister
AELVFIDIRELVKNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCAYAATYWGGEDH
120

*************:*********:************************************

Cop
PTSNSLNALFVMLEMLNYVDYNIIFRAMN
149

WR
PTSNSLNALFVMLEMLNYVDYNIIFRAMN
149

Tian
PTSNSINALFVMMEMLNYVDYNIIERRMN
149

Wyeth
PTSNSLNALFVKLEMLNYVDYNIIERRMN
149

Lister
PTSNSLNALEVKLEMLNYVDYNIIERRMN
149

*****************************

B ORF E

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 611-612, respectively, in

order of appearance)

Cop
MINSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMVETSPVSSSICTKSDDGRNISDGEL
60

Tian
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMVETSPVSSSICTKSDDGRNISDGEM
60

************************************************************

Cop
LIRYITTDDFCTIFDIIPRHIFYQLANVDEH
91

Tian
LIRYITTDDFCTIFDIIPRHIFYQLANVDEH
91

*******************************

B16R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 613-617, respectively, in

order of appearance)

Cop
MSILPVIFLPIFFISSFVQTFNASECIDKGXYFASFMELENEPVILPCPQINTISSGYNI
60

WR
MSILPVIELSIFFYSSFVQTFNAPECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

Tian
------------------------------------MELENEPVILPCPQINTLSSGYNI
24

Wyeth
MSILPVIELSIFFYSSFVQTFNASECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

Lister
MSILPVIFLPIFFYSSFVQTFNAPECIDKGQYFASFMELENEPVILPCPQINTLSSGYNI
60

***********************

Cop
LDILWEKRGADNDRIIPIDNGSNMIIINPTQSDSGIYICITTNETYCDMMELNLTIVSVS
120

WR
LDILWEKRGADNDRIIPIDNGSNMIILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

Tian
LDILWEKAGADNDRIIPIDNGSNMMILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVM
84

Wyeth
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

Lister
LDILWEKRGADNDRIIPIDNGSNMLILNPTQSDSGIYICITTNETYCDMMSLNLTIVSVS
120

************************************************************

Cop
ESNIDFISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGERRLANKRIKQRTPGIIT
180

WR
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLANKRLKQRTPGIIT
180

Tian
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHARLANKRLKQRTPGIIT
144

Wyeth
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHRRLANKRLKQRTPGIIT
180

Lister
ESNIDLISYPQIVNERSTGEMVCPNINAFIASNVNADIIWSGHARLANKRIKQRTPGIIT
180

*****:******************************************************

Cop
IEDVRKNDAGYITCVLEYIYGGKTYNVTRIVKLEVRDKIIHPTMQLPEGVVTSIGSNITI
240

WR
IEDVEKNDAGYITCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVISIGSNLTI
240

Tian
IEDVRKNDAGYITCVLEYIYEGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVTSIGSNLTI
204

Wyeth
IEDVRKNDAGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVISIGSNITI
240

Lister
IEDVRKNDAGYITCVLEYIYRGKTYNVTRIVKLEVRDKIIPSTMQLPDGIVISIGSNITI
240

******************** ******************* *****:*:**********

Cop
ACRVSLAPPTIDADVFWISNGMYYEEDDGDGDGRISVANKIYMTDKRRVITSRININPVX
300

WR
ACRVSLAPPTTDADVFWISNGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNINPVK
300

Tian
ACAVSLAPPTTDADVFWISNGMYYEEDDGDGNGAISVANKIYMTDKAAVITSALNINPVE
264

Wyeth
ACAVSLAPPTTDTDVFWISNGMYYEEDDGDGDGAISVANKIYMIDKAAVITSALNINPVK
300

Lister
ACAVSLAPPTTDADVFWISNGMYYEEDDGDGNGRISVANKIYMTDKAAVITSAININPVK
300

************:******************:****************************

Cop
EEDATTFTCMAFTIPSISKTVTVSIT
326

WA
EEDATTFTCMAFTIPSISKTVTVSIT
326

Tian
EEDATTFTCMAFTIPSISKTVTVSI-
289

Wyeth
EEDATTFTCMAFTIPSISKTVTVSIT
326

Lister
EEDATTFTCMAFTIPSISKTVTVSIT
326

**************************

B ORF F

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 618-619, respectively, in

order of appearance)

Cop
MVIIPGVRCISLLFLARACPLHIISAFTLLAINALILGHTISPVDISFTICGYEIKSIFD
60

Tian
MVIIPGVACLSLLFLAARCPLHIISAFTLLAINALILGHTISPVDLSFTICGYEIRSIFD
60

*******************************************************:****

Cop
SETDTIVAFNDIMSQ
75

Tian
SKTDTIVKFNDIMSQ
75

*:*************

B17L

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 620-624, respectively, in

order of appearance)

Cop
MSAKFMQVYEYDREQYLDEFIEDAYNDSFITSPEYYSAEKYMCAYTTINHNCVNVRACAL
60

WR
MSAKFMQYEYDREQYYLDEFIEDAYNDSFITSPEYYSAEKYMCAYTTINHNCINVARCAL
60

Tian
MSRKFMQVYEYDREQYLDEFIEDAYNDSFITSPEYYSAEKYMCAYTTLNHNCVNVRACAL
60

Wyeth
MSAKFMQVYEYDREQYLDEFIEDAYNDSFITSPEYYSAEKYMCAYTTLNUNCVNVARCAL
60

Lister
MSAKFMONYEYDREQYLDEFIEDAYNDSFITSPEYYSAEKYMCAYTTINHNCINVRACAL
60

****************************************************:*******

Cop
DSKLIHDIITNCKIYNNIELVAATKFVYYLDLIKCNWVSKVGDSVIYAVIFITHTSTRNL
120

WR
DSKILHDIITNCKIYNNIELVAATKFVYYLDLIKCNWVSKVGDSVLYAVIFITHTSTANL
120

Tian
DSKILHDIITNCKIYNNIELVAATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTANL
120

Wyeth
DSKLLHDIITNCKIYNNIELVAATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTANL
120

Lister
DSKLLHDIITNCKIYNNIELVAATKFVYYLDLIKCNWVSKVGDSVLYPVIFITHTSTANL
120

************************************************************

Cop
DKVSVKTYKGVKVKKINACADHAIVINPFVAFKITLANKTSHAINIVTFCKLATDITAVE
180

WR
DKVSVKTYKGVAVKKLNACADHAIVINPFVKFKITLANKTSHAINIVTFCKLKTDITAVE
180

Tian
DKVSVKTYKGVKVAELNACADHAIVINPFVKFKITLANKTSHAEVIVTFCKLRTDITQIE
180

Wyeth
DKVSVKTYKGVKVKKINACADHAIVINPFVAFKLTIPNKTSHAKVIVTFCKLRTDITQIE
180

Lister
DKVSVATYKGVAVKKINRCADHAIVINPFVKFKITLPNRTSHARVINTFCKLRTDITQIE
180

****************************************************:**** :*

Cop
APLAGNVIVYTFADINKAIAGYIHVNIEGCIDGMIYINSSKFACVIKLHASMYRIPPFPI
240

WR
APLAGNVIVYTFADINKAIAGYIHINIEGCIDGMIYINSSKFACVLKIHRSMYRIPPFPI
240

Tian
APLSGNVIVYTFPNINKAIAGYIHVNIEGCIDGMIYINSSKFACVIKLHASMYRIPPFPI
240

Wyeth
AFISGNVIVYTFADINKAIAGYIHVNIEGCIDGMIYINSSKFACVLKLHASMFAIPPFPI
240

Lister
APLSGNVINYTIPDINKRIPGYTHVNIEGCIDGMIYINSSKFACVLKLHASMYRIPPFPI
240

*** *********:**********:***********************************

Cop
DICSCCSQYTNDDIEIPIHDLIKDVAIFANAETVYYLKINNKTIAAFTYFENIDTAITQE
300

WR
DICSCCSQYINYDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIAAFTYFNNIDTAITQE
300

Tian
DICSCCSQYTNGDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNIDTAITQE
300

Wyeth
DICSCCSQYTNDDIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIAAFTYFNNIDTAITQE
300

Lister
DICSCCSOYTNDDIEIPIHDLIKDVAIFKNKETVYYLKINNKTIAAFTYFNNIDTAITQE
300

********* * ************************************************

Cop
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

WR
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

Tian
HEYVKIALGIVCKLMINNMHSIVGVNRSNTFVNCLLEDNV
340

Wyeth
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

Lister
HEYVKIALGIVCKLMINNMHSIVGVNHSNTFVNCLLEDNV
340

****************************************

B18R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 625-628, respectively, in

order of appearance)

Cop
MSRRLIYVLNINRKSTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQHVTGYTA
60

WR
MSRRLIYVLNINRESTHKIGENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQPVTGYTA
60

Tian
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRHPVTGYTA
60

Wyeth
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTSTELDFVVKNYDLNRRQPVTGYTA
60

*************:**************************************: ******

Cop
LHCYLYNNYFTNDVIKILLNHDVNVTMETSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

WR
LHCYLYNNYFTNDVLKILLNHGVDVIMKTSSGRMPVYILLTRCCNISHDVVIDMIDKDEN
120

Tian
LHCYLYNNYFTNDVLKILLNHGVDVTMETSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

Wyeth
LHCYLYNNYFTNDVLKILLNHGVDVTMETSSGRMPVYILLTRCCNISHDVVIDMIDKDKN
120

*********************.*:************************************

Cop
HISHRDYSNLLLEYIKSRYMILKEEDIDENIVSTLIDKGIDPNETQDGYTALHYYYLCLA
180

WR
HLIHRDYSNILLEYIKSRYMILKEEDIDENIVSTILDKGIDPNFKQDGYTALHYYYLCLA
180

Tian
HLLHRDYSMILLEYIKSRYMILKEEDIDENIVSTILDKGIDPNETQDGYTALHYYYLCLA
180

Wyeth
HLSHRDYSNILLEYIKSRYMLIKEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCLA
180

** *********************************************************

Cop
HVYKPGECRKPITIKKAKRIISIFIQHGANINALDNCGNTPFHLYLSIEMCNNIHMTKMI
240

WR
HVYKPGECRKPITIKEAKRIISIFIQHGANINALDNCONTPFHLYLSIEMCNNIHMTKMI
240

Tian
HVYKPGECRKPITIKKAKRIISIFIQHGANLNALDNCONTPFHLYLSIEMCNNIHMTKMI
240

Wyeth
HVYKPGECRKPITIKKAKRIISLFIQHGANINALDNCGNTPFHLYLSIEMCNNIHMTKMI
240

************************************************************

Cop
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRMIVFEFIK
300

WR
LTFNPNFEICNNHOLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRIIVFEFIK
300

Tian
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPIDERRIIVFEFIK
300

Wyeth
LTFNPNFKICNNHGLTPILCYITSDYIQHDILVMLIHRYETNVGEMPIDERRIIVFEFIK
300

*******:*********************************************:******

Cop
TYSTRPADSITYLMNRFKNINIYTRYEGKTLIHVACEYNNTQVIDYLIRINGDINALTDN
360

WR
TYSTRPADSITYLMNRFKNIDIYTRYEGKTILHVACEYNNTHVIDYLIRINGDINALTDN
360

Tian
TYSTRPADSITYLMNRFKNINIYTRYEGKTILHVACEYNNTQVIDYLIRINGDINALTDN
360

Wyeth
TYSTRPADSITYLMNRFKNINIYTRYEGKTILHVACEYNNTHVIDYLIRINGDINALTDN
360

******************:**********************:******************

Cop
NKHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQLPSLPIFDIKSFEKFISYC
420

WR
NKHATQLIIDNKENSPYTINCLLYILRYIVDENVIRSLVDQLPSLPIFDIKSFEKFISYC
420

Tian
NKHATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDOLPSLPIFDIKSFEKFISYC
420

Wyeth
NKHAIQLIIDNKENSPYTIDCLLYILRYIVDKNVIRSLVDQLPSLPIFDIKSFEKFISYC
420

**** **************:****************************************

Cop
ILLDDTFYDRHVENRDSKTYRYAFSKYMSFDKYDGIITKCHDETMILKLSTVLDTTLYAV
480

WR
ILIDDTFYNRHVENRDSKTYRYAFSKYMSFDKYDGIITKCHKETILLKISTVLDTTLYAV
480

Tian
ILLDDTFYDRHVENRNSKTYRYAFSKYMSFDKYDGIITKCHDETMLLKISTVIDTTLYAV
480

Wyeth
ILIDDTFYNRHVENRNSKTYRYAFSKYMSFDKYDGIITKCHDETMILKLSTVLDTTLYAV
480

********:***:**:*************************.**:***************

Cop
LRCHNSRKLARYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

WR
LACHNSKKIRRYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVETDK
540

Tian
LACHNSRKLARYLTELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

Wyeth
LRCHNSKKLRRYLNELKKYNNDKSFKIYSNIMNERYLNVYYKDMYVSKVYDKLFPVFTDK
540

******:******.**********************************************

Cop
NCLITLLPSEIIYEILYMITINDLYNISYPPTIN
574

WR
NCLITILPSEIIYEILYMITINDLYNISYPPTKV
574

Tian
NCLLTLLPSEIIYEILYMITINDLYNISYPPTKV
574

Wyeth
NCLITLIPSEIIYEILYMLTINDLYNISYPPTKV
574

----------------------------------

B19R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 629-632, respectively, in

order of appearance)

Cop
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGT
60

WR
MTMKMMVHIYFVL--LLLLLFHSYAIDIENEITEFFNKMRDTIPAKDSKWLNPACMFGGT
58

Tian
MTMKNMVYIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWLNPACMFGGT
60

Wyeth
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFNKMRDTLPAKDSKWINPACMFGGT
60

************** *********************************************

Cop
MNDMATLGERFSAKCPPIEDSLLSHRYKDYVVKWERLEKNARRQVSNKRVNEGDLWIANY
120

WR
MNDIAALGEPFSAKCPPIEDSLLSHRYKDYVVXWERLEKNARRQVSNKRVKHGDLWIANY
118

Tian
MNDIAALGEPFSANCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSNERVEHGDLWIANY
120

Wyeth
MNDIATLGEPFSAKCPPIEDSLLSERYKDYVVKWERLEKNRRRQVSNKRVENGDLWIANY
120

***:*:******************************************************

Cop
TSKESNRRYLCTVTTKNGDCVOGIVRSHIKEPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

WR
TSKESNRRYLCTVTTENGDCVQGIVRSHIREPPSCIPKTYELGTHDKYGIDLYCGILYAK
178

Tian
TSEFSNRRYLCTVTTRNGDCVQGIVRSHIKEPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

Wyeth
TSKESNRRYLCTVTTKNGOCVQGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILYAK
180

*****************************:******************************

Cop
HYNNITWYKDRKEINIDDIKYSOTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
240

WR
HYNNITWYKDNEEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVHYDDVRIKNDIVVSR
238

Tian
HYNNITWYKDRKEINIDDIKYSQTGKELIIHNPELEDSGRYDCYVEYDDVRIENDIVVSR
240

Wyeth
HYNNITWYKDRKEINIDDIKYSQTGKKLIIHNPELEDSGRYDCYVHYDDVRIENDIVVSR
240

**************************:*********************************

Cop
CKILTVIPSQDHREKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
300

WR
CKILTVIPSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
298

Tian
CKILTVIRSQDHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIEWENPSGWLIGF
300

Wyeth
CKILVTVIPSQDHRFKLKRNCGYASN----------------------------------
265

***************** : .

Cop
DEDVYSVLTSRGGITEATLYFENVTEEYIGNTYNCRGHNYYFEKTLTTTVVLE
353

WR
DEDVYSVITSRGGITEATLYFENVTEEYIGNTYKCRGENYYFEKTLTTTVVLE
351

Tian
DEDVYSVITSRGGITEATLYFENVTEEYIGNTYKCRGENYYREKTLTTTVVIE
353

Wyeth
-----------------------------------------------------

B21R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 633-634, respectively, in

order of appearance)

Cop
MSLESFIITTFNNNSSTNIDNMCHLYVKVCPSSILFRLETECCDINKLVEGTTPLHCYLM
60

Wyeth
MSLESFIITTFNNNSSTNIDNMCHLYVKVCPSSLLFRIFVECCDINELVEGTTPLHCYLM
60

************************************************************

Cop
NEGFESSVLKNLIKEYVMNTFNVHDIHYTNI
91

Wyeth
NEGFESSVLMNLLKEYVMTSITQIFNS----
87

*******************.::.

B22R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 635-637, respectively, in

order of appearance)

Cop
MISLSFLIHNPLEKWKLKPSISINGYRSTFTMAFPCAQFRPCHCHATKDSLNTVADVREC
60

Wyeth
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCAQFRPCHCHATKDSLNTVADVRHC
60

Lister
----------------------------- MASPCAKFRPCHCHATKDSLYTVADVRIIC
29

** ***:***********************

Cop
LTEYILWVSHRWTHRESAGSLYRLLISFRTDATELFGGELKDSLPWDNIDNCVEIIKCEI
120

Wyeth
LTEYILWVSHRWTHRETAGPLYRLLISFRTDATELEGGELKDSLPWDNIDNCVEIIKCEI
120

Lister
LTEYILWVSHRWTHRESAGSLYRLLISFRTDATELFGGELKDSLPWD---NCVEIIKCFI
86

****************:** *************************** **********

Cop
RNDSMKTAEELRAIIGLCTQSAIVSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTAK
180

Wyeth
RNDSZECAEELRAIIGLCTQSAIVSGRVFNDKYIDILLMCRKILNENDYLTLLDHIRTAK
180

Lister
RNDSMKTAEELRAIIGLCTOSAIVSGRVFNDKYIDILLMLRKILNENDYLTLLDHIRTAK
146

************************************************************

Cop
Y
181

Wyeth
Y
181

Lister Y
147

*

B23R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 638-639, respectively, in

order of appearance)

Cop
MIAFIIFREIGIISTRIAMDYCGRECTILCRLLDEDVTYKKIKLEIETCHNLSKHIDRRG
60

Wyeth
MIAFIIFREIGIISTRIAMDCT----CILCRLLDEDVTYKKIKLEIETCHNLSKHIDRRG
56

******************** *********************************

Cop
NNALHCYVSNKCDTDIKIVALLLSRGVERLCRNNEGLTPLGAYSKHRYVKSQIVHILISS
120

Wyeth
NNALHCYVFNKCDTDIKIVRLLLSRGVERLCANNEGLTPLGVYSKHRYVKSQIVHLLISS
116

******** ********************************.******************

Cop
YSNSSNELKSNINDFDLSSDNIDLALLKYLIVDKRIRPSKNTNYAINGLGLVDIYVTTPN
180

Wyeth
YSNSSNELKSNINDFDLSSDNIDLALLKYLIVDKRIRPSKRTNYAINSLGLVDIYVTTPN
176

***********************************************.************

Cop
PRPEVLLWLLKSECYSTGYVESTCMYNSDMCKNSLHYYISSHRESQSLSKDVIKCLINNN
240

Wyeth
PRPEVLLWLLKSECYSTGYVFRTCMYNSDMCKNSLHYYISSHRESQSLSKDVIKCLINNW
236

************************************************************

Cop
VSIHGRDEGGSLPIQYYWSFSTIDIEIVKILLIKDVDTCRVYDVSPILEAYYLNKRFRVT
300

Wyeth
VSIHGRDEGGSLPIQYYWSFSTIDIEIVKLLLIKDVDTCRVYDVSPILEAYYLNKRFRVT
296

************************************************************

Cop
PYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYNHYTIDNILKRFRQQDESIVQAMLIN
360

Wyeth
PYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYNHYIIDNILKRFRQQDESIVQAMLIN
356

************************************************************

Cop
YLHYGDMVVRCMLDNGQQLSSARLLC
386

Wyeth
YLHYGDMVVRCMLDNGQQLSSARLLC
382

**************************

B24R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 640-641, espectively, in

order of appearance)

Cop
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDDIDMIDNENKTLLYYAVDVNNIQFAKR
60

Wyeth
MYGLILSRENNCGYHCYETILIDVFDILSKYMDNIDMIDNENKTLLYYAVDVNNIQFAKR
60

*********************************:**************************

Cop
LLEYGASVTTSRSIINTAIQKSSYQRENKTRIVDLLLSYHPTLETMIDAFNRDIRYLYPE
120

Wyeth
LLEYGASVTTSRSIINTAIQKSSYRRENKTKLVDLLLSYHPTLETMIDAFERDIRYLYPE
120

************************:*****::****************************

Cop
PLFACIRYALILDDDETSKVSMISPVIIRN------------------------------
150

Wyeth
PLFACIRYALILDDDFPSKVKYDISGRHKELKRYRVDINRMKNAYISGVSMFDILFKRSK
180

********************. ::

Cop
------------------

Wyeth
RHRLRYAKNPTSNGTKKN
198

B25R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 642-644, respectively, in

order of appearance)

Cop
MBRINITKKIYCSVFLFLFLFLSYISNYEKVNDEMYEMGEMDEIVSIVRDSMWYIPNVFM
60

WR
----------------------------------------MDEIVRIVRDSMWYIPNVFM
20

Wyeth
MBRINITKKIYCSVFLF--LFLSYISNYEKVNDEMYEMGEMDEIVSIVRDSMWYIPNVFM
58

***** **************

Cop
DDGKNEGHVSVNNVCHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
120

WR
DDGKNEGHVSVNNVCHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
80

Wyeth
DDGKNEGHVSVNNVOMMYFTFEDVDTSSHLFKLVIKHCDLNKRGNSPLHCYTMNTRFNPS
118

************************************************************

Cop
VLKILLHHOMRNFDSKDEKGHHYLIHSLSIDNKIFDILTDTIDDFSKSSDLLLCYLRYKF
180

WR
VLKILLHHGMANFDSKDEKGHHYQSITRSLIY----------------------------
112

Wyeth
VLKILLHHGMRNFDSKD---DHYQSITRSLIY----------------------------
147

***************** .** : *:

Cop
NGSLNYYVLYKGSDPNCADEDELTSLHYYCKHISTFYKSNYYKLSHTFMRAEKRFIYAII
240

WR
------------------------------------------------------------

Wyeth
------------------------------------------------------------

Cop
DYGANINAVTHLPSTVYQT
259

WR
-------------------

Wyeth
-------------------

B26R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 645-648, respectively, in

order of appearance)

Cop
MEQTLTRLETYLQQYTKHSPRVVYALLSRGYVIILIVITSWNDCATGHILIMLINWEEQK
60

WR
-------------------MIFYLEEPIRGYVIILIVEPSWNDCATCHILIMILNWHEQK
41

Wyeth
MEQTLTRLETYLQQYTKHSPRVVYALLSRGYVIILIVITSWNDCATGHILIMLLNWHEQK
60

Lister
MEQTLTRUITYLQQYTKHSPRVVYALLSRGYVIILIVHPSWNDCATGHILIMLINWEEQK
60

. ********************************

Cop
EEGQHLLYLFIKENQGYTLNILRYLLDRFDIQKDEYIYRLSEL-----------------
103

WR
EEGQHLLYLFIKHNQGYTLNILRYLLDREDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
101

Wyeth
EEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
120

Lister
EEGQHLLYLFIKENQGYTLNILRYLLDREDIQKDEYYNTAFQNCNNNVASYIGYDINLPT
120

************************************ :

Cop
--------

WR
KDGIRLGV
109

Wyeth
KDGIRLGV
128

Lister
KDGIRLGV
128

B27R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 649-652, respectively, in

order of appearance)

Cop
MLPHTSDTTSTFRLKTVEDLVFENRNIIYKADVVNDIIKHRLKVSLPMIKSLKYKMSEFS
60

WR
MLPHTSDTTSTFRLKTVEDLVFENRNITYKADVVNDIIHHRLKVSLPMIKSLEYKMSLPT
60

Wyeth
MLPHTSDTTSTFRLKTVFDLVFENRNITYKADVVNDIIHHRLK--VPMIKSLEYKMSEFS
58

Lister
MLPHTSDTTSTFRLKTVEDLVFENRNITYKADVVNDIIHHRLKVSLPMIKSLEYMKSLPT
60

******************************************* :********* :

Cop
PYDDYYVKKILAYCLLRDESFAELHSKFCLNEDYKSVFMKNISETKIDSIIVT
113

WR
TITT-------------------------------------------------
64

Wyeth
PYDDYYVKKILAYCLLRDESFAELKSKFCLNEDYKSVFMKNISEDKIDSIIVT
111

Lister
TITT-------------------------------------------------
64

B28R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 653-655, respectively,

appearance)

Cop
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCKDNEYKRENLCPGTYASALCDSKTNTQC
60

WR
MKSVLYSYILELSCIIINGRDIAPHAPSDGKCKDNEYKRENLCPGTYASRLCDSKTNTQC
60

Wyeth
-----------------------MHHPMESVKTTN--TNAIICV---REHTLPDYANTQC
32

* * :. . * .. :* .: . :****

Cop
TPCGSGTETSRNNKLPACLSCNGRADPVTLLTIESVNALPDIIVFSKDHPDARHVFPKQN
120

WR
TPCGSGTFTSRNNHLPACLSCNGRRDRVTRLTIESVNALPDIIVFSKDHPDARHVFPKQN
120

Wyeth
TPCGSGTFTSANNELPACLSCNGRRDRVTLLTIESVNALPDIIVFSKDHPDARHVFPKQN
92

***************************** ******************************

Cop
VE 122

WR
VE 122

Wyeth
V- 93

*

C23L/B29R

CLUSTAL 0(1.2.4) multiple sequence alignment (SEQ ID NOS 656-660, respectively, in

order of appearance)

Cop
--------------MEVPASLQQSSSSSSSCTEEENKEHMGIDVIIKVTKQDQTPTNDKI
46

WR
--------------MEVPASLQQSSSSSSSCTEEENKHEMGIDVIIKVTKQDQTFINDKI
46

Tian
--------------MEVPASLQQSSSSSSSCTEEENKHHMGIDVIIINTKQDQTPTNDKI
46

Wyeth
--------------MEVPASLQQ---SSSSCTEEENKEHMGIDVIIKVTKQDQTPINDKI
43

Lister
MKQYIVLACMCLAAAAMPASLQQSSSSSSSCTEEENKHEMGIDVIIKVTKQDQTFINDKI
60

:****** **********************************

Cop
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPITYYSIIGGGLRMNFGETKCPQIKSIS
106

WR
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPITYYSIIGGGLRMNFGETKCPQIKSIS
106

Tian
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPTTYYSIIGGGIRMNFGFTKCPQIKSIS
106

Wyeth
CQSVTEITESESDPDPEVESEDDSTSVEDVDIPTTYYSIIGGGIRMNFGETKCPQIKSIS
103

Lister
CQSVTEITESESDPDPEVESEDDSTSVEDVDPPITYYSIIGGGLRMNFGETKCPQIKSIS
120

******************************* ****************************

Cop
ESADGNTVNARLSSVSPGQGKDSPAITREEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
166

WR
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIEDCEVSIDIRCSEEEKDSDIKTHPV
166

Tian
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
166

Wyeth
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
163

Lister
ESADGNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPV
180

***************************:********************************

Cop
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVEDGFKYVDGSASE
226

WR
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVXDGFKYVDGSASE
226

Tian
LGSNISHKKVSYEDIIGSTIVDTKCVENLEFSVRIGDMCKESSELEVEDGFEYVDGSASE
226

Wyeth
LGSNISHKKVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVKDGEKYVDGSASE
223

Lister
LGSNISHKEVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCKESSELEVEDGEKYVDGSASE
240

************************************************************

Cop
GATDDTSLIDSTKLEACV
244

WR
GATDDTSLIDSTKLEACV
244

Tian
GATDDISLIDSTKLKACV
244

Wyeth
GATDDISLIDSTKIKACV
241

Lister
GATDDTSLIDSTKLEACV
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 aid 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′)

EAILNGINYHAFESLLDYIRWKKINITINNYEMILVA
Deletion

AIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGI

KKLYNASMSEIINNITAVISDPEFGKLSKDELTTILS

HENVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETICKWKTVTNMSS

LKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIICKSPMPRHHSIVYAVEYD

GDIYVITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEKQ

FLQ

SEQ ID
C1L
AAA48000.1
MVICNNKISNSCRMIMSTNPNNILMRHLKNLTDDEF
Inside

NO:24

KCIIHRSSDFLYLSDSDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFICKIDSYRTRAI

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

CHIIKEFMTYEQMAIDHYGEYVNAILYQIRICRPNQH

HTIDLFICKIKRTPYDTFKVDPVEFVICKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFINYVETKYF

SEQ ID
MIL
AAA48003.1
MIFVIESKLLQIYRNRNRNINFYTTMDNIMSAEYYLS
Inside

NO:27

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGAKINNSVDEEGCCPLLACTDPSER

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPICASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYE

TMVDYLLFNHFSVDSVVNGHTCMSECVRLNNPVIL

SKLMLHNPTSETMYLTMICAIEKDKLDKSIIIPFIAYF

VLMHPDFCKNRRYFTSYICRFVTDYVHEGVSYEVFD

DYF

SEQ ID
M2L
AAA48004.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO:28

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNICLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTIUNPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIICELSYDIC

YRE

SEQ ID
HR/K1L
AAA48005.1
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO:29

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGMDDSQFDDKGNTALYYAVDSG

NMQTVKLFVKKNWRLMFYGKTGWKTSFYHAFML

NDVSIVSYFLSEIPSTFDLAILLSCIHTTIKNGHVDMM

ILLLDYMTSTNTNNSLLFIPDIKLAIDNICDIEMLQAL

FKYDINIYSVNLENVLLDDAEITKMIIEKHVEYKSDS

YTKDLDIVKNNICLDEIISKNKELRLMYVNCVKKN

SEQ ID
SPI-
AAA48006.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO:30
3/K2L

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPLYYQQYHRFGLYRLNFRRDAVNIUNSIVERRSG

MSNVVDSNMLDNNTLWAIINTIYFKGTWQYPFDIT

KTRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDE

EYDMVRLPYKDANISMYLAIGDNMTHFIDSITAAK

LDYWSFQLGNKVYNLICLPICFSIENKRDIKSIAEMM

APSMFNPDNASFKHMTRDPLYIYICMFQNAKIDVDE

QGTVAEASTIMVATARSSPEKLEFNTPFVFIIRHDITG

FILFMGKVESP

SEQ ID
K ORF A
AAA48007.1
MGHIITYCQVHTNISILIRKAHHIIFFVIDCDCISLQFS
Inside

NO:31

NYVHHGNRFRTVLISKTSIACFSDIKRILPCTFICIYSI
Deletion

NDCP

SEQ ID
K ORF B
AAA48008.1
MGTVFVPYLLVICLALRVLVISNGYCHVPLKYIVLMI
Inside

NO:32

AHRVLLSSILESTTLDIPDLRSTIELILLTASRLICFNLY
Deletion

RPNL

SEQ ID
K ORF B
AAA48009.1
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPH
Inside

NO:33

SEAILAESVICMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYICRMCRHQ

SEQ ID
K4L
AAA48010.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO:34

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPICRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPIIYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNIDIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
K5L
AAA48011.1
MGATISILASYDNPNLFTAMILMSPLVNADAVSRLN
Inside

NO:35

LLAAKLMGTITPNAPVGKLCPESVSRDMDKVYKYQ
Deletion

YDPLINHEKIKAGFASQVLKATNKVRKIISKINTPRL

SYSREQTMRLVMFQVHIISCNMQIVIE

MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH

SEQ ID
K6L
AAA48012.1
SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Inside

NO:36

DDFGTARGNY
Deletion

SEQ ID
K7R
AAA48013.1
MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO:37
LN

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
F1L
AAA48014.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO:38

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

ICDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
DUT/F2
AAA48015.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO:39
L

SAYDYTIPPGERQLIKTDISMSMPKICYGRIAPRSGLS
Deletion

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTNRGDQGFSTGLR

SEQ ID
F3L
AAA48016.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO:40
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFITYTCI

NFILRDFRICEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYATGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

RDNPELIIVDNICLLLIGGFYRGSYIDTIEVYNHHTY

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
VACWR
AA089305.1
MESVIFSINGEIIQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO:41
026

EMILNGINYHAFESLLDYIRWICKINITINNVEMILVA
Deletion

(26% 5′)

AIIIDVPPVVDLCVKTMIHNINSTNCIRMFNFSKRYGI

KKLYNASMSEIINNITAVISDPEFGKLSKDELITTILS

HENVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

CLYIIGGMINNRHVYSVSRVDLETIUCWKTVTNMSS

LKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYVITGITHETRNYLYKYIVICEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETPKCNVTHKSLPSFLSNCEKQ

FLQ

SEQ ID
VACWR
AA089306.1
MVKNNKIQKNKISNSCRMIMSTDPNNILMRHLKNL
Inside

NO:42
027

TDDEFKCIIHRSSDFLYLSDSDYTSITKETLVSEIVEE
Deletion

YPDDCNKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAI

LDKMTEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSY

RTRAINKYSICELGLATEYFNKYGHLMFYTLPIPYNR

FFCRNSIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKF

KKELMSK

SEQ ID
VACWR
AA089307.1
MRTLLIRYILWRNDNDQTYYNDNFICKLMLLDELVD
Inside

NO:43
028

DGDVCTLIICNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
VACWR
AA089308.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIEMD
Inside

NO:44
029

CINRHINMCIQRTYSSSIIAILDRFLMMNICDELNNTQ
Deletion

CHIIICEFMTYEQMAIDHYGGYVNAILYQIRICRPNQH

HTIDLFKRIKRTRYDTFKVDPVEFVICKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFINYVETKYF

SEQ ID
VACWR
AA089309.1
MIFVIESKLLQIYRNRNRNINFYTTNIDNIMSAEYYLS
Inside

NO:45
030

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDICHNKTPLYYLSGTDDE

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNICIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIICEDAMYYAVLSEYE

TMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVIL

SKLMLHNPTSETMYLTMKAIEKDRLDKSIIIPFIAYF

VLMHPDFCICNRRYFTSYKRFVTDYVHEGVSYEVFD

DYF

SEQ ID
VACWR
AA089310.1
MVYKLVLLFCIASLGYSVEYKNTICPPRQDYRYWY
Inside

NO:46
031

FAAELTIGVNYDINSTIIGECHMSESSYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGICDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYSMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
VACWR
AA089311.1
MDLSRINTWKSKQLKSFLSSKDAFKADVHGHSALY
Inside

No:47
032

YAIADNNVRLVCTLLNAGALICNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVICKNVVRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNICDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISICNKELRLMYVNCVKKN

SEQ ID
SPI-3
AA089312.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO:48

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPSYYQQYHRFGLYRLNFRRDAVNKINSIVERRSG

MSNVVDSNMLDNNTLWAIINTIYFKGIWQYPFDITK

TRNASFTNKYGTKTVPMMNVVTKLQGNTITIDDEE

YDMVRLPYICDANISMYLAIGDNMTHFTDSITAAKL

DYWSFQLGNKVYNLKLPKFSIENKRDIKSIAEMMAP

SMFNPDNASFKHMTRDPLYIYKMFQNAKIDVDEQG

TVAEASTIMVATARSSPEICLEFNTPFVFIIRHDITGFI

LFMGKVESP

SEQ ID
VACWR
AA089313.1
MLAFCYSLPNAGDVIKGRVYEKDYALYIYLFDYPH
Inside

NO:49
34

FEAILAESVICMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
VACWR
AA089314.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO:50
035

TTKTLDISSFYWSLSDEVGTNFGTHLNKIVQLPICRG
Deletion

VRVRVAVNKSNKPLICDVERLQMAGVEVRYIDITNI

LGGVLHTICFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPHYSKAGNI

LFWPYIEDELRRAAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCRLLK

NMKQCINDIYCDEIQPEICEIPEYSLE

SEQ ID
VACWR
AA089315.1
MQHANCNREIKIYEGAKHHLHICETDEVICKSVMKEI
Outside

NO:51
036

ETWIFNRVK
Deletion

SEQ ID
VACWR
AA089316.1
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMIL
Inside

NO:52
037

MSPLVNADAVSKLNLLAAKLMGTITLNAPVGKLCP
Deletion

ESVSRDMDKVYKYQYDPLINHEKHCAGFASQVLKA

TNKVRKIISKINTPRLSYSREQTIRLAMF

SEQ ID
VACWR
AA089317.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO:53
038

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
VACWR

MATKLDYEDAVFYFVDDDKICSRDSIIDLIDEYITW
Inside

NO:54
039
AA089318.1
RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGICAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEICK

LN

SEQ ID
VACWR
AA089319.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO:55
040

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NICGLGVRLATISFITELGRRCMNPVETIKMFTLLSHT

ICDDYFVDYITDISPPDNTIPNTSTREYLICIIGITAIMF

ATYKTLKYMIG

SEQ ID
DUT
AA089320.1
MFNMNINSPVRFVKETNRAKSPTROQSPYAAGYDLY
Inside

NO:56

SAYDYTIPPGERQLIKTDISMSMPKFCYGRIAPRSGL
Deletion

SLKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTG

DRIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGL

R

SEQ ID
VACWR

MPIFVNTVYCKNILALSMTKICFKTIIDAIGGNIIVNST
Inside

NO:57
042

ILKKLSPYFRTHLRQKYTKNKDPVTWVCLDLDIHSL
Deletion

(75% 3′)

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTICNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYIICRRNICLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGFtRLFLVGRNAEFYCESSNTWTLIDDPIY

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

MESVIFSINGEHQVNKEIITASPYNFFKRIQDHHLKD
Inside

NO:58
(26% 5′)
AAF33878.I
EAIILNGINYHAFESLLDYIRWKKTNITINNVEMILVA
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

NIULAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
TNIL
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

N0:62

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Deletion

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKTPLYYLSGTDDE

VIERINLLVQYGAKINNSVDEEGCGPLLACTDPSER

VFKKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTI

SWMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIID

LLLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLS

TSNVITDQTVNKIFYDRDDVLEIINDKGKQYDSTDF

KMAVEVGSIRCVKYLLDNDIKEDAMYYAVLSEYE

TMVDYLLFNHFSVDFVVNGHTCMSECVRLNNPVIL

SKLMLHNLTSETMYLTMKAIEKDRLDKSIIIPFIAYF

VLMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFD

DYF

SEQ ID
TM2L
AAF33883.1
MSSSTRLPVLVLAAELTIGVNYDINSTIIGECHMSES
Inside

NO:63

YIDRNANIVLTGYGLEINMTIMDTDQRFVAAAEGV
Deletion

GKDNKLSVLLFTTQRLDKVHHNISVTITCMEMNCG

TTKYDSDLPESIHKSSSCDITINGSCVTCVNLETDPT

KINPHYLHPKDKYLYHNSEYGMRGSYGVTFIDELN

QCLLDIKELSYDKYRE

SEQ ID
TK1L
AAF33884.1
MLQALFKYDINIYSANLENVLLDDAEIAKMIIEKHV
Inside

NO:64

EYKSDSYTKDLDIVKNNKLDEIISKNKELRLMYVNC
Deletion

VKKN

SEQ ID
TK2L
AAF33885.1
MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO:65

YAIADNNVRLVCTLLNSGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNVVRLMFYGKTGWKTSFYHAVMLN

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.I
MTLVQHVVTIKSTYWVIPWELASYDNPNLFTAMIL
Inside

N0:69

MSPLVNADAVSKLNLLAAKLMGTITLNAPVGKLCP
Deletion

ESVSRDMDKVYKYQYDPLINHEKIKAGFASQVLKA

TNKVRKIISKINTPRLSYSREQTIRLAMF

SEQ ID
TK8R
AAF33890.1
MATKLDYEDAVFYFVDDDKKSRDSIIDLIDEYITW
Inside

NO:70

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGKAKITE

HVVGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
TR1L
AAF33891.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDR
Inside

NO:71

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

KDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

SEQ ID
TF2L
AAF33892.1
SAYDYTIPPGERQLIKTDISMSMPKKYGRIAPRSGLS
Deletion

N0:72

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTDRGDQGFGSTGLR

SEQ ID
TF3L
AAF33903.1
MPIFVNTVYCKNILALSMTIUCFKTIIDAIGGNIIVNST
Inside

NO: 73
(75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFHYTCI

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 CopN1D5p vector

Protein

SEQ ID

Accession

NO
Gene
ID
Amino Acid Sequence
Location

SEQ ID
VAC_D
AEY74729.1
MESVIFSINGEHQVNKEHTASPYNFFKRIQEHHINDE
Inside

NO:74
PP20_03

VIILNGINYHAFESLLDYMRWKKINITINNVEMILVA
Deletion

5

AVIIDVTPVVDLCVKTMIHNINSTNCIRMFNFSKRYG

(26% 5′)

IKKLYNASMSEIINNITAVTSDPEFGKLSKDELTTILS

HEDVNVNHEDVTAMILLKWIHKNPNDVDIINILHPK

FMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVIK

NSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLHN

LYIIGGMINNRHVYSVSRVDLETKKWKTVTNMSS

LKSEVSTCVNNGKLYVIGGLEFSISTGVAEYLKHGT

SKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTYE

KYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEYD

GDIYAITGITHETRNYLYKYIVKEDKWIELYMYFNH

VGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFDM

STRNIEYYDMFTKDETHKSLPSFLSNCEKQFLQ

SEQ ID
VAC_D
AEY74730.1
MVKNNKISNSCRMIMSTNPNNILMRFILKNLTDDEF
Inside

NO:75
PP10_03

KCIIHRSSDFLYLSDRDYTSITKETLVSEIVEEYPDDC
Deletion

6

NKILAIIFLVLDKDIDVDIKTKLKPKPAVRFAILDKM

TEDIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNFtFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFIUCEL

MSK

SEQ ID
NIL
AEY74731A
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO:76

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
VAC_D
AEY74732.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIIDC
Inside

NO:77
PP11_03

NRHINMCIQRTYSSSIIAILDRFLTMNKDELNNTQC
Deletion

8

HIIKEFMTYEQMAIDHYGGYVNAILYQIRKRPNQHH

TIDLFKKIKRTRYDTFKVDPVEFVKKVIGFVSILNKY

KPVYSYVLYENVLYDEFKCFIDYVETKYF

SEQ ID
VAC_D
AEY74733.1
MLHNPTSETMYLTMNAIKKDKLDKSIIIPFIAYFVLM
Not

NO:78
PP11_03

HPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDDYF
Present

9

SEQ ID
VAC_D
AEY74734.1
MSIGFEARIVDKFGKNH1HRHLMSDNPKASTISWM
Inside

NO:79
PP12_04

MKLGISPSKPDHDGNTPLHIVCSKTVKYVDIIDLLLP
Deletion

0

STDVNKQNFGDSPLTLLIKTLSLAHINKLLSTSNV

ITDQTVNICIFYDRDDVLEIINDKGKQYDFKMAVEV

GSIKCVKYLLDNDIKEDAMYYAVLSEYKTMVDYL

LFNHFSVDSVVNGHTCMSECVKLNNRHFIEADVT

SEQ ID
VAC_D
AEY74735.1
MIFVLESKLLQIYRNRNRNINFYTTMDNIMSAEYYLS
Not

NO:80
PP12_04

LYAKYNSKNLDVFRNMLQAIEPSGNNYHILHAYCG
Present

1

IKGLDERFVEELLHRGYSPNETDDDGNYPLHIASKIN

NNRIVAMLLTHGADPNACDKHNKTPLYYLSGTDDE

VIERINLLVQYGAKINN

SEQ ID
VAC_D
AEY74736.1
MVYKLVLLFCIASLGYSVEYKNTKPPRQDYRYWY
Inside

NO:81
PP12_04

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

2

YGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGITKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDIC

YRE

SEQ ID
VAC_D

MDLSRINTWKSKQLKSFLSSKDTFKADVHGHSALY
Inside

NO:82
PP10_04 AEY74737.1

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

3

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISKNKELRLMYVNCVKKN

SEQ ID
VAC_D
AEY74738.1
MIALLILSLTCSVSTYRLQGFTNAGIVAYKNIQDDNI
Inside

NO:83
PP20_04

VFSPFGYSFSMFMSLLPASGNTRIELLKTMDLRKRD
Deletion

4

LGPAFTELISGLAKLKTSKYTYTDLTYQSFVDNTVCI

KPSYYQQYHRLNFRRDAVNKINSIVERRSGMSNVV

DSNMLDNNTLWAIINTIYFKGIWQYPFDITKTRNAS

FTNKYGTKTVPMMNVVTKLQGNTITIDDKEYDMV

RLPYKDANISMYLAIGDNMTHFTDSITAAKLDYWS

FQLGNKVYNLKLPKFSIENKRDIKSIAEMMAPSMFN

PDNASFKHMTRDPLYIYKMFQNAKIDVDEQGTVAE

ASTIMVATARSSPEKLEFNTPFVFIIRHDITGFILFMG

KVESP

SEQ ID
VAC_D
AEY74739.1
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPH
Inside

NO:84
PP10_04

FEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

5

VDYTKGYIDVNYKRMCRHQ

SEQ ID
K4L
AEY74740.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

NO:85

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKFVYVSVMNFIPHYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
VAC_D
AEY74741.1
MGATISILASYDNPNLFTAMILMSPLVNADAVSKLN
Inside

NO:86
PP20_04

LLAAKLMGTITPNAPVGKLCPESVSRDMDKVYKYQ
Deletion

7

YDPLINHEKIKAGFASQVLKATNKVRIUISKINTPPT

LILQGTNNEISDVLGAYYFMQHANCNREIKIYEGAK

HHLHKETDEVECKSVMKEIETWIFNRVK

SEQ ID
List034/
AEY74742.1
SANCMFNLDNDYIYWKPITYPKAINFISHGAGKH
Inside

NO:87
VAC_D

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

PP20_04

DDFGTARGNY

8

SEQ ID
K7R/VA
AEY74743.1
MATKLDYEDAVFYFVDDDKKSRDSIIDLIDEYITW
Inside

NO:88
C_DPP2

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

0-49

INEIVEAMSEGDHYINFTKVHDQESLFATIGKAKITE

HWGYKKISESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
LIVPclo
AEY74744.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO:89
ne14_04

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

6/VAC

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

DPP20_0

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

47

NKGLGVRLATISFITKLGRRCMNPVKTIKMFTLLSH

TKDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIM

FATYKTLKYMIG

SEQ ID
F2L/VA
AEY74745.1
MFNMNINSPVREVKETNRAKSPTRQSPGAAGYDLY
Inside

NO:90
C_DPP2

SAYDYTIPPGERQLIKTDISMSMPKKYGRIAPRSGLS
Deletion

0_051

LKGIDIGGGVIDEDYRGNIGVILINNGKCTFNVNTGD

RIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGLR

SEQ ID
F3L
AEY74746.1
MPIFVNTVYCKNILALSMTKKEKTIIDAIGGNIIVNST
Inside

NO:91
75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSYTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSMKLILESDELNVPDEDYV

VDFVIKWYEKRRNKLGNLLLLIKNVIRSNYLSPRGIN

NVKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQI

IDIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVN

YISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNP

TSVERWFHGDAAWVNMPSLLKPRCNPAVASINNVI

YVMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHY

KSCALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIY

PRDNPELIIVDNKILLIGGFYRESYIDTIEVYNHHTYS

WNIWDGK

TABLE 35

Examples of proteins encoded by Lister Vaccinia genes equivalent to

those deleted in CopMD5pvector

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

GIIKKLYNASMSEIINNITAVTSDPEFGKLSKDELTTIL

SHEDVNVNHEDVTAMILLKWIHKNPNDVDIINILHP

KFMTNTMRNAISLLGLTISKSTKPVTRNGIKHNIVVI

KNSDYISTITHYSPRTEYWTIVGNTDRQFYNANVLH

NCLYIIGGM1NNRHVYSVSRVDLKTKKWKTVTNMS

SLKSEVSTCVNDGKLYVIGGLEFSISTGVAEYLKHG

TSKWIRLPNLITPRYSGASVFVNDDIYVMGGVYTTY

EKYVVLNDVECFTKNRWIKKSPMPRHHSIVYAVEY

DGDIYVITGITHETRNYLYKYIVKEDKWIELYMYFN

HVGKMFVCSCGDYILIIADAKYEYYPKSNTWNLFD

MSTRNIEYYDMFT1CDETPKCNVTHKSLPSFLSNCEK

QFLQ

SEQ ID
C1L/List
ABD52474.1
MVKNNKISNSCRMIMSTNPNNILMRHLKNLTDDEF
Inside

NO:93
024

KCIIHRSSDFLYLSDSDYTSITKETLVSEIVEEYPDDC
Deletion

NKILAIIFLVLDKDIDVDIETKLKPKPAVRFAILDKM

TADIKLTDLVRHYFRYIEQDIPLGPLFKKIDSYRTRAI

NKYSKELGLATEYFNKYGHLMFYTLPIPYNRFFCRN

SIGFLAVLSPTIGHVKAFYKFIEYVSIDDRRKFKKEL

MSK

SEQ ID
NIL/List
ABD52475.1
MRTLLIRYILWRNDNDQTYYNDDFKKLMLLDELVD
Inside

NO:94
025

DGDVCTLIKNMRMTLSDGPLLDRLNQPVNNIEDAK
Deletion

RMIAISAKVARDIGERSEIRWEESFTILFRMIETYFDD

LMIDLYGEK

SEQ ID
List026
ABD52476.1
MTSSAMDNNEPKVLEMVYDATILPEGSSMDPNIMD
Inside

NO:95

CINRHINMCIQRTYSSSIIAILDRFLTMNKDELNNTQ
Deletion

CHIIKEFMTYEQMAIDHYGEYVNAILYQIRKRPNQH

HTIDLFKKIKRTRYDTEKVDPVEFVKKVIGFVSILNK

YKPVYSYVLYENVLYDEFKCFIDYVETKYF

SEQ ID
List027
ABD52477.1
MIFVIESKLLQIYRNRNINFYTTMDNIMSAEYYLSLY
Inside

N0:96

AKYNSKNLDVFRNMLQAMPSGNNYHILHAYCGIK
Deletion

GLDERFVEELLHRGYSPNETDDDGNYPLHIASKINN

NRIVAMLLTHGADPNACDKQHKTPLYYLSGTDDEV

IERINLLVQYGAKINNSVDEEGCGPLLACTDPSERVF

KKIMSIGFEARIVDKFGKNHIHRHLMSDNPKASTIS

WMMKLGISPSKPDHDGNTPLHIVCSKTVKNVDIIDL

LLPSTDVNKQNKFGDSPLTLLIKTLSPAHLINKLLST

SNVITDQTVNKIFYDRDDVLEIINDKGKQYDSTDFK

MAVEVGSIRCVKYLLDNDIKEDAMYYAVLSEYET

MVDYLLFNHFSVDSVVNGHTCMSECVRLNNPVILS

KLMLHNPTSETMYLTMKAIEKDRLDKSIIIPFIAYFV

LMHPDFCKNRRYFTSYKRFVTDYVHEGVSYEVFDD

YF

SEQ ID
List028
ABD52478.1
MVYKLVLLFCIASLGYSVEYKNTKPPRQDYRYWY
Inside

NO:97

FAAELTIGVNYDINSTIIGECHMSESYIDRNANIVLTG
Deletion

YGLEINMTIMDTDQRFVAAAEGVGKDNKLSVLLFT

TQRLDKVHHNISVTITCMEMNCGTTKYDSDLPESIH

KSSSCDITINGSCVTCVNLETDPTKINPHYLHPKDKY

LYHNSEYGMRGSYGVTFIDELNQCLLDIKELSYDK

YRE

SEQ OD
K1L/List029
ABD52479.1
MDLSRINTWKSKQLKSFLSSKDAFKADINGHSALY
Inside

NO:98
029

YAIADNNVRLVCTLLNAGALKNLLENEFPLHQAAT
Deletion

LEDTKIVKILLFSGLDDSQFDDKGNTALYYAVDSGN

MQTVKLFVKKNWRLMFYGKTGWKTSFYHAVMLN

DVSIVSYFLSEIPSTFDLAILLSCIHITIKNGHVDMMIL

LLDYMTSTNTNNSLLFIPDIKLAIDNKDIEMLQALFK

YDINIYSANLENVLLDDAEIAKMIIEKHVEYKSDSYT

KDLDIVKNNKLDEIISKNKELKLMYVNCVKKN

SEQ ID
List030
ABD52480.1
IELLKTMDLRKRDLGPAFTELISGLAKLKTSKYTYT
Inside

NO:99

DLTYQSFVDNTVCIKPSYYQQYHRFGLYRLNFRRD
Deletion

AVNKINSIVERRSGMSNVVDSNMLDNNTLWAIINTI

YFKGIWQYPFDITKTRNASFTNKYGTKTVPMMN

TKLQGNTITIDDEEYDMVRLPYKDANISMYLAIGDN

MTHFTDSITAAKLDYWSSQLGNKVYNLKLPKFSIEN

KRDIKSIAEMMAPSMFNPDNASFKHMTRDPLYIYK

MFQNAKIDVDEQGTVAEASTIMVATARSSPEKLEFN

TPFVFIIRHDITGFILFMGKVESP

SEQ ID
K3L/List
ABD52483.1
MLAFCYSLPNAGDVIKGRVYENDYALYIYLFDYPH
Inside

NO:100
031

SEAILAESVKMHMDRYVEYRDKLVGKTVKVKVIR
Deletion

VDYTKGYIDVNYKRMCRHQ

SEQ ID
K4L/List
ABD52484.1
MNPDNTIAVITETIPIGMQFDKVYLSTFNMWREILSN
Inside

N0:101
032

TTKTLDISSFYWSLSDEVGTNFGTIILNEIVQLPKRG
Deletion

VRVRVAVNKSNKPLKDVERLQMAGVEVRYIDITNI

LGGVLHTKFWISDNTHIYLGSANMDWRSLTQVKEL

GIAIFNNRNLAADLTQIFEVYWYLGVNNLPYNWKN

FYPSYYNTDHPLSINVSGVPHSVFIASAPQQLCTME

RTNDLTALLSCIRNASKEVYVSVMNFIPHYSKAGKI

LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRN

FLRSIAMLKSKNINIEVKLFIVPDADPPIPYSRVNHAK

YMVTDKTAYIGTSNWTGNYFTDTCGASINITPDDGL

GLRQQLEDIFMRDWNSKYSYELYDTSPTKRCKLLK

NMKQCTNDIYCDEIQPEKEIPEYSLE

SEQ ID
List033
ABD52485.1
MGHSMGATISILASYDNPNLFTAMILMSPLVNADA
Outside

NO:102

VSRLNLLAAKLMGTITPNAPVGKLCPESVSRDMDK
Deletion

VYKYQYDPLINHEKIKAGFASQVLKATNKVRKIISKI

NTPPTLILQGTNNIUSDVLGAYYFMQHANCNREIKI

YEGAKHHLHKETDEVKKSVMKEIETWIFNRVK

SEQ ID
List034
ABD52486.1
MSANCMFNLDNDYIYWKPITYPKALVFISHGAGKH
Inside

NO:103

SGRYDELAENISSLGILVFSHDHIGHGRSNGEKMMI
Deletion

DDFGTARGNY

SEQ ID
K7R/List
ABD52487.1
MATKLDYEDAVFYFVDDDKKSRDSIIDLIDEYITW
Inside

NO:104
035

RNHVIVFNKDITSCGRLYKELMKFDDVAIRYYGIDK
Deletion

INEIVEAMSEGDHYINFTKVHDQESLFATIGKAKITE

HWGYKIUSESRFQSLGNITDLMTDDNINILILFLEKK

LN

SEQ ID
F1L/List
ABD52489.1
MLSMFMCNNIVDYVDDIDNGIVQDIEDEASNNVDH
Inside

NO:105
036

DYVYPLPENMVYRFDKSTNILDYLSTERDHVMMA
Deletion

VRYYMSKQRLDDLYRQLPTKTRSYIDIINIYCDKVS

NDYNRDMNIMYDMASTKSFTVYDINNEVNTILMD

NKGLGVRLATISFITELGRRCMNPVKTIKMFTLLSHT

KDDCFVDYITDISPPDNTIPNTSTREYLKLIGITAIMF

ATYKTLKYMIG

SEQ ID
List037
ABD52490.1
MFNMNINSPVRFVKETNRAKSPTRQSPGAAGYDLY
Inside

NO:106

SAYDYTIPPGERQLIKTDISMSMPKFCYGRIAPRSGL
Deletion

SLKGIDIGGGV1DEDYRGNIGVILINNGKCTFNVNTG

DRIAQLIYQRIYYPELEEVQSLDSTNRGDQGFGSTGL

R

SEQ ID
List038
ABD52491.1
MPIFVNTVYCKNILALSMTKKFKTIIDAIGGNIIVNST
Inside

NO:107
75% 3′)

ILKKLSPYFRTHLRQKYTKNKDPVTRVCLDLDIHSL
Deletion

TSIVIYSTGKVYIDSHNVVNLLRASILTSVEFIIYTCI

NFILRDFRKEYCVECYMMGIEYGLSNLLCHTKNFIA

KHFLELEDDIIDNFDYLSIKLILESDELNVPDEDYVV

DFVIKWYIKRRNKLGNLLLLIKNVIRSNYLSPRGINN

VKWILDCTKIFHCDKQPRKSYKYPFIEYPMNMDQII

DIFHMCTSTHVGEVVYLIGGWMNNEIHNNAIAVNY

ISNNWIPIPPMNSPRLYASGIPANNKLYVVGGLPNPT

SVERWFHGDAAWVNMPSLLKPRCNPAVASINNVIY

VMGGHSETDTTTEYLLPNHDQWQFGPSTYYPHYKS

CALVFGRRLFLVGRNAEFYCESSNTWTLIDDPIYPR

DNPELIIVDNKLLLIGGFYRESYIDTIEVYNIIHTYSW

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′)

EMVDVSMMSMYGELFNHASVKESFGNFSHELPYV
Deletion

GDTSMMVILPDKIDGLESIEQNLTDTNFKKWCNSLD

AMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGSTG

DYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAATC

ALVSDCASTITNEFCVDHPFIYVIRHVDGKILFVGRY

CSPTTNC

SEQ ID
B15R
AAA49212.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYVV
Inside

NO:109

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
B ORF E
AAA48213.1
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMV
Inside

NO:110

FTSPVSSSKTKSDDGRNLSDGFLLIRYITTDDFCTIF
Deletion

DIIPRHIFYQLANVDEH

SEQ ID
B16R
AAA48214.1
MSILPVIFLPIFFYSSFVQTFNASECIDKGXYFASFME
Inside

NO:111

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYKITTNETYCDMMSLN

LTIVSVSESNIDFISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIHPTM

QLPEGVVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGDGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
B ORF F
AAA48215.1
MVIIPGVRCLSLLFLRRRCPLHIISAFTLLAINALILGH
Inside

NO:112

TISPVDLSFTKGYEIKSIFDSETDTIVKFNDIMSQ
Deletion

SEQ ID
B17L
AAA48216.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO:113

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

CKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITP

VEAPLPGNVLVYTFPDINKRIPGYIHVNIEGCIDGMI

YINSSKFACVLKLHRSMYRIPPFPIDKSCCSQYTND

DIEIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYF

NNIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVN

HSNTFVNCLLEDNV

SEQ ID
B18R
AAA48217.1
MSRRLIYVLNINRKSTHKIQENEIYTYFSHCNIDHTS
Inside

NO:114

TELDFVVKNYDLNRRQHVTGYTALHCYLYNNYFT
Deletion

NDVLKILLNHDVNVTMKTSSGRMPVYILLTRCCNIS

HDVVIDMIDKDKNHLSHRDYSNLLLEYIKSRYMLL

KEEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLC

LAHVYKPGECRKPITIKKAKRIISLFIQHGANLNALD

NCGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKKN

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

TEEYIGNTYKCRGHNYYFEKTLITTVVLE

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

IKIVRLLLSRGVERLCRNNEGLTPLGAYSKHRYVKS

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

MGEMDEIVSIVRDSM1ArYIPNVFMDDGKNEGHVSV
Deletion

NNVCIIMYFTFFDVDTSSHLFKLVIKHCDLNKRGNS

PLHCYTMNTRFNPSVLKILLHHGMRNFDSKDEKGH

HYLIHSLSIDNKIFDILTDTIDDFSKSSDLLLCYLRYK

FNGSLNYYVLYKGSDPNCADEDELTSLHYYCKHIST

FYKSNYYKLSHTKMRAEKRFIYAIIDYGANINAVTH

LPSTVYQT

SEQ ID
B26R
AAA48226.1
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVHLI
Inside

NO:123

VHPSWNDCATGHILIMLLNWHEQKEEGQHLLYLFI
Deletion

KHNQGYTLNILRYLLDRFDIQKDEYIYRLSKL

SEQ ID
B27R
AAA48227.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO:124

IHHRLKVSLPMIKSLFYKMSEFSPYDDYYVKKILAY
Deletion

CLLRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSII

VT

SEQ ID
B28R
AAA48228.I
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCI(DNEY
Inside

NO:125

KRHNLCPGTYASRLCDSKTNTQCTPCGSGTFTSRNN
Deletion

HLPACLSCNGRRDRVTLLTIESVNALPDIIVFSKDHP

DARHVFPKQNVE

SEQ ID
C23L/B2
AAA48229.1
MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTK
Inside

NO:126
9R (44%

QDQTPTNDKKQSVTEITESESDPDPEVESEDDSTSV
Deletion

5,)

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-
AO89474.1
MDIFREIASSMKGENVFISPASISSVLTILYYGANGST
Inside

NO:127
VACWR

AEQLSKYVEKEENMDKVSAQNISFKSINKVYGRYS
Deletion

195

AVFKDSFLRKIGDKFQTVDFTDCRTIDAINKCVDIFT

(26% 3′)

EGKINPLLDEPLSPDTCLLAISAVYFKAKWLTPFEKE

FTSDYPFYVSPTEMVDVSMMSMYGKAFNHASVKE

SFGNFSIIELPYVGDTSMMVILPDKIDGLESIEQNLTD

TNFKKWCNSLEATFIDVHIPKFKVTGSYNLVDTLVK

SGLTEVFGSTGDYSNMCNSDVSVDAMIHKTYIDVN

EEYTEAAAATCALVSDCASTITNEFCVDHPFIYVIRH

VDGKILFVGRYCSPTTNC

SEQ ID
VACWR
AAO89475.1
MTANFSTHVFSPQHCGCDRLTSIDDVRQCLTEYIYW
Inside

NO:128
196

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
VACWR
AAO89476.1
MSILPVIFLSIFFYSSFVQTFNAPECIDKGQYFASFME
Inside

NO:129
197

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYKITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
VACWR
AAO89477.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO:130
198

SAEKYMCRYTTLNHNCINVRRCALDSKLLHDIITNC
Deletion

KIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYP

VIFITHTSTRNLDKVSVKTYKGVKVKKLNRCADHAI

VINPFVKFKLTLPNKTSHAKVLVTFCKLKTDITPVEA

PLPGNVLVYTFPDINKRIPGYIHLNIEGCIDGMIYINS

SKFACVLKLHRSMYRIPPFPIDKSCCSQYINYDIEIPI

HDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNID

TAITQEHEYVIUALGIVCKLMINNMHSIVGVNHSNT

FVNCLLEDNV

SEQ ID
VACWR
AAO89478.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO:131
199

ELDFVVKNYDLNRRQPVTGYTALHCYLYNNYFTN
Deletion

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLLHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPITIKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFEKNN

HGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNIDIYTRYE

GKTLLHVACEYNNTHVIDYLIRINGDINALTDNNKH

ATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYNRHVRNRDSKT

YRYAFSKYMSFDKYDGIITKCHKETILLKLSTVLDT

TLYAVLRCHNSKURRYLTELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
B18R/V
AAO89479.1
MTMKMMVHIYFVSLLLLLFHSYAIDIENEITEFFNK
Inside

NO:132
ACWR2

MRDTLPAKDSKWLNPACMFGGTMNDIAALGEPFS
Deletion

00

AKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSN

KRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDCV

QGIVSHIRKPPSCIPKTYELGTHDKYGIDLYCGILY

AKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPEL

EDSGRYDCYVHYDDVRIKNDIVVSRCIULTVIPSQD

HRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLIE

WENPSGWLIGFDFDVYSVLTSRGGITEATLYFENVT

EEYIGNTYKCRGHNYYFEKTLTTTVVLE

SEQ ID
VACWR
AAO89480.1
MHVIDVDVRLYMSTFIIIDQSTENTSIDTTVTINIIYL
Not

NO:133
201

AIMKIIMNIIMMIMIELV
Present

SEQ ID
VACWR
AAO89481.1
MNSESDNISIKTEYEFYDETQDQSTQLVGYDIKLKT
Not

NO:134
202

NEDDFMANIDQWVSMII
Present

SEQ ID
VACWR
AAO89482.1
MEMYPRHRYSKHSVFKGFSDKVRKNDLDMNVVKE
Inside

NO:135
203

LLSNGASLTIKDSSNKDPITVYFRRTIMNLEMIDERK
Deletion

YIVHSYLKNYKNFDYPFFRKLVLTNKHCLNNYYNIS

DSKYGTPLHILASNKKLITPNYMKLLVYNGNDINAR

GEDTQMRTPLHKYLCKFVYHNIEYGIRYYNEKIIDA

FIELGADLTIPNDDGMIPVVYCIHSNAEYGYNNITNI

KIIRKLLNLSRRASHNLFRDRVMHDYISNTYIDLECL

DIIRSLDGFDINGYFEGRTPLHCAIQHNFTQIAKYLL

DRGADIVVPNTLIIHQYIQ

SEQ ID
VACWR
AAO89483.1
MLNFSLCLYPVFILNKLVLRTQSIILHTINNASIKNR
Not

NOS 677
204

\\\\\\ and \\\\\\
Present

and 136

MEEDTNISNKVIRYNTVNNIWETLPNFWTGTINPGV

VSHKDDIYVVCDIKDEKNVKTCIFRYNTNTYNGWE

LVTTTESRLSALHTILYNNTIMMLHCYESYMLQDTF

NVYTREWNHMCHQHSNSYIMYNILPIY

SEQ ID
1/VACW
SPI-
MDIFKELILKHTDENVLISPVSILSTLSILNHGAAGST
Outside

NO:137

AAO89484.1
AEQLSKYIENIVINENTPDDNNDMDVDIPYCATLATA
Deletion

NKIYGSDSIEFHASFLQKIKDDFQTVNFNNANQTKE

LINEWVKTMTNGKINSLLTSPLSINTRMTVVSAVHF

KAMWKYPFSKHLTYTDKFYISKNIVTSVDMMVSTE

NNLQYVHINELFGGFSIIDIPYEGNSSMVIILPDDIEGI

YNIEKNITDEKFKKWCGMLSTKSIDLYMPKFKVEM

TEPYNLVPILENLGLTNIFGYYADFSKMCNETITVEK

FLHTTFIDVNEEYTEASAVTGVFMTNFSMVYRTKV

YINHPFMYMIKDNTGRILFIGKYCYPQ

SEQ ID
Cl3L/V
AAO89485.1
MMIYGLIACLIFVTSSIASPLYIPVIPPISEDKSFNSVE
Outside

NO:138
ACWR2

VLVSLFRDDQKDYTVTSQFNNYTIDTKDWTIGVLST
Deletion

06

PDGLDIPLTNITYWSRFTIGRALFKSESEDIFQKKMSI

LGVSIECKKSSTLLTFLTVRKMTRVFNKFPDMAYYR

GDCLKAVYVTMTYKNTKTGETDYTYLSNGGLPAY

YRNGVDG

SEQ ID
VACWR
AAO89486.1
MKLFTQNDRYFGLLDSCTHIFCITCINIWHKTRRETG
Outside

NO:139
207

ASDNCPKRTRFRNITMSKFYKLVN
Deletion

SEQ ID
p28/VA
AA089487.1
MEFDPAKINTSSIDHVTILQYIDEPNDIRLTVCIIRNIN
Outside

NO:140
CWR208

NITYYINITKINTHLANQFRAWKKRIAGRDYMTNLS
Deletion

RDTGIQQSKLTETIRNCQKNRNIYGLYIHYNLVINVV

IDWITDVIVQSILRGLVNWYIANNTYTPNTPNNTTTI

SELDIIKILDKVEDVYRVSKEKECGKYEVVYSKR

SEQ ID
C10L/V
AAO89488.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO:141
ACWR2

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDSTIT
Deletion

09

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPICGGKIKLYHPTGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSMTTIHYNDDVDIDIHTDK

NGKELCYCYITIDDHYLVD'VETIGVI'VNRSGKCLLV

NNHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFS

LTNDDNRNIAWDTDKLDDDTDIWTPVTEDDYKFLS

RLVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRF

YFNMPK

SEQ ID
VGF-
AAO89489.1
MSMKYLMLLFAAMIIRSFADSGNAIETTSPEITNATT
Outside

NO:142
1/VACW

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

R210

GYTGIRCQHVVLVDYQRSENPNTTTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTKLPIQDMWP

SEQ ID
VACWR
AAO89490.1
MDEIVRIVRDSMWYIPNVFMDDGKNEGHVSVNNV
Inside

NO:143
211

CHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHC
Deletion

YTMNTRFNPSVLIULLHHGMRNFDSKDEKGHHYQS

ITRSLIY

SEQ ID
C20LJV
AAO89491.1
MLFYLEEPIRGYVIILIVHPSWNDCATGHILIMLLNW
Inside

NO:144
ACWR2

HEQKEEGQHLLYLFIKHNQGYTLNILRYLLDRFDIQ
Deletion

12

KDEYYNTAFQNCNNNVASYIGYDINLPTKDGIRLG

V

SEQ ID
VACWR
AAO89492.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO:145
213

IHHRLKVSLPMIKSLFYKMSLPTTITT
Deletion

SEQ ID
VACWR
AAO89493.1
MYDDLIEQCHLSMERKSKLVDKALNKLESTIGQSRL
Outside

NO:146
214

SYLPPEIMRNII
Deletion

SEQ ID
B28R/V
AAO89494.1
MKSVLYSYILFLSCIIINGRDIAPHAPSDGKCKDNEY
Inside

NO:147
ACWR2

KRHNLCPGTYASRLCDSKTNTQCTPCGSGTFTSRNN
Deletion

15

HLPACLSCNGRRDRVTRLTIESVNALPDIIVFSKDHP

DARHVFPKQNVE

SEQ ID
VACWR
AAO89495.1
MDSLRPVVVVNWIQINFHIDIVKGITGYGFAFKGRD
Outside

NO:148
216

GVRKSETTRRTDDVSGYSVSYSTFCLGNTCLASG
Deletion

SEQ ID
VACWR
AAO89496.1
MWKLKIQLTTTTGLSESISTSELTITMNHKDCNPVF
Outside

NO:149
217

REEYFSVLNKVATSGFFTGERCAL
Deletion

SEQ ID
B29R/V
AA089497.1
MHVPASLQQSSSSSSSCTEEENKHHMGIDVIIKVTK
Inside

NO:150
AC2R2

QDQTPTNDKKQSVTEITESESDPDPEVESEDDSTSV
Deletion

18(44%

EDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSISESAD

5′)

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 SequenceLocation

SEQ ID
TF3L
AAF34083.1
MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPT
Inside

NO:151
(41% 3′)

EMVDVSMMSMYGKAFNHASVKESFGNFSIIELPYV
Deletion

GDTSMMVILPDKIDGLESIEQNLTDTNFKIONCDFM

DAMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGST

GDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAA

TCALVSDCASTITNEFCVDHPFIYVIRHVDGIULFVG

RYCSPTTNC

SEQ ID
TB15R
AAF34084.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO:152

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
ORFL
AAF34085.1
MYNSSIHTPEYDVIIHVIEHLKHHKQCVQTVTSGMV

NO:153

FTSPVSSSKTKSDDGRNLSDGFLLIRYITTDDFCTIF

DIIPRHIFYQLANVDEH

SEQ ID
TB16R
AAF34086.1
MELENEPVILPCPQINTLSSGYNILDILWEKRGADND
Inside

N0:154

RIIPIDNGSNMLILNPTQSDSGIYKITTNETYCDMMS
Deletion

LNLTIVSVLESNIDLISYPQIVNERSTGEMVCPNINAF

IASNVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRK

NDAGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPS

TMQLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWIS

NGMYYEEDDGDGNGRISVANKIYMTDKRRVITSRL

NINPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
ORFL
AAF34087.1
MVIIPGVRCLSLLFLRRRCPLHIISAFTLLAINALILGH

NO:155

TISPVDLSFTKGYEIRSIFDSKTDTIVKFNDIMSQ

SEQ ID
TB17L
AAF34088.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO:156

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

CKINNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQ

IEAPLSGNVLVYTFPNINKRIPGYIHVNIEGCIDGMIY

INSSKFACVLKLHRSMYRIPPFPIDKSCCSQYTNGDI

EIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFN

NIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVNH

SNTFVNCLLEDNV

SEQ ID
TB18R
AAF34089.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO:157

ELDFVVKNYDLNRRHPVTGYTALHCYLYNNYFTN
Deletion

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLLHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPMKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKKNN

HGLTPILCYITSDYIQHDILVMLIHHYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNINIYTRYE

GKTLLHVACEYNNTQVIDYLIR1NGDINALTDNNKH

ATQLIIDNKENSPYTINCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYDRHV1CNRNSKT

YRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDT

TLYAVLRCHNSRKLRRYLTELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
TB19R
AAF34090.1
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFN
Inside

NO:158

KMRDTLPAKDSKWLNPACMFGGTMNDIAALGEPF
Deletion

SAKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVS

NKRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDC

VQGIVRSHIKKPPSCIPKTYELGTHDKYGIDLYCGIL

YAKHYNNITWYKDNKEINIDDIKYSQTGKELIIHNPE

LEDSGRYDCYVHYDDVRIKNDIVVSRCKILTVIPSQ

DHRFKLILDPKINVTIGEPANITCTAVSTSLLIDDVLI

EWENPSGWLIGFDFDVYSVLTSRGGITEATLYFENV

TEEYIGNTYKCRGHNYYFEKTL1TTVVLE

SEQ ID
ORFR
AAF34091.1
MHVIDVDVRLYMSTFIIIDQSTENTSIDTTVTINHYL

NO:159

AIMIUIMNIIMMIMIELV

SEQ ID
TB21R
AAF34092.1
LKNVECVDIDSTITFMKYDPNDDNKRTCSNWVPLT

NO:160

NNYMEYCLVIYLETPKGGKIKLYHPTGNIKSDKDI

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′)

QDQTPTNDKKQSVTEITESESDPDPEVESEDDSTSV
Deletion

EDVDPPTTYYSIIGGGLRMNFGFTKCPQIKSISESAD

GNTVNARLSSVSPGQGKDSPAITHEEALAMIKDCEV

SIDIRCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGST

VDTKCVKNLEFSVRIGDMCKESSELEVKDGFKYVD

GSASEGATDDTSLIDSTKLKACV

TB2OR

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_D
AEY74905.1
MNHCLLAISAVYFKAKWLTPFEKEFTSDYPFYVSPT
Inside

NO:163
PP20_20

EMVDVSMMSMYGKAFNHASVKESFGNFSHELPYV
Deletion

7

GDTSMMVILPDKIDGLESIEQNLTDTNFKKWCDFM

DAMFIDVHIPKFKVTGSYNLVDTLVKSGLTEVFGST

GDYSNMCNLDVSVDAMIHKTYIDVNEEYTEAAAA

TCALVSDCASTVTNEFCADHPFIYVIRHVDGKILFV

GRYCSPTTNC

SEQ ID
VAC_D
AEY74906.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO:164
PP10_20

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

8

KHMPWDDVKDCAEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
VAC_D
AEY74907.1
MSILPVIFLSIFFYSSFVQTFNASECIDKGQYFASFME
Inside

NO:165
PP12_20

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

9

PIDNGSNMLILNPTQSDSGIYKITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLIINKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYGGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDTDVFWISNG

MYYEEDDGDGDGRISVANKIYMTDKRRVITSRLNI

NPVKEEDATTFTCMAFTIPSISKTVTVSIT

SEQ ID
VAC_D
AEY4908.1
MSRKFMQVYEYDREQYLDEFIEDRYNDSFITSPEYY
Inside

NO:166
PP20_21

SAEKYMCRYTTLNHNCVNVRRCALDSKLLHDIITN
Deletion

0

CKIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVL

YPVIFITHTSTRNLDKVSVKTYKGVKVKKLNRCAD

HAIVINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQ

IEAPLSGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIY

INSSKFACVLKLHRSMYRIPPFPIDKSCCSQYTNDDI

EIPIHDLIKDVAIFKNKETVYYLKLNNKTIARFTYFN

NIDTAITQEHEYVKIALGIVCKLMINNMHSIVGVNH

SNTFVNCLLEDNV

SEQ ID
VAC_D
AEY74909.1
MSRRLIYVLNINRESTHKIQENEIYTYFSHCNIDHTST
Inside

NO:167
PP20_21

ELDFWKNYDLNRRQPVTGYTALHCYLYNNYFTN
Deletion

1

DVLKILLNHGVDVTMKTSSGRMPVYILLTRCCNISH

DVVIDMIDKDKNHLSHRDYSNLLLEYIKSRYMLLK

EEDIDENIVSTLLDKGIDPNFKQDGYTALHYYYLCL

AHVYKPGECRKPITIKKAKRIISLFIQHGANLNALDN

CGNTPFHLYLSIEMCNNIHMTKMLLTFNPNFKKNN

HGLTPILCYITSDYIQHDILVMLIHEIYETNVGEMPID

ERRIIVFEFIKTYSTRPADSITYLMNRFKNINIYTRYE

GKTLLHVACEYNNTHVIDYLIRINGDINALTDNNKH

AIQLIIDNKENSPYTIDCLLYILRYIVDKNVIRSLVDQ

LPSLPIFDIKSFEKFISYCILLDDTFYNRHVRNRNSKT

YRYAFSKYMSFDKYDGIITKCHDETMLLKLSTVLDT

TLYAVLRCHNSKKLRRYLNELKKYNNDKSFKIYSNI

MNERYLNVYYKDMYVSKVYDKLFPVFTDKNCLLT

LLPSEIIYEILYMLTINDLYNISYPPTKV

SEQ ID
VAC_D
AEY74910.1
MTMKMMVHIYFVSLSLLLLLFHSYAIDIENEITEFFN
Inside

NO:168
PP20_21

KMRDTLPAKDSKWLNPACMFGGTMNDIATLGEPFS
Deletion

2

AKCPPIEDSLLSHRYKDYVVKWERLEKNRRRQVSN

KRVKHGDLWIANYTSKFSNRRYLCTVTTKNGDCV

QGIVRSHIRKPPSCIPKTYELGTHDKYGIDLYCGILY

AKHYNNITWYKDNKEINIDDIKYSQTGKKLIIHNPEL

EDSGRYDCYVHYDDVRIKNDIWSRCKILTVIPSQD

HRFKLKRNCGYASN

SEQ ID
VAC_D
AEY74911.1
MRQIKINGTDMLTVMYMLNKPTKKRYVNNPIFTD
Not

NO:169
PP10_21

WANKQYKFYNQIIYNANKLIEQSKKIDDMIEEVSID
Present

7

DNRLSTLPLEIRHL1FSYAFL

SEQ ID
VAC_D
AEY74912.1
MSSKGGSGGMWSVFIHGHDGSNKGSKTYTSGGGG
Outside

NO:170
PP10_21

MWGGGSSSGVNGGVKSGTGKI
Deletion

8

SEQ ID
VAC_D
AEY74913.1
MFDYLENEEVALDELKQMLRDRDPNDTRNQFKNN
Outside

NO:171
PP10_21

ALHAYLFNEHCNNVEVVKLLLDSGTNPLRKNWRQ
Deletion

9

LPH

SEQ ID
VAC_D
AEY74914.1
MLKLKDIAMALLEATGFSNINDFNIFSYMKSKNVD
Outside

NO:172
PP10_22

VDLIKVLVEHGFDLSVKCENHRSVIENYVMTMILFI
Deletion

0

ENGCSVLYEDEY

SEQ ID
VAC_D
AEY74915.1
MKGIDNTAYSYIDDLTCCTRGIMADYLNSDYRYNK
Outside

NO:173
PP10_22

DVDLVKLFLENGKPHGIMCSIVPLVVRNDKETIFLILK
Deletion

1

TMNSDVLQHILIEYMTFGDIPLVEYGTWNKEAIHG

YFRNINIDSYTMKYLL1UCEGRCHQLSRLDTYVNPT

MDVIISTLIHTKRVFVTCLMLAQFLVL

SEQ ID
VAC_D
AEY74916.1
MPSIISIGHLCKSNYGCYNFYTYTYKKGLCDMSYAC
Outside

NO:174
PP10_22

PILSTINKLPYLKDINMIDKRGETLLHKAVRYNKQS
Deletion

2

LVSLLLESGSDVNIRSNNGYTCIAIAINESKNIELLKM

LLCHKPTLDYVIDSLREISNIVDNDYAIKQCIKYAMII

DDCTSSKIPEFISQRYNDYIDLCN

SEQ ID
VAC_D
AEY49171.1
MKKIMVGGNTMFSLIFTDHGAIUIHRYANNPELREY
Outside

NO:175
PP10_22

YELKQNKIYVEAYDIISNAIVKHDRIHKTIESVDDNT
Deletion

3

YISNLPYTIKYIUFEQQ

SEQ ID
VAC_D
AEY74918.1
MRILFLIAFMYGCVHSYVNAVETKCSNLDIVTSSGE
Outside

NO:176
PP10_22

FHCSGCVEHMPNFSYMYWLAKDMRSDEDAKFIEH
Deletion

4

LGEGIKEDETVRTIDGRIVTLQKVLHVTDTNKFAHY

RFTCVLTTIDGVSKKNIWLK

SEQ ID
VAC_D
AEY74919.1
MKLFTQNDRYFGLLDSCNHIFCITCINIWFIKTRRET
Outside

NO:177
PP10_22

GASDNCPKRTRFRNITMSKFYKLVN
Deletion

5

SEQ ID
VAC_D
AEY74920.1
MHYPKYYINITKINPHLANQFRAWKKRIAGRDYMT
Outside

NO:178
PP10_22

NLSKDTGIQQSKLYVTVKKIETYMVYIYTTI
Deletion

6

SEQ ID
VAC_D
AEY74921.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO:179
PP20_22

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDNTIT
Deletion

7

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPKGGKIKLYHPTGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSITTIHYNDDVDIDIHTDKN

GKELCYCYITIDDHYLVDVETIGVIVNRSGKCLLVN

NHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFSL

TNDDNRNIAWDTDKLDDDTDIWTPVTENDYKFLSR

LVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRFy

FNMFK

SEQ ID
VAC_D
AEY74922.1
MLINYLMLLFAAMIIRSFADSGNAIETTLPEITNATT
Outside

NO:180
PP20_22

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

8

GYTGIRCQHVVLVDYQRSEKPNITTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTNKLPLQDMVVP

SEQ ID
VAC_D
AEY74923.1
MDIFKELIVKHPDENVLISPVSILSTLSILNHGAAGST
Outside

NO:181
PP20_22

AEQLSKYIENMNENTPDDIUCDDNNDMDVDIPYCAT
Deletion

9

LATANKIYGSDSIEFHASFLQKIKDDFQTVNFNNAN

QTKELINEWVKTMTNGKINSLLTSPLSINTRMTVVS

AVHFKAMWKYPFSKHLTYTDKFYISKNIVTSVDMM

VGTENNLQYVHINELFGGFSIIDIPYEGNSSMVIILPD

DIEGIYNIEKNITDEKFIUCWCGMLSTKSIDLYMPKF

KVEMTEPYNLVPILENLGLTNIFGYYADFSKMCNET

ITVEKFLHTTFIDVNEEYTEASAVTGVFMTNFAMVY

RTKVYINHPFMYMIKDTTGRILFIGKYCYPQ

MMIYGLIACLIFVTSSIASPLYIPVIPPITEDKSFNSVE
Outside

SEQ ID
C13L/V
AEY74924.1
VLVSLFRDDQKDYTVISQFNNYTIDTKDWTIGVLST
Deletion

NO:182
AC_DPP

PDGLDIPLTNITYWSRFTIGRALFKSESEDIFQKKMSI

20_230

LGVSIECKKSSTLLTFLTVRKMTRVFNKFPDMAYYR

GDCLKAVYVTMTYKNTKTGETDYTYLSNGGLPAY

YRNGVDG

SEQ ID
VAC_D
AEY74925.1
MNLQKLSLAIYLTATCSWCYETCIRKTALYHDIQLE
Outside

NO:183
PP20_23

HVEDNKDSVASLPYK
Deletion

1

SEQ ID
VAC_D
AEY74926.1
MSLESFIITITNNNSSTNIDNMCHLYVKVCPSSLLFR
Inside

NO:184
PP20_23

LFVECCDINKLVEGTTPLHCYLMNEGFESSVLKNLL
Deletion

2

KEYVMTSITQIFNS

SEQ ID
VAC_D
AEY74927.1
MISLSFLIHNPLKKWKLKPSISINGYRSTFTMAFPCA
Inside

NO:185
PP20_23

QFRPCHCHATKDSLNTVADVRHCLTEYILWVSHRW
Deletion

3

THRETAGPLYRLLISFRIDATELFGGELKDSLPWDNI

DNCVEIIKCFIRNDSMKTAEELRAIIGLCTQSAIVSGR

VFNDKYIDILLMLRKILNENDYLTLLDHIRTAKY

SEQ ID
VAC_D
AEY74928.1
MIAFIIFREIGHSTRIAMDCTCILCRLLDEDVTYKKIK
Inside

NO:186
PP20_23

LEIETCHNLSKIIIDRAGNNALHCYVFNKCDTDIKIV
Deletion

4

RLLLSRGVERLCANNEGLTPLGVYSKHRYVKSQIVH

LLISSYSNSSNELKSNINDFDLSSDNIDLRLLKYLIVD

KRIRPSKNTNYAINSLGLVDIYVTTPNPRPEVLLWLL

KSECYSTGYVFRTCMYNSDMCKNSLHYYISSHRES

QSLSKDVIKCLINNNVSIHGRDEGGSLPIQYYWSFST

IDIEIVKLLLIKDVDTCRVYDVSPILEAYYLNKRFRV

TPYNVDMEIVNLLIERRHTLVDVMRSITSYDSREYN

HYHDNILKRFRQQDESIVQAMLINYLHYGDMVVRC

MLDNGQQLSSARLLC

SEQ ID
VAC_D
AEY74929.1
MYGLILSRFNNCGYHCYETILIDVFDILSKYMDNID
Inside

NO:187
PP20_23

MIDNENKTLLYYAVDVNNIQFAKRLLEYGASVTTS
Deletion

5

RSIINTAIQKSSYRRENKTKLVDLLLSYHPTLETMID

AFNRDIRYLYPEPLFACIRYALILDDDFPSKVKYDIS

GRHKELKRYRVDINRMKNAYISGVSMFDILFKRSK

RHRLRYAKNPTSNGTKKN

SEQ ID
VAC_D
AEY74930.1
MSRINITKKIYCSVFLFLFLSYISNYEKVNDEMYEMG
Inside

NO:188
PP20_23

EMDEIVSIVRDSMWYIPNVFMDDGKNEGHVSVNNV
Deletion

6

CHMYFTFFDVDTSSHLFKLVIKHCDLNKRGNSPLHC

YTMNTRFNPSVLKILLHHGMRNFDSKDDHYQSITRS

LIY

SEQ ID
VAC_D
AEY74931.1
MEQTLTRLHTYLQQYTKHSPRVVYALLSRGYVIILI
Inside

NO:189
PP20_23

VHPSWNDCATGHILIMLLNWHEQKEEGQHLLYLFI
Deletion

7

KHNQGYTLNILRYLLDRFDIQKDEYYNTAFQNCNN

NVASYIGYDINLPTKDGIRLGV

SEQ ID
VAC_D
AEY74932.1
MLPHTSDTTSTFRLKTVFDLVFENRNIIYKADVVNDI
Inside

NO:190
PP20_23

IHHRLKVPMIKSLFYKMSEFSPYDDYYVKKILAYCL
Deletion

8

LRDESFAELHSKFCLNEDYKSVFMKNISFDKIDSIIV

T

SEQ ID
VAC_D
AEY74933.1
MHHPMESVKTTNTNAIKVREHTLPDYANTQCTPC
Inside

NO:191
PP20_23

GSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNAL
Deletion

9

PDIIVFSKDHPDARHVFPKQNVE

SEQ ID
VAC_D
AEY74934.1
MHVPASLQQSSSSCTEEENKHHMGIDVIIKVTKQDQ
Inside

NO:192
PP20-24

TPTNDKKQSVTEITESESDPDPEVESEDDSTSVEDV
Deletion

1(43%

DLPTTYYSIIGGGLRMNFGFIXCPQIKSISESADGNT

5′)

VNARLSSVSPGQGKDSPAITHEEALAMIKDCEVSIDI

RCSEEEKDSDIKTHPVLGSNISHKKVSYEDIIGSTIVD

TKCVKNLEFSVRIGDMCKESSELEVKDGFKYVDGS

ASEGATDDTSLIDSTKLKACV

SEQ ID
VAC_D
AEY74919.1
MKLFTQNDRYFGLLDSCNHIFCITCINIVVHKTRRET
Outside

NO:193
PP10_22

GASDNCPKRTRFRNITMSKFYKLVN
Deletion

5

SEQ ID
VAC_D

MHYPKYYINITKINPHLANQFRAVVKKRIAGRDYMT
Outside

NO:194
PP10_22
AEY74920.1
NLSKDTGIQQSKLYVTVKKIETYMVYIYTTI
Deletion

6

SEQ ID
VAC_D
AEY7492.1
MDIYDDKGLQTIKLFNNEFDCIRNDIRELFKHVTDS
Outside

NO:195
PP20_20

DSIQLPMEDNSDIIENIRKILYRRLKNVECVDIDNTIT
Deletion

7

FMKYDPNDDNKRTCSNWVPLTNNYMEYCLVIYLE

TPKGGKIKLYHPIGNIKSDKDIMFAKTLDFKSKKVL

TGRKTIAVLDISVSYNRSITTIHYNDDVDIDIHTDKN

GKELCYCYITIDDHYLVDVETIGVIVNRSGKCLLVN

NHLGIGIVKDKRISDSFGDVCMDTIFDFSEARELFSL

TNDDNRNIAWDTDKLDDDTDIWTPVTENDYKFLSR

LVLYAKSQSDTVFDYYVLTGDTEPPTVFIFKVTRFY

FNMPK

SEQ ID
VAC_D
AEY74922.1
MLINYLMLLFAAMIIRSFADSGNAIETTLPEITNATT
Outside

NO:196
PP20:22

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSH
Deletion

8

GYTGIRCQHVVLVDYQRSEKPNTTTSYIPSPGIMLV

LVGIIIITCCLLSVYRFTRRTN1KLPLQDMVVP

SEQ ID
VAC_D
AEY74933.1
MHHPMESVKTTNTNAIKVREHTLPDYANTQCTPC
Inside

NO:197
PP20_23

GSGTFTSRNNHLPACLSCNGRRDRVTLLTIESVNAL
Deletion

9

PDIIVFSKDHPDARHVFPKQNVE

SEQ ID
VAC_D
AEY74934.1
MHVPASLQQSSSSCTEEENKHHMGIDVIIKVTKQDQ
Inside

NO:198
PP20-24

TPTNDKKQSVTEITESESDPDPEVESEDDSTSVEDV
Deletion

1(43%

DLPTTYYSIIGGGLRMNFGFTKCPQIKSISESADGNT

5′)

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
B15RfLi
ABD52695.1
MTANFSTHVFSPQHCGCDRLTSIDDVKQCLTEYIYW
Inside

NO:200
st191

SSYAYRNRQCAGQLYSTLLSFRDDAELVFIDIRELV
Deletion

KNMPWDDVKDCTEIIRCYIPDEQKTIREISAIIGLCA

YAATYWGGEDHPTSNSLNALFVMLEMLNYVDYNII

FRRMN

SEQ ID
List192
ABD52696.1
MSILPVIFLPIFFYSSFVQTFNAPECIDKGQYFASFME
Inside

NO: 201

LENEPVILPCPQINTLSSGYNILDILWEKRGADNDRII
Deletion

PIDNGSNMLILNPTQSDSGIYKITTNETYCDMMSLN

LTIVSVSESNIDLISYPQIVNERSTGEMVCPNINAFIAS

NVNADIIWSGHRRLRNKRLKQRTPGIITIEDVRKND

AGYYTCVLEYIYRGKTYNVTRIVKLEVRDKIIPSTM

QLPDGIVTSIGSNLTIACRVSLRPPTTDADVFWISNG

MYYEEDDGDGNGRISVANKIYMTDKRRVITSRLNI

SEQ ID
B17L/Lis
ABD52698.1
NPVKEEDATTFTCMAFTIPSISKTVTVSIT
Inside

NO:202
t193

MSRKFMQVYEYDREQYLDEFIEDRYNDSF1TSPEYY
Deletion

SAEKYMCRYTTLNHNCINVRRCALDSKLLHDIITNC

KIYNNIELVRATKFVYYLDLIKCNWVSKVGDSVLYP

VIFITHTSTRNLDKVSVKTYKGVKVKKLNRCADHAI

VINPFVKFKLTLPNKTSHAKVLVTFCKLRTDITQIEA

PLSGNVLVYTFPDINKRIPGYIHVNIEGCIDGMIYINS

SKFACVLKLHRSMYRIPPFPIDKSCCSQYTNDDIEIPI

HDLIKDVAIFKNKETVYYLKLNNKTIARFTYFNNID

TAITQEHEYVKIALGIVCKLMINNMHSIVGVNHSNT

FVNCLLEDNV

SEQ ID
crmE/Lis
ABD52700.1
MTKVIIILGFLIINTNSLSMKCEQGVSYYNSQELKCC
Not

NO:203
t195

KLCKPGTYSDHRCDKYSDTKGHCPSDTFTSIYNRSP
Present

WCHSCRGPCGTNRVEVTPCTPTTNRKHCDSNSYCL

LKASDGNCVTCAPKTKCGRGYGKKGEDEMGNTK

KKCRKGTYSDIVSDSDQCKPMTR

SEQ ID
L6/List1
ABD52701.1
MAMPSLSACSSIEDDFNYGSSVASASVHIRMAFLRK
Not

NO:204
96

VYGILCLQFLLTTATTAVFLYFDCMRTFIQGSPVLIL
Present

ASMFGSIGLIFALTLHRHKHPLNLYLLCGFTLSESLT

LASVVTFYDVHVVMQAFMLTTAAFLALTTYTLQSK

RDFSKLGAGLFAALWILILSGLLGIFVQNETVKLVLS

AFGALVFCGFIIYDTHSLIHKLSPEEYVLASINLYLDII

NLFLHLLQLLEVSNKK

SEQ ID
List197
ABD52704.1
MASPCAKFRPCHCHATKDSLNTVADVRHCLTEYIL
Inside

NO:205

WVSHRWTHRESAGSLYALLISFRTDATELFGGELKD
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

IHHRLKVSLPM1KSLFYKMSLPTTITT
Deletion

SEQ ID
C23L/Lis
ABL63827.1
MKQYIVLACMCLAAAAMPASLQQSSSSSSSCTEEE
Inside

NO:208
t201

NKHHMGIDVIIKVTKQDQTPTNDKKQSVTEITESES
Deletion

(47% 5′)

DPDPEVESEDDSTSVEDVDPPTTYYSIIGGGLRMNF

GFTKCPQIKSISESADGNTVNARLSSVSPGQGKDSPA

ITHEEALAMIKDCEVSIDIRCSEEEKDSDIKTHPVLGS

NISHIUCVSYEDIIGSTIVDTKCVKNLEFSVRIGDMCK

ESSELEVKDGFKYVDGSASEGATDDTSLIDSTKLKA

CV

List198

A

List198

B

List199

A

List199

B

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 CopMDp3p “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, 786-0, 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). llumina 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 32 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. 35, 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 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 29-34.

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, 786-0, 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 72h 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 2h, the media was changed for overlay media as done for a plaque assay. At 48h 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 24h 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-Foxn1nu) 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) 24h 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 (Fluc) 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 24h 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 1×10⁷Vaccinia 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 its 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 (24h apart, first three dashed lines) 1×10⁷PFU doses into the tumor (intra-tumor). Mice treated with Anti-CTLA4 received five (24h apart, first five dashed lines) 100 μg doses of antibody i.p. Mice treated with Anti-PD1 received five (24h apart, last five dashed lines) 100 μg doses of antibody i.p. 24h 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, B7L, 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 inteterferon 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. 38. 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 107 pfu 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. 36, 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 (786-0). FIG. 37 Confluent monolayers of 1 million cells were infected at an MOI of 3 (3×10⁶PFU) for 18h 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 (CopMD5p3p-B8R+-IL2TM-FLT3-antiCTLA4) expressing three transgenes was evaluated in terms of transgene expression kinetics. Confluent monolayers of 786-0 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. 39.

Example 23 SKV Expressing Murine IL-12 p35 Membrane Bound (SKVm-3) has Greater Efficacy in Controlling Murine Tumors

The survival of mice treated with either SKV (CopMD5p3p-B8R+) or SKVm-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 SKVm-3. Although SKV virus extends survival of mice bearing CT26 colon tuomurs. SKVm-3 expression of IL-12 is able to induce remissions that lead to durable cures. See FIG. 40.

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 HeLa cells when compared to their parental wildtype and 3p major deleted strains. See FIG. 41. FIG. 42 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. 43. Mice were also examined for pox lesions 6 days post-injection. 5p3p vaccinia strains do not induce pox lesions compared to wildtype strains. FIG. 44.

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

MODIFIED ORTHOPOXVIRUS VECTORS

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

PCT Information

Provisional Applications (2)