Use of Triplex CMV Vaccine in CAR T Cell Therapy

Abstract

A method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE I (e4) and exon 5 of CMV protein IE2 (e5) either prior to or subsequent to administering the composition comprising a population of T cells to the patient is described.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 16/343,701, filed Apr. 19, 2019 which is a National Stage application under 35 U.S.C. .sctn. 371 of International Application No. PCT/US2017/057433, filed on Oct. 19, 2017, which claims the benefit of U.S. Provisional Application No. 62/410,372, filed on Oct. 19, 2016. The disclosures of the prior applications are incorporated by reference in their entirety.

BACKGROUND

[0002] Tumor-specific T cell based immunotherapies, including therapies employing engineered T cells, have been investigated for anti-tumor treatment. In some cases, the T cells used in such therapies do not remain active in vivo for a long enough period.

[0003] Over 70,000 new cases of non-Hodgkin lymphoma (NHL) are diagnosed each year in the United States with about 20,000 deaths due to NHL each year, representing the 5th leading cause of cancer deaths. The majority of these patients have widespread disease at the time of diagnosis and over two-thirds will suffer a recurrence after remission induction with cytotoxic chemotherapy and rituximab. Efforts to improve the survival of patients with recurrent lymphoma have focused mainly on the use of autologous hematopoietic cell transplant (HCT), which is curative in approximately half of good-risk patients, but confers a less than 15% 5-year event-free survival in patients with poor prognostic features. Allogeneic HCT provides a tumor-free stem cell graft, cells that have not been damaged by prior chemotherapy and the opportunity for graft-versus-lymphoma (GVL) effect, and has been increasingly applied in patients with relapsed NHL. Although relapse rates are improved over autologous HCT, allogeneic HCT is associated with both significant risks of transplant-related complications and also disease recurrence. Thus, there is an important need for the development of new therapies that can consolidate the tumor cytoreduction achieved with autologous or allogeneic HCT by eradicating the limited number of tumor cells surviving after autologous myeloablative and reduced intensity allogeneic conditioning. A Phase I clinical trial using ex vivo-expanded autologous central memory-enriched T cells (TCM) transduced with lentivirus expressing CD19-specific CAR has demonstrated the data safety and feasibility of CD19 CAR T cell therapy after HCT Wang et al., Blood 127:2980, 2016).

SUMMARY

[0004] Described herein is the use of a cytomegalovirus (CMV) Triplex Vaccine in combination with engineered T cells that both recognize a CMV antigen and express a chimeric antigen receptor target to an antigen expressed on normal B cells as well as on cancerous cells (CMV/CAR T cells) to treat a variety of cancers. The methods entail administering CMV/CAR T cells which recognize a tumor antigen (e.g., CD19) in addition to a CMV antigen to a patient. Subsequent to administration of the CMV/CAR T cells, a CMV Triplex Vaccine is administered to the patient. The vaccine can promote proliferation of the CMV/CAR T cells and enhance their anti-tumor activity. Thus, the methods can improve T cell resistance and provide a means by which to re-stimulate CAR T cells after relapse. In addition, the methods can provide more reliable engraftment and persistence in a low target-antigen setting (e.g., post-myeloablative HCT) with re-expansion of CAR T cells by CMV vaccine administration. The methods described herein also permit in vivo expansion of CMV-specific CAR T cells, instead of or in addition to ex vivo expansion, avoiding excessive T cell exhaustion that results in some cases from ex vivo manufacturing.

[0005] The CMV/CAR T cells can be prepared by a method comprising: (a) providing PBMC from a cytomegalovirus (CMV)-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen (e.g., pp65 or a mixture of IE1/IE2 overlapping peptides); (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV (e.g., treating them to create a population of cells that is enriched for stimulated cells specific for CMV relative to the untreated population of cells); (d) transducing at least a portion of the enriched population of cells with a vector (e.g., a lentiviral vector) expressing a CAR, thereby preparing T cells specific for CMV and expressing a CAR. In some cases, a CMV vaccine, for example, the CMV Triplex Vaccine, can be administered to the donor prior to harvest of the PBMC in order to increase the frequency of CMV-positive T cells.

[0006] The CMV Triplex Vaccine is a recombinant MVA expressing a fusion protein of two CMV antigens, IE1-exon4 and IE2-exon5 and CMV antigen pp65. The sequence encoding the fusion protein is inserted in the MVA deletion-II locus and the sequence encoding the CMV pp65 antigen is inserted into the MVA deletion-III locus. The CMV Triplex Vaccine is described in greater detail in U.S. Pat. No. 8,580,276, hereby incorporated by reference.

[0007] The methods described herein include: a method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) either prior to or subsequent to administering the composition comprising a population of T cells to the patient.

[0008] Described herein is a method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) either prior to or subsequent to administering the composition comprising a population of T cells to the patient.

[0009] In various embodiments: the step of administering a viral vector to the patient comprises administering recombinant MVA virus; expression of (i) CMV pp65 and (ii) the fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) is under the control of mH5 promoter; the patient is immunocompromised; the patient is immunocompetent; the patient is CMV-seronegative prior to treatment; the patient is CMV-seropositive prior to treatment; the patient received hematopoietic stem cells (HSC) from a CMV-positive or CMV-negative donor prior to administering the comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; and the CAR is targeted to CD19; administration of the viral vector occurs at least 5 days after treatment with the composition comprising a population of T cells; the viral vector is administered to the patient both prior to and subsequent to the administration of the composition comprising a population of T cells; the viral vector is administered to the hematopoietic stem cell donor prior to harvesting the stem cells; the viral vector is administered to the patient only prior to the administration of the composition comprising a population of T cells; the viral vector is administered to the patient only subsequent to the administration of the composition comprising a population of T cells; the viral vector is administered to the patient at least four times; the patient is suffering from non-Hodgkin's Lymphoma.

[0010] The CAR is selective can be selective for any antigen, for example: CD19, CS1, CD123, 5T4, 8H9, .alpha.v.beta.6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-.alpha., G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor 2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1), mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (RORI), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.

[0011] In certain embodiments: the CAR is selective for an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13.alpha.2, PSA, HER-2, EGFRv3, CEA, and C7R; the CAR comprises: a scFv selective for the selected non-CMV antigen; a hinge/linker region; a transmembrane domain; a co-signaling domain; and CD3 .xi. signaling domain; the co-signaling domain is selected from a CD28 co-signaling domain and a 4-IBB co-signaling domain; transmembrane domain is selected from a CD28 transmembrane domain and a CD4 transmembrane domain.

[0012] In various embodiments of the treatment method: the population of human T cells is autologous to the patient; the population of human T cells is allogeneic to the patient; the method reduces the risk of CMV infection; the method reduces CMV viremia and/or disease; the patient was CMV-immune prior to treatment and the method reduces the risk of CMV infection; the patient was not CMV-immune prior to treatment and the method reduces CMV viremia or disease.

[0013] In some embodiments, the step of providing a population of T cells expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) providing PBMC or a T cell subpopulation from a CMV-seropositive human donor; (b) exposing the PBMC or the T cell subpopulation to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR. In some embodiments, the step of treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV comprises treating the stimulated cells to produce a population of cells enriched for cells expressing an activation marker.

[0014] In some embodiments, the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a human donor to convert a CMV-seronegative human donor to one containing T cells responsive to CMV antigens pp65, IE1 and IE2; (b) obtaining PBMC from the CMV-seropositive human donor; (c) exposing the PBMC to at least one CMV antigen; (d) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (e) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

[0015] In some embodiments, the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

[0016] In the case of patients who have received HSC transplant, in some embodiments, the viral vector is administered to the patient or the hematopoietic stem cell transplant donor at least twice subsequent to the administration of the composition comprising a population of T cells, the hematopoietic stem cells were autologous to the patient; and the hematopoietic stem cells were allogenic to the patient.

[0017] Also described is a method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen, the method comprising: (a)) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

[0018] Also described is a method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen, the method comprising: a)) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

[0019] In various embodiments of the of the methods for producing T cells, the method further comprises expanding the population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

[0020] In various embodiments of the of the methods for producing T cells: the activation marker is IFN-.gamma. or other activation marker such as CD137, CD107 or other cytokines; the CMV antigen is pp65 protein or an antigenic portion thereof; the CMV antigen comprises two or more different antigenic CMV pp65 peptides; the step of transducing the enriched population of cells does not comprise CD3 stimulation; the step of transducing the enriched population of cells does not comprise CD28 stimulation; the step of transducing the enriched population of cells does not comprise CD28 stimulation or CD3 stimulation; the step of transducing the enriched population of cells does not comprise exposing the cells to an anti-CD28 antibody or an anti-CD3 antibody; the enriched population of cells is at least 40% IFN-.gamma. positive, at least 20% CD8 positive, and at least 20% CD4 positive; the enriched population of cells are cultured for fewer than 10 days prior to the step of transducing the enriched population of cells with a vector encoding a CAR; the method further comprises expanding the CMV specific T cells expressing a CAR cells by exposing them to an antigen that binds to the CAR; the step of expanding the CMV-specific T cells expressing a CAR comprises exposing the cells to T cells expressing the antigen that binds the CAR; and the expansion takes place is the presence of at least one exogenously added interleukin.

[0021] In some cases, the method includes a step of preparing T cells specific for cytomegalovirus (CMV) and expressing a chimeric antigen receptor (CAR), the method comprising: (a) providing T cells (e.g., PBMC) from a cytomegalovirus CMV seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby preparing T cells specific for CMV and expressing a CAR. In various cases: the step of treating the exposed cells (e.g., using a selection step) to produce a population of cells enriched for stimulated cells specific for CMV comprises treating the stimulated cells to produce a population of cells enriched for cells expressing an activation marker (e.g., IFN-.gamma. of IL-13); the PBMC are cultured for less than 5 days (less than 4, 3, 2, 1 days) prior to exposure to the CMV antigen; the cells are exposed to the CMV antigen for fewer than 3 days (fewer than 48 hrs, 36 hrs, 24 hrs) the CMV antigen is pp65 protein or an antigenic portion thereof; the CMV antigen comprises two or more different antigenic CMV pp65 peptides; the step of transducing the enriched population of cells does not comprise CD3 stimulation; the step of transducing the enriched population of cells does not comprise CD28 stimulation; the step of transducing the enriched population of cells does not comprise CD3 stimulation or CD28 stimulation; the enriched population of cells is at least 40% (e.g., 50%, 60%, 70%) IFN-.gamma. positive, at least 20% (e.g., 25%, 30%, 35%) CD8 positive, and at least 20% (e.g., 25%, 30%, 35%) CD4 positive; the enriched population of cells are cultured for fewer than 10 (fewer than 9, 8, 7, 5, 3, 2) days prior to the step of transducing the enriched population of cells with a vector encoding a CAR. In some cases, the T cells are from a CMV positive donor and are exposed to a CMV antigen such as CMV pp65 or a mixture of CMV protein peptides (for example 10-20 amino acid peptides that are fragments of pp65) in the presence of IL-2 to create a population of stimulated cells. In some cases, the population of stimulated cells is treated to prepare a population of cells that express IFN-.gamma.. In some cases, the CMV/CAR T cells do not recognize an antigen from a second virus. For example, they do not recognize an Epstein-Barr virus antigen or an influenza virus antigen or an Adenovirus antigen.

[0022] In some cases, the method further comprises expanding the CMV specific T cells expressing a CAR (CMV/CAR T cells) by exposing them an antigen that binds to the CAR.

[0023] In some cases, the CMV/CAR T cells are not expanded ex vivo by exposure to an antigen that binds the CAR, by a CMV antigen or by exposure to exogenously added cytokines.

[0024] In some cases, the step of expanding the CMV-specific T cells expressing a CAR comprises exposing the cells to T cells expressing the antigen that bind the CAR (e.g., the expansion takes place is the presence of at least one exogenously added interleukin (e.g., one or both of IL-1 and IL-15) and a T cell expressing the antigen recognized by the CAR.

[0025] In various cases: the CAR is selective for an antigen selected from: CD19, CS1, CD123, 5T4, 8H9, .alpha.v.beta.6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor-.alpha., G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor 2, KDR, k-light chain, Lewis Y (LeY), L1 cell adhesion molecule, melanoma-associated antigen (MAGE-A1), mesothelin, Murine CMV infected cella, mucin-1 (MUC1), mucin-16 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-1, Oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (RORI), TAA targeted by mAb IgE, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.

[0026] In some cases, the CAR is selective for an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13.alpha.2, PSA, HER2, EGFRv3, CEA, and C7R.

[0027] In some cases: the CAR comprises: a scFv selective for the selected non-CMV antigen; a hinge/linker region; a transmembrane domain; a co-signaling domain; and CD3 .xi. signaling domain; the chimeric antigen receptor further comprises a spacer sequence located between the co-signaling domain and the CD3 .xi. signaling domain; the co-signaling domain is selected from a CD28 co-signaling domain and a 4-IBB co-signaling domain; the transmembrane domain is selected from a CD28 transmembrane domain and a CD4 transmembrane domain; the vector expressing the CAR expresses a truncated human EGFR from the same transcript encoding the CAR, wherein the truncated human EGFR lacks a EGF ligand binding domain and lacks a cytoplasmic signaling domain; the spacer sequence comprises or consists of 3-10 consecutive Gly; the hinge/linker region comprises at least 10 amino acids of an IgG constant region or hinge region; the IgG is IgG4; the hinge/linger region comprises an IgG4 CD3 domain; the hinge/linger region comprises an IgG4 Fc domain or a variant thereof; the hinge/linker region comprises or consists of 4-12 amino acids; and hinge/linker region is selected from the group consisting of: the sequence ESKYGPPCPPCPGGGSSGGGSG and the sequence GGGSSGGGSG.

[0028] In some cases, the CMV/CAR T cell population is a population in which at least 20% of the cells in the population are CD4+, in which at least 20% of the cells in the population are CD8+, or in which at least 60% of the cells in the population are IFN.gamma.+.

[0029] In various cases: the T cells are specific for CMV pp65; and the CAR binds an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13.alpha.2, PSA, HER2, EGFRv3, CEA, and C7R.

[0030] Also described is a method of treating a patient suffering from cancer comprising administering a composition comprising CMV/CAR T cells followed by administration of CMV Triplex Vaccine. In various cases: the population of human T cells are autologous to the patient; the population of human T cells are allogenic to the patient; the population of human T cells are autologous to the patient; the method further comprises administering to the patient at least two or at least three doses of a CMV Triplex Vaccine.

[0031] An amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence. An "amino acid substitution" or "substitution" refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

DESCRIPTION OF DRAWINGS

[0032] FIGS. 1A-1D depict the development of clinically feasible platform for derivation of CMV/CAR T cells and the schematic structure of a lentiviral vector expressing a CD19 CAR. (A) CMV-specific T cells from CMV immune HLA A2 donors were selected using IFN.gamma. capture after overnight stimulation with cGMP grade CMVpp65 protein. After selection, the cells were stained with antibodies specific to IFN.gamma., CD4, and CD8. The frequency of each population is presented after exclusion of dead cells with DAPI. (B) The selected cells were transduced with the second generation CD19CAR with a double mutation in the spacer, 24 hours after the IFN.gamma. capture. 7-10 days later, the transduced cells were stimulated with irradiated CD19 expressing NIH3T3 cells at 10:1 ratio (3T3:T cells) and the stimulation was repeated 7 days post the first stimulation. CAR expression was defined by cetuximab-biotin and streptavidin (SA) APC-Cy7 staining. Percentages of CAR.sup.+ cells are indicated in each histogram (filled gray), and based on subtraction of that stained with SA-APC-Cy7 alone (black line). (C) Growth of total cell number was determined by Guava Viacount at different time points. (D) Schematic diagram of 10039 nt lentiviral vector encoding a CD19 CAR. Within the 3183 nucleotide long CD19R:CD28:z(CO)-T2A-EGFRt construct, the CD19-specific scFv, IgG4 Fc spacer, the CD28 transmembrane and cytoplasmic signaling domains, three-glycine linker, and CD3z cytoplasmic signaling domains of the CD19R:CD28:z(CO) CAR containing the 2 point mutations, L235E and N297Q, in the CH2 portion of the IgG4 spacer (CD19R(EQ)), as well as the T2A ribosome skip and truncated EGFR sequences are indicated. The human GM-CSF receptor alpha signal sequences that drive surface translocation of the CD19R:CD28:z(CO) CAR and EGFRt are also indicated.

[0033] FIGS. 2A-2C depict the results of studies demonstrating that CMV/CAR T cells exhibit specific effector function after engagement with CD19' and CMVpp65.sup.+ tumors. (A) 7 days after the second CD19 Ag stimulation, T cells were stained with HLA A2 restricted pp65 tetramer, cetuximab-biotin, anti-CD8 and antibodies specific to central memory T cell surface markers. Percent positive cells are indicated after dead cell exclusion with DAPI, gating based on pp65 tetramer and cetuximab double-positivity, and isotype-matched stained samples. (B) Four-hour .sup.51Cr release assays were performed using the CMV/CAR T cells and indicated .sup.51Cr-labeled target cells at different effector: target (E:T) ratios. OKT3-expressing LCLs were used as positive controls, KG1A and U251T as negative controls. CD19' LCL and engineered pp65U251T cells were used as target for CD19 and CMV-specific T cells, respectively. Data from a representative donor is presented. (C) CMV/CAR T cells (105) were activated overnight with 105 LCL-OKT3, LCL, or KG1a in 96-well tissue culture plates and 10.sup.5 U251T and engineered pp65 expressing U251T cells (pp65U251T) in 24-well tissue culture plates. Supernatants were collected after overnight co-incubation of CMV/CAR T cells and stimulators. Cytokine levels with indicated stimulators (means.+-.SEM of triplicate wells) were determined using cytometric bead array.

[0034] FIG. 2D depicts the results of studies examining cytokine levels in the serum of CMV/CAR T cell treated tumor bearing mice. NSG mice were injected i.v. on day 0 with 2.5.times.106 GFPffluc+ LCL cells. Three days after tumor inoculation, recipient mice were administered i.v. with 2.times.106 CMV/CAR cells that underwent 2 rounds of CD19 stimulation. Vaccine was given by i.v. injection of peptide (pp65 or MP1) pulsed autologous T cells on day 14. Thirteen days post vaccine, serum of recipient mice was collected and levels of human cytokines were determined by cytometric bead array. Cytokine levels in the serum of untreated mice was used as baseline. Mean and SEMs from triplicates are presented.

[0035] FIGS. 3A-3C depicts the mH5-IEfusion-pZWIIA (GUS) plasmid DNA sequence (SEQ ID NO: A.

[0036] FIGS. 4A-4C depicts the mH5-pp65-pLW51(GUS) plasmid DNA sequence (SEQ ID NO: B.

[0037] FIGS. 5A-5C depicts an IE1 antigen and the IE2 antigen fusion protein (SEQ ID NO:C).

DETAILED DESCRIPTION

[0038] Described below are T cells specific for CMV and CD19. These CMV/CAR T cells were generated using a rapid and efficient method for generating and selecting CMV-specific T cells. The method, which employs IFN.gamma. capture of CMV-specific T cells, consistently and efficiently enriched CMV-specific T cells while preserving the broad spectrum of CMV repertoires. Moreover, the cells remained amenable to gene modification after a brief CMVpp65 stimulation, avoiding the need for CD3/CD28 bead activation prior to transduction. This is significant because CD3/CD28 activation can cause activation-induced cell death (AICD) of CMV-specific T cells. Engineering the bulk IFN.gamma.-captured T cells with a CD19CAR lentivirus followed by stimulation with CD19 antigen resulted in 50 to 70% of the CAR.sup.+ T cells responding to pp65 stimulation, representing the subset of functional CMV/CAR T cells. The CMV/CAR T cells exhibited specific cytolytic activity and secreted IFN.gamma., as well as proliferating vigorously after engagement of endogenous CMVpp65 T cell receptors or engineered CD19 CARs. Upon transfer into tumor bearing mice, the CMV/CAR T cells mediated cytokine released syndrome (CRS), which has been found to correlate with anti-tumor efficacy in the clinic.

[0039] While the CMV/CAR T cells described herein express a CAR targeted to CD19, the same methods can be used to generate CMV CAR T cells targeted to any desired antigen.

[0040] Efficient in vivo activation of virus-specific T cells through the TCR demands that viral antigens are processed and presented in a human leukocyte antigen (HLA)-dependent manner. This can be achieved by administering CMV Triplex Vaccine to the patient subsequent to administration of the CMV/CAR T cells.

[0041] The antitumor activity of CMV/CAR T cells can be enhanced as a consequence of proliferation following CMV peptide vaccination. This suggests that the cell dose of CMV/CAR T cells could be significantly decreased as compared to conventional CAR T cells, due to their potential to proliferate in vivo in response to vaccine, avoiding prolonged culture times and the risk of terminal differentiation.

[0042] In some cases, such as in the CMV/CAR T cells targeted to CD19 described herein, the CMV/CAR T cells also express a truncated EGFR (EGFRt). Cells expressing EGFRt can be killed by administration of an antibody, such a cetuximab, targeted to EGFR. This permits control and reduction of potential on/off-target toxicity.

[0043] The administration of CMV Triplex Vaccine subsequent to treatment with CMV/CD19 CAR T cells can augment the antitumor activity of adoptively transferred CMV/CD19 CAR T cells in several scenarios: 1) to salvage patients not achieving complete remission or relapsing after CAR T cell therapy, 2) vaccine boost when CD19 CAR T cells are failing to persist regardless of tumor responses at that time, 3) planned vaccination on days post-administration CD19 CAR T cells. There is also potential benefit of using the CMV/CAR T cells pre-emptively post-allogeneic HCT, both to eliminate minimal residual disease (MRD) and control CMV, potentially preventing reactivation of virus or undergoing expansion in response to latent CMV reactivation.

[0044] Moreover, administration of CMV Triplex Vaccine has the potential to profoundly impact the general field of adoptive T cell therapy, since by transducing a variety of tumor-directed CARs into CMV-specific T cells, it is possible to tailor this strategy to a wide range of malignancies and tumor targets.

Triplex Vaccine

[0045] CMV Triplex Vaccine is a recombinant MVA that expresses three CMV antigens, i.e., at least a portion or Immediate-Early Gene-1 (IE1), at least a portion of Immediate-Early Gene-2 (IE2) and at least a portion of pp65. The IE1 antigen and the IE2 antigen can be expressed a fusion protein, for example, a protein encoded by the nucleotide sequence of SEQ ID NO: C. Expression of the CMV antigens can be under the control of a modified H5 (mH5) promoter. A CMV Triplex Vaccine is fully described in U.S. Pat. No. 8,580,276 and in Wang et al. (Vaccine 28:1547, 2010)

[0046] The CMV Triplex Vaccine can express CMV pp65 and an CMV IE fusion protein (IEfusion). The IEfusion can include an antigenic portion of IE1 (e.g., Exon 4) and an antigenic portion of 1E2 (e.g., Exon 5), wherein the antigenic portions elicit an immune response when expressed by a vaccine. In one aspect, the IEfusion is has the sequence encoded by SEQ ID NO: C or another nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: C.

[0047] As explained in U.S. Pat. No. 8,580,276, the CMV Triplex Vaccine includes three of the best recognized antigens in the CD8 subset: pp65, IE1, and IE2. There is no region of homology greater than 5 amino acids between the major exons of both proteins. Individually, both antigens are recognized broadly by almost 70% of the general population (Sylwester et al. 2005). The divergent sequence of both IE/e4 and IE2/e5 used here predicts an entirely different subset of HLA binding peptides using publicly available Class I and II motif algorithms (Peters and Sette 2007). Human subjects that were evaluated for recognition of both IE1 and IE2 antigens were found in many instances to recognize one or the other but not both. Among the research subjects analyzed, 24% recognized IE2 with or without pp65 to the exclusion of IE. This result strongly suggests that the recognition elements for both antigens are unique, and by including both of them in the vaccine, the breadth of individuals with disparate HLA types that will recognize and develop an immune response to the vaccine is extended. The fusion of major exons from both antigens achieves the dual goal of reducing the number of separate inserts and eliminating the need for a third insert promoter. The advantages of this approach include placement of all vaccine antigens in one vector, and diminishing the dose of virus needed to attain sufficient immunity simultaneously against all of the included antigens.

[0048] Also as explained in U.S. Pat. No. 8,580,276, prior to conducting experiments with rMVA in clinical samples, the capacity for stimulation of both CD4+ and CD8+ T cells was assessed using the commercially available pp65 and IE1 library and a newly designed IE2 peptide library. Relationships among the T cell populations were similar to prior results: pp65 promotes a substantial CD4 and CD8 response in over 70% of participants, while IE1 and IE2 are recognized less frequently and mainly in the CD8+ T cell compartment. MVA expressing the IEfusion antigen with or without the pp65 antigen was evaluated in PBMC from healthy volunteers to establish their recognition properties using a fully human system. The results showed that the memory T cell expansion stimulated by the rMVA for both the IEfusion and pp65 antigens, followed the proportions found ex vivo for the same volunteers using the peptide library approach. While there was substantial amplification of the relevant T cell populations, the stimulation did not skew the population towards a particular subset or antigen specificity. The data also confirms that the IEfusion protein is processed and presented appropriately to stimulate existing T cell populations in a manner that maintains the phenotypic distribution as expected in the ex vivo analysis. The most rigorous evaluation of the processing of the rMVA for T cell response is using PBMC from transplant patients. PBMC from HCT recipients in all three risk categories were evaluated and an equivalently strong recognition of both rMVAs was found. In some cases, it was even more vigorous than in the PBMC of healthy adults. No interference with the recognition of the IE antigen by the co-expressed pp65 antigen was found from the same rMVA, which further confirms that the recognition of both antigens can take place at the same time and derived from the same vector.

[0049] In one embodiment, the nucleic acid sequence encoding vaccinia mH5 promoter has a sequence containing nucleotides 3075-3168 of SEQ ID NO: A or 3022-3133 of SEQ ID NO: B

Components of Chimeric Antigen Receptors

[0050] A wide variety of CAR have been described in the scientific literature. In general CAR include an extracellular antigen-binding domain (often a scFv derived from variable heavy and light chains of an antibody), a spacer domain, a transmembrane domain and an intracellular signaling domain. The intracellular signaling domain usually includes the endodomain of a T cell co-stimulatory molecule (e.g., CD28, 4-1BB or OX-40) and the intracellular domain of CD3.xi..

Spacer Region

[0051] The CAR described herein can include a spacer region located between the cancer antigen targeting domain (e.g., a CD19 ScFv, e.g., the scFv portion can include the CD19 targeted scFv sequence of a CD19-targeted CAR such as that described in Wang et al. 2016 Blood 127:2980-2990) and the transmembrane domain. A variety of different spacer regions can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can be used in the CARs described herein.

TABLE-US-00001 TABLE 1 Examples of Spacer Regions Name Length Sequence a3 3 aa AAA linker 10 aa GGGSSGGGSG (SEQ ID NO: 2) IgG4 hinge (S.fwdarw.P) 12 aa ESKYGPPCPPCP (SEQ ID NO: 3) (S228P) IgG4 hinge 12 aa ESKYGPPCPSCP (SEQ ID NO: 4) IgG4 hinge 22 aa ESKYGPPCPPCPGGGSSGGGSG (S228P) + linker (SEQ ID NO: 5) CD28 hinge 39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKP (SEQ ID NO: 6) CD8 hinge-48 aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACD (SEQ ID NO: 7) CD8 hinge-45 aa 45 aa TTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACD (SEQ ID NO: 8) IgG4 (HL-CH3) 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPR (includes EPQVYTLPPSQEEMTKNQVSLTCLVK 5228P in hinge) GFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9) IgG4 (L235E, 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPK N297Q) PKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHQAKTKPREEQFQ STYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10) IgG4 (S228P, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPK L235E, N297Q) PKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHQAKTKPREEQFQ STYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11) IgG4 (CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 12)

[0052] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one ore more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0053] The spacer region can also comprise a IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3).

[0054] The spacer region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K (SEQ ID NO:12). Thus, the entire spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:11). In some cases, the spacer has 1, 2, 3, 4 or 5 single amino acid changes (e.g., conservative changes) compared to those shown in Table 1. In some cases, the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).

[0055] Transmembrane Domain

[0056] A variety of transmembrane domains can be used in the. Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain is located carboxy terminal to the spacer region.

TABLE-US-00002 TABLE 2 Examples of Transmembrane Domains Name Accession Length Sequence CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 13) CD28 NM_006139 27 aa FWVLVVVGGVLACYSLLVTVAFI IFWV (SEQ ID NO: 14) CD28(M) NM_006139 28 aa MFWVLVVVGGVLACYSLLVTVAF IIFWV (SEQ ID NO: 15) CD4 M35160 22 aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 16) CD8tm NM_001768 21 aa IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 17) CD8tm2 NM_001768 23 aa IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 18) CD8tm3 NM_001768 24 aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 19) 41BB NM_001561 27 aa IISFFLALTSTALLFLLFFLTLR FSVV (SEQ ID NO: 20)

Costimulatory Domain

[0057] The costimulatory domain can be any domain that is suitable for use with a CD3 .xi. signaling domain. In some cases, the costimulatory domain is a CD28 costimulatory domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:23; LL to GG amino acid change double underlined). In some cases, the CD28 co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative and preferably not in the underlined GG sequence) compared to SEQ ID NO:23. In some cases the co-signaling domain is a 4-1BB co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

TABLE-US-00003 (SEQ ID NO: 24) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

In some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:24.

[0058] The costimulatory domain(s) are located between the transmembrane domain and the CD3.xi. signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3.xi. signaling domain.

TABLE-US-00004 TABLE 3 CD4 Domain and Examples of Costimulatory Domains Name Accession Length Sequence CD3.zeta. J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO: 21) CD28 NM_006139 42 aa RSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS (SEQ ID NO: 22) CD28gg* NM_006139 42 aa RSKRSRGGHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS (SEQ ID NO: 23) 41BB NM_001561 42 aa KRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24) OX40 42 aa ALYLLRRDQRLPPDAHKPPGGGS FRTPIQEEQADAHSTLAKI (SEQ ID NO: 25)

[0059] In various embodiments: the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present. In some embodiments there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. The costimulatory domain is amino terminal to the CD3.xi. signaling domain and in some cases a short linker consisting of 2-10, e.g., 3 amino acids (e.g., GGG) is positioned between the costimulatory domain and the CD3.xi. signaling domain.

CD3.xi. Signaling Domain

[0060] The CD3.xi. Signaling domain can be any domain that is suitable for use with a CD3.xi. signaling domain. In some cases, the CD3.xi. signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO:21). In some cases, the CD3 .xi. signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21.

Truncated EGFR

[0061] The CD3.xi. signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRE CVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDG PHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTN GPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO:28). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:28.

Example 1: Enrichment of CMV-Specific T Cells from PBMC of Healthy Donors after Stimulation with cGMP Grade CMVpp65 Protein

[0062] CMV-specific T cells were prepared from PBMC of healthy donors by stimulating the PBMC with cGMP grade CMVpp65 protein. Briefly, PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) from peripheral blood of consented healthy, HLA-A2 CMV-immune donors under a City of Hope Internal Review Board-approved protocol. PBMC were frozen for later use. After overnight rest in RPMI medium containing 5% Human AB serum (Gemini Bio Products) without cytokine, the PBMC were stimulated with current good manufacturing practice (cGMP) grade CMVpp65 protein (Miltenyi Biotec, Germany) at 10 ul/10.times.10.sup.6 cells for 16 hours in RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 2 mM L-glutamine (Irvine Scientific), 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, Irvine Scientific), 100 U/mL penicillin, 0.1 mg/mL streptomycin (Irvine Scientific) in the presence of 5U/ml IL-2 and 10% human AB serum. CMV-specific T cells were selected using the IFN.gamma. capture (Miltenyi Biotec, Germany) technique according to the manufacturer's instructions.

[0063] To demonstrate the consistency of this clinically feasible process, the selection was repeated five times using PBMC from three different donors. IFN.gamma.-positive T cells were consistently enriched from a baseline mean of 3.8% (range 1.8-5.6) to a post-capture mean of 71.8% (range 61-81) and contained polyclonal CD8+(34%) and CD4.sup.+ T cells (37%) after selection (FIG. 1A and FIG. 1C). Moreover, the selected CMV-specific T cells included both CD4 and CD8 subsets and represented the entire spectrum of CMV-specificity, showing responsiveness to CMVpp65 pepmix stimulation with broad recognition.

Example 2: Genetic Modification of Enriched CMV-Specific T Cells to Express CD19 CAR and In Vitro Expansion of the CMV/CAR T Cells

[0064] In the clinically adaptable procedure, IFN.gamma.-captured CMV-specific T cells were transduced 2 days after the selection, without OKT3 activation, using the second generation CD19RCD28EGFRt lentiviral construct containing the IgG4 Fc hinge region mutations (L235E; N297Q) that improve potency due to distortion of the FcR binding domain. The complete amino sequence of the this CD19 CAR is depicted in FIGS. 3A-3C. Starting seven days post lenti-transduction, the cells were stimulated on a weekly basis with 8000 cGy-irradiated, CD19-expressing NIH3T3 cells at a 1:10 ratio (T cells:CD19NIH 3T3). The percentage of CAR.sup.+ cells detected by cetuximab increased from 8% post transduction to 46% after 2 rounds of stimulation with a 120-150-fold total cell increase (FIG. 1B and FIG. 1D). Further details regarding the lentiviral construct, the CD19-expressing NIH3T3 cells and other materials and techniques used in the studies described herein are presented below.

Example 3: CMV/CAR T Cells Exhibited Specific Effector Function after Stimulation Through Pre-Defined Viral TCR and CD19CAR

[0065] Recapitulating our previous studies (23), the ex vivo expanded CMV-specific T cells possessed an effector phenotype and no longer expressed the central memory markers of the originally selected cells, such as CD62L, CD28, and IL-7Ra (FIG. 2A and FIG. 2D). However, levels of CD27 remained high, suggesting a greater proliferative potential that has been associated with greater clinical efficacy (24). To investigate CMV/CAR T cell effector function via signaling by both the endogenous CMV-specific TCR and the introduced CD19CAR, we evaluated response to engineered pp65-expressing U251T cells from HLA-A2 donors, and also allogeneic CD19.sup.+ LCLs, based on cytotoxicity, cytokine production and proliferation profiles. As expected, the expanded CMV/CAR T cells specifically lysed CD19.sup.+ LCLs with the same maximum killing levels as the OKT3-expressing LCL used as positive controls. Likewise, specific killing was also observed when pp65U25IT cells were used as targets as compared to parental U251T cells (FIG. 2B). Accordingly, after overnight stimulation, elevated IFN.gamma. secretion was observed after either CD19 or pp65 antigen stimulation as compared to antigen-negative stimulators such as KG1a and U251T parental cells (FIG. 2C).

[0066] Antibodies and Flow Cytometry: Fluorochrome-conjugated isotype controls, anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-CD45, anti-CD27, anti-CD62L, anti-CD127, anti-IFN.quadrature., and streptavidin were obtained from BD Biosciences. Biotinylated cetuximab was generated from cetuximab purchased from the City of Hope pharmacy. The IFN-.quadrature. Secretion Assay--Cell Enrichment and Detection Kit and CMVpp65 protein were purchased from Miltenyi Biotec (Miltenyi Biotec, Germany). Phycoerythrin (PE)-conjugated CMV pp65 (NLVPMVATV)-HLA-A2*0201 iTAg MHC tetramer, PE-conjugated multi-allele negative tetramer was obtained from Beckman Coulter (Fullerton, Calif.). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Invitrogen (Carlsbad, Calif.). All monoclonal antibodies, tetramers and CFSE were used according to the manufacturer's instructions. Flow cytometry data acquisition was performed on a MACSQuant (Miltenyi Biotec, Germany) or FACScalibur (BD Biosciences), and the percentage of cells in a region of analysis was calculated using FCS Express V3 (De Novo Software).

[0067] Cell lines: EBV-transformed lymphoblastoid cell lines (LCLs) were made from peripheral blood mononuclear cells (PBMC) as previously described (16). To generate LCL-OKT3, allogeneic LCLs were resuspended in nucleofection solution using the Amaxa Nucleofector kit T, OKT3-2A-Hygromycin_pEK plasmid was added to 5 .mu.g/107 cells, the cells were electroporated using the Amaxa Nucleofector I, and the resulting cells were grown in RPMI 1640 with 10% FCS containing 0.4 mg/ml hygromycin. To generate firefly luciferase+ GFP+ LCLs (fflucGFPLCLs), LCLs were transduced with lentiviral vector encoding eGFP-ffluc. Initial transduction efficiency was 48.5%, so the GFP+ cells were sorted by FACS for >98% purity. To generate CD19 NIH3T3 cells, parental NIH3T3 cells (ATCC) were transduced with a retrovirus encoding CD80, CD54 and CD58 (17). The established cell line was further engineered to express CD19GFP by lentiviral transduction. GFP+ cells were purified by FACS sorting and expanded for the use of stimulation of CMV/CAR T cells. To generate pp65 stimulator cells, U251T cells derived from human glioblastoma cells from an HLA A2 donor (ATCC) were transduced with a lentiviral vector encoding full length pp65 fused to green fluorescent protein (GFP). pp65U251T cells were purified by GFP expression using flow cytometry. Banks of all cell lines were authenticated for the desired antigen/marker expression by flow cytometry prior to cryopreservation, and thawed cells were cultured for less than 6 months prior to use in assays.

[0068] Peptides: The pp65 peptide NLVPMVATV (HLA-A 0201 CMVpp65) at >90% purity was synthesized using automated solid phase peptide synthesis in (Department of Experimental Therapeutics, Beckman Research Institute of City of Hope). MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, Calif.). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany). All peptides were used according to the manufacturer's instructions.

[0069] Lentivirus vector construction: The lentivirus CAR construct was modified from the previously described CD19-specific scFvFc:.xi. chimeric immunoreceptor(18), to create a third-generation vector. The CD19CAR containing a CD28.quadrature. co-stimulatory domain carries mutations at two sites (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers to ensure enhanced potency and persistence after adoptive transfer.

[0070] The lentiviral vector also expressed a truncated human epidermal growth factor receptor (huEGFRt), which includes a cetuximab (Erbitux.TM.) binding domain but excludes the EGF-ligand binding and cytoplasmic signaling domains. A T2A ribosome skip sequence links the codon-optimized CD19R:CD28:.xi. sequence to the huEGFRt sequence, resulting in coordinate expression of both CD19R:CD28:.xi. and EGFRt from a single transcript (CD19CARCD28EGFRt) (19). The CD19RCD28EGFRt DNA sequence (optimized by GeneArt) was then cloned into a self-inactivating (SIN) lentiviral vector pHIV7 (gift from Jiing-Kuan Yee, Beckman Research Institute of City of Hope) in which the CMV promoter was replaced by the EF-1.mu. promoter.

[0071] Enrichment of CMV-specific T cells after CMVpp65 protein stimulation: PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) from peripheral blood of consented healthy, HLA-A2 CMV-immune donors under a City of Hope Internal Review Board-approved protocol. PBMC were frozen for later use. After overnight rest in RPMI medium containing 5% Human AB serum (Gemini Bio Products) without cytokine, the PBMC were stimulated with current good manufacturing practice (cGMP) grade CMVpp65 protein (Miltenyi Biotec, Germany) at 10 .mu.l/10.times.10.sup.6 cells for 16 hours in RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 2 mM L-glutamine (Irvine Scientific), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, Irvine Scientific), 100 U/mL penicillin, 0.1 mg/mL streptomycin (Irvine Scientific) in the presence of 5U/ml IL-2 and 10% human AB serum. CMV-specific T cells were selected using the IFN.gamma. capture (Miltenyi Biotec, Germany) technique according to the manufacturer's instructions.

[0072] Derivation and expansion of CMV/CAR T cells: The selected CMV-specific T cells were transduced on day 2 post IFN.gamma. capture with lentiviral vector expressing CD19CARCD28EGFRt at MOI 3. Seven to ten days after lenti-transduction, the CMV/CAR T cells were expanded by stimulation through CAR-mediated activation signals using 8000 cGy-irradiated CD19-expressing NIH 3T3 cells at a 10:1 ratio (T cells:CD19 NIH3T3) once a week as described (17) in the presence of IL-2 50U/ml and IL-15 1 ng/ml. After 2 rounds of expansion, the growth and functionality of the CMV/CAR T cells was evaluated in vitro and in vivo.

[0073] Intracellular cytokine staining: CMV/CAR T cells (105) were activated overnight with 105 LCL-OKT3, LCL, or KG1a cells in 96-well tissue culture plates, and with 10.sup.5 U251T and engineered pp65-expressing U251T cells (pp65U251T) in 24-well tissue culture plates in the presence of Brefeldin A (BD Biosciences). The cell mixture was then stained using anti-CD8, cetuximab and streptavidin, and pp65Tetramer to analyze surface co-expression of CD8, CAR and CMV-specific TCR, respectively. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences). After fixation, the T cells were stained with an anti-IFN.gamma..

[0074] CFSE Proliferation assays: CMV/CAR T cells were labeled with 0.5 .mu.M CFSE and co-cultured with stimulator cells LCL-OKT3, LCLs, and pp65 U251T for 8 days. Co-cultures with U251T and KG1a cells were used as negative controls. Proliferation of CD3- and CAR-positive populations was determined using multicolor flow cytometry.

[0075] Cytokine production assays: T cells (105) were co-cultured overnight in 96-well tissue culture plates with 105 LCL-OKT3, LCL, or KG1a cells and in 24-well tissue culture plates with 105 U251T and engineered pp65-expressing U251T cells. Supernatants were then analyzed by cytometric bead array using the Bio-Plex Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according to the manufacturer's instructions.

[0076] Cytotoxicity assays: 4-hour chromium-release assays (CRA) were performed as previously described (20) using effector cells that had been harvested directly after 2 rounds of CD19 Ag stimulations.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 31 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker <400> SEQUENCE: 2 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 1 5 10 <210> SEQ ID NO 3 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4 hinge with mutation (S228P) <400> SEQUENCE: 3 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> SEQ ID NO 4 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4 hinge with mutation (S228P) + linker <400> SEQUENCE: 5 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly 20 <210> SEQ ID NO 6 <211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 hinge <400> SEQUENCE: 6 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> SEQ ID NO 7 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8 hinge <400> SEQUENCE: 7 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> SEQ ID NO 8 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8 hinge <400> SEQUENCE: 8 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> SEQ ID NO 9 <211> LENGTH: 129 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(HL-CH3)(includes S228P in hinge) <400> SEQUENCE: 9 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 20 25 30 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 35 40 45 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 50 55 60 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 65 70 75 80 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 85 90 95 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 100 105 110 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 115 120 125 Lys <210> SEQ ID NO 10 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(L235E,N297Q) <400> SEQUENCE: 10 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> SEQ ID NO 11 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(S228P, L235E,N297Q) <400> SEQUENCE: 11 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> SEQ ID NO 12 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(CH3) <400> SEQUENCE: 12 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> SEQ ID NO 13 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD3z J04132.1 <400> SEQUENCE: 13 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu 20 <210> SEQ ID NO 14 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 NM_006139 <400> SEQUENCE: 14 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> SEQ ID NO 15 <211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28(M) NM_006139 <400> SEQUENCE: 15 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> SEQ ID NO 16 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD4 M35160 <400> SEQUENCE: 16 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 1 5 10 15 Gly Leu Gly Ile Phe Phe 20 <210> SEQ ID NO 17 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm NM_001768 <400> SEQUENCE: 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> SEQ ID NO 18 <211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm2 - NM_001768 <400> SEQUENCE: 18 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr 20 <210> SEQ ID NO 19 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm3 - NM_001768 <400> SEQUENCE: 19 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> SEQ ID NO 20 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 41BB - NM_001561 <400> SEQUENCE: 20 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu 1 5 10 15 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val 20 25 <210> SEQ ID NO 21 <211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD3 J04132.1 <400> SEQUENCE: 21 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> SEQ ID NO 22 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 NM_006139 <400> SEQUENCE: 22 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> SEQ ID NO 23 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28gg* NM_006139 <400> SEQUENCE: 23 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> SEQ ID NO 24 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 41BB NM_001561 <400> SEQUENCE: 24 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> SEQ ID NO 25 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: OX40 <400> SEQUENCE: 25 Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His 1 5 10 15 Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln 20 25 30 Ala Asp Ala His Ser Thr Leu Ala Lys Ile 35 40 <210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ribosomal skip sequence <400> SEQUENCE: 27 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro Arg 20 <210> SEQ ID NO 28 <211> LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: truncated EGFR <400> SEQUENCE: 28 Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu 1 5 10 15 Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys 20 25 30 Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys 35 40 45 Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly 50 55 60 Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile 65 70 75 80 Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp 85 90 95 Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile 100 105 110 Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser 115 120 125 Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp 130 135 140 Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr 145 150 155 160 Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile 165 170 175 Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys 180 185 190 His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp 195 200 205 Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys 210 215 220 Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu 225 230 235 240 Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr 245 250 255 Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile 260 265 270 Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu 275 280 285 Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 290 295 300 Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu 305 310 315 320 Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met 325 330 335 Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu 340 345 350 Phe Met <210> SEQ ID NO 29 <211> LENGTH: 9388 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: mH5-IEfusion-pZWIIA (GUS) plasmid (SEQ ID NO:A) <400> SEQUENCE: 29 cctcctgaaa aactggaatt taatacacca tttgtgttca tcatcagaca tgatattact 60 ggatttatat tgtttatggg taaggtagaa tctccttaat atgggtacgg tgtaaggaat 120 cattatttta tttatattga tgggtacgtg aaatctgaat tttcttaata aatattattt 180 ttattaaatg tgtatatgtt gttttgcgat agccatgtat ctactaatca gatctattag 240 agatattatt aattctggtg caatatgaca aaaattatac actaattagc gtctcgtttc 300 agacatggat ctgtcacgaa ttaatacttg gaagtctaag cagctgaaaa gctttctctc 360 tagcaaagat gcatttaagg cggatgtcca tggacatagt gccttgtatt atgcaatagc 420 tgataataac gtgcgtctag tatgtacgtt gttgaacgct ggagcattga aaaatcttct 480 agagaatgaa tttccattac atcaggcagc cacattggaa gataccaaaa tagtaaagat 540 tttggctatt cagtggactg gatgattcga ggtaccgact attgttctat attatatatg 600 gttgttgatg gatctgtgat gcatgcaata gctgataata gaacttacgc aaatattagc 660 aaaaatatat tagacaatac tacaattaac gatgagtgta gatgctgtta ttttgaacca 720 cagattagga ttcttgatag agatgagatg ctcaatggat catcgtgtga tatgaacaga 780 cattgtatta tgatgaattt acctgatgta ggcgaatttg gatctagtat gttggggaaa 840 tatgaacctg acatgattaa gattgctctt tcggtggctg ggtaccaggc gcgcatttca 900 ttttgttttt ttctatgcta taaatggtac gtcctgtaga aaccccaacc cgtgaaatca 960 aaaaactcga cggcctgtgg gcattcagtc tggatcgcga aaactgtgga attgatcagc 1020 gttggtggga aagcgcgtta caagaaagcc gggcaattgc tgtgccaggc agttttaacg 1080 atcagttcgc cgatgcagat attcgtaatt atgcgggcaa cgtctggtat cagcgcgaag 1140 tctttatacc gaaaggttgg gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc 1200 attacggcaa agtgtgggtc aataatcagg aagtgatgga gcatcagggc ggctatacgc 1260 catttgaagc cgatgtcacg ccgtatgtta ttgccgggaa aagtgtacgt atcaccgttt 1320 gtgtgaacaa cgaactgaac tggcagacta tcccgccggg aatggtgatt accgacgaaa 1380 acggcaagaa aaagcagtct tacttccatg atttctttaa ctatgccgga atccatcgca 1440 gcgtaatgct ctacaccacg ccgaacacct gggtggacga tatcaccgtg gtgacgcatg 1500 tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt ggtggccaat ggtgatgtca 1560 gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc actagcggga 1620 ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc tatgaactgt 1680 gcgtcacagc caaaagccag acagagtgtg atatctaccc gcttcgcgtc ggcatccggt 1740 cagtggcagt gaagggcgaa cagttcctga ttaaccacaa accgttctac tttactggct 1800 ttggtcgtca tgaagatgcg gacttgcgtg gcaaaggatt cgataacgtg ctgatggtgc 1860 acgaccacgc attaatggac tggattgggg ccaactccta ccgtacctcg cattaccctt 1920 acgctgaaga gatgctcgac tgggcagatg aacatggcat cgtggtgatt gatgaaactg 1980 ctgctgtcgg ctttaacctc tctttaggca ttggtttcga agcgggcaac aagccgaaag 2040 aactgtacag cgaagaggca gtcaacgggg aaactcagca agcgcactta caggcgatta 2100 aagagctgat agcgcgtgac aaaaaccacc caagcgtggt gatgtggagt attgccaacg 2160 aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc gccactggcg gaagcaacgc 2220 gtaaactcga cccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 2280 ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggatggtatg 2340 tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 2400 aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 2460 tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 2520 atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 2580 attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaaaggg atcttcactc 2640 gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 2700 tcggtgaaaa accgcagcag ggaggcaaac aatgagagct cggttgttga tggatctgtg 2760 atgcatgcaa tagctgataa tagaacttac gcaaatatta gcaaaaatat attagacaat 2820 actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag gattcttgat 2880 agagatgaga tgctcaatgg atcatcgtgt gatatgaaca gacattgtat tatgatgaat 2940 ttacctgatg taggcgaatt tggatctagt atgttgggga aatatgaacc tgacatgatt 3000 aagattgctc tttcggtggc tggcggcccg ctcgagaaaa attgaaaata aatacaaagg 3060 ttcttgaggg ttgtgttaaa ttgaaagcga gaaataatca taaataagcc accaccgttt 3120 aaacgccacc acaatggtca aacagattaa ggttcgagtg gacatggtgc ggcatagaat 3180 caaggagcac atgctgaaaa aatataccca gacggaagag aaattcactg gcgcctttaa 3240 tatgatggga ggatgtttgc agaatgcctt agatatctta gataaggttc atgagccttt 3300 cgaggagatg aagtgtattg ggctaactat gcagagcatg tatgagaact acattgtacc 3360 tgaggataag cgggagatgt ggatggcttg tattaaggag ctgcatgatg tgagcaaggg 3420 cgccgctaac aagttggggg gtgcactgca ggctaaggcc cgtgctaaaa aggatgaact 3480 taggagaaag atgatgtata tgtgctacag gaatatagag ttctttacca agaactcagc 3540 cttccctaag accaccaatg gctgcagtca ggccatggcg gcactgcaga acttgcctca 3600 gtgctcccct gatgagatta tggcttatgc ccagaaaata tttaagattt tggatgagga 3660 gagagacaag gtgctcacgc acattgatca catatttatg gatatcctca ctacatgtgt 3720 ggaaacaatg tgtaatgagt acaaggtcac tagtgacgct tgtatgatga ccatgtacgg 3780 gggcatctct ctcttaagtg agttctgtcg ggtgctgtgc tgctatgtct tagaggagac 3840 tagtgtgatg ctggccaagc ggcctctgat aaccaagcct gaggttatca gtgtaatgaa 3900 gcgccgcatt gaggagatct gcatgaaggt ctttgcccag tacattctgg gggccgatcc 3960 tctgagagtc tgctctccta gtgtggatga cctacgggcc atcgccgagg agtcagatga 4020 ggaagaggct attgtagcct acactttggc caccgctggt gtcagctcct ctgattctct 4080 ggtgtcaccc ccagagtccc ctgtacccgc gactatccct ctgtcctcag taattgtggc 4140 tgagaacagt gatcaggaag aaagtgagca gagtgatgag gaagaggagg agggtgctca 4200 ggaggagcgg gaggacactg tgtctgtcaa gtctgagcca gtgtctgaga tagaggaagt 4260 tgccccagag gaagaggagg atggtgctga ggaacccacc gcctctggag gcaagagcac 4320 ccaccctatg gtgactagaa gcaaggctga ccagggtgac atcctcgccc aggctgtcaa 4380 tcatgccggt atcgattcca gtagcaccgg ccccacgctg acaacccact cttgcagcgt 4440 tagcagcgcc cctcttaaca agccgacccc caccagcgtc gcggttacta acactcctct 4500 ccccggggca tccgctactc ccgagctcag cccgcgtaag aaaccgcgca aaaccacgcg 4560 tcctttcaag gtgattatta aaccgcccgt gcctcccgcg cctatcatgc tgcccctcat 4620 caaacaggaa gacatcaagc ccgagcccga ctttaccatc cagtaccgca acaagattat 4680 cgataccgcc ggctgtatcg tgatctctga tagcgaggaa gaacagggtg aagaagtcga 4740 aacccgcggt gctaccgcgt cttccccttc caccggcagc ggcacgccgc gagtgacctc 4800 tcccacgcac ccgctctccc agatgaacca ccctcctctt cccgatccct tgggccggcc 4860 cgatgaagat agttcctctt cgtcttcctc ctcctgcagt tcggcttcgg actcggagag 4920 tgagtccgag gagatgaaat gcagcagtgg cggaggagca tccgtgacct cgagccacca 4980 tgggcgcggc ggttttggtg gcgcggcctc ctcctctctg ctgagctgcg gccatcagag 5040 cagcggcggg gcgagcaccg gaccccgcaa gaagaagagc aaacgcatct ccgagttgga 5100 caacgagaag gtgcgcaata tcatgaaaga taagaacacc cccttctgca cacccaacgt 5160 gcagactcgg cggggtcgcg tcaagattga cgaggtgagc cgcatgttcc gcaacaccaa 5220 tcgctctctt gagtacaaga acctgccctt cacgattccc agtatgcacc aggtgttaga 5280 tgaggccatc aaagcctgca aaaccatgca ggtgaacaac aagggcatcc agattatcta 5340 cacccgcaat catgaggtga agagtgaggt ggatgcggtg cggtgtcgcc tgggcaccat 5400 gtgcaacctg gccctctcca ctcccttcct catggagcac accatgcccg tgacacatcc 5460 acccgaagtg gcgcagcgca cagccgatgc ttgtaacgaa ggcgtcaagg ccgcgtggag 5520 cctcaaagaa ttgcacaccc accaattatg cccccgttcc tccgattacc gcaacatgat 5580 catccacgct gccacccccg tggacctgtt gggcgctctc aacctgtgcc tgcccctgat 5640 gcaaaagttt cccaaacagg tcatggtgcg catcttctcc accaaccagg gtgggttcat 5700 gctgcctatc tacgagacgg ccgcgaaggc ctacgccgtg gggcagtttg agcagcccac 5760 cgagacccct cccgaagacc tggacaccct gagcctggcc atcgaggcag ccatccagga 5820 cctgaggaac aagtctcagt aaaataaagg cgcgccataa aaatttttat actagtgtac 5880 cgcggtcgaa tcgatttaat taacgatgct agcattgtcg acggtggtgg cgcggccgcc 5940 agtgtgatgg atatctgcag aattcggctt ggggggctgc aggtggatgc gatcatgacg 6000 tcctctgcaa tggataacaa tgaacctaaa gtactagaaa tggtatatga tgctacaatt 6060 ttacccgaag gtagtagcat ggattgtata aacagacaca tcaatatgtg tatacaacgc 6120 acctatagtt ctagtataat tgccatattg gatagattcc taatgatgaa caaggatgaa 6180 ctaaataata cacagtgtca tataattaaa gaatttatga catacgaaca aatggcgatt 6240 gaccattatg gagaatatgt aaacgctatt ctatatcaaa ttcgtaaaag acctaatcaa 6300 catcacacca ttaatctgtt taaaaaaata aaaagaaccc ggtatgacac ttttaaagtg 6360 gatcccgtag aattcgtaaa aaaagttatc ggatttgtat ctatcttgaa caaatataaa 6420 ccggtttata gttacgtcct gtacgagaac gtcctgtacg atgagttcaa atgtttcatt 6480 gactacgtgg aaactaagta tttctaaaat taatgatgca ttaatttttg tattgattct 6540 caatcctaaa aactaaaata tgaataagta ttaaacatag cggtgtacta attgatttaa 6600 cataaaaaat agttgttaac taatcatgag gactctactt attagatata ttctttggag 6660 aaatgacaac gatcaaaccg ggcatgcaag cttgtctccc tatagtgagt cgtattagag 6720 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 6780 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 6840 actcacatta attgcgttgc gctcactgcc cgctttcgag tcgggaaacc tgtcgtgcca 6900 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6960 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 7020 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7080 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7140 cgataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7200 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7260 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7320 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7380 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7440 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7500 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7560 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7620 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7680 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7740 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7800 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 7860 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7920 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7980 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 8040 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 8100 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 8160 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttggcattg ctacaggcat 8220 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 8280 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 8340 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 8400 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 8460 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 8520 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 8580 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 8640 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 8700 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 8760 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 8820 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 8880 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 8940 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 9000 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 9060 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 9120 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 9180 ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 9240 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 9300 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgga 9360 tttaggtgac actatagaat acgaattc 9388 <210> SEQ ID NO 30 <211> LENGTH: 8152 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: mH5-pp65-pLW51(GUS) plasmid (SEQ ID NO:B) <400> SEQUENCE: 30 gaattcgttg gtggtcgcca tggatggtgt tattgtatac tgtctaaacg cgttagtaaa 60 acatggcgag gaaataaatc atataaaaaa tgatttcatg attaaaccat gttgtgaaaa 120 agtcaagaac gttcacattg gcggacaatc taaaaacaat acagtgattg cagatttgcc 180 atatatggat aatgcggtat ccgatgtatg caattcactg tataaaaaga atgtatcaag 240 aatatccaga tttgctaatt tgataaagat agatgacgat gacaagactc ctactggtgt 300 atataattat tttaaaccta aagatgccat tcctgttatt atatccatag gaaaggatag 360 agatgtttgt gaactattaa tctcatctga taaagcgtgt gcgtgtatag agttaaattc 420 atataaagta gccattcttc ccatggatgt ttcctttttt accaaaggaa atgcatcatt 480 gattattctc ctgtttgatt tctctatcga tgcggcacct ctcttaagaa gtgtaaccga 540 taataatgtt attatatcta gacaccagcg tctacatgac gagcttccga gttccaattg 600 gttcaagttt tacataagta taaagtccga ctattgttct atattatata tggttgttga 660 tggatctgtg atgcatgcaa tagctgataa tagaacttac gcaaatatta gcaaaaatat 720 attagacaat actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag 780 gattcttgat agagatgaga tgctcaatgg atcatcgtgt gatatgaaca gacattgtat 840 tatgatgaat ttacctgatg taggcgaatt tggatctagt atgttgggga aatatgaacc 900 tgacatgatt aagattgctc tttcggtggc tgggtaccag gcgcgcattt cattttgttt 960 ttttctatgc tataaatggt acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc 1020 gacggcctgt gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg 1080 gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc 1140 gccgatgcag atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata 1200 ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc 1260 aaagtgtggg tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa 1320 gccgatgtca cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt ttgtgtgaac 1380 aacgaactga actggcagac tatcccgccg ggaatggtga ttaccgacga aaacggcaag 1440 aaaaagcagt cttacttcca tgatttcttt aactatgccg gaatccatcg cagcgtaatg 1500 ctctacacca cgccgaacac ctgggtggac gatatcaccg tggtgacgca tgtcgcgcaa 1560 gactgtaacc acgcgtctgt tgactggcag gtggtggcca atggtgatgt cagcgttgaa 1620 ctgcgtgatg cggatcaaca ggtggttgca actggacaag gcactagcgg gactttgcaa 1680 gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc tctatgaact gtgcgtcaca 1740 gccaaaagcc agacagagtg tgatatctac ccgcttcgcg tcggcatccg gtcagtggca 1800 gtgaagggcg aacagttcct gattaaccac aaaccgttct actttactgg ctttggtcgt 1860 catgaagatg cggacttgcg tggcaaagga ttcgataacg tgctgatggt gcacgaccac 1920 gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa 1980 gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac tgctgctgtc 2040 ggctttaacc tctctttagg cattggtttc gaagcgggca acaagccgaa agaactgtac 2100 agcgaagagg cagtcaacgg ggaaactcag caagcgcact tacaggcgat taaagagctg 2160 atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 2220 acccgtccgc aaggtgcacg ggaatatttc gcgccactgg cggaagcaac gcgtaaactc 2280 gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca caccgatacc 2340 atcagcgatc tctttgatgt gctgtgcctg aaccgttatt acggatggta tgtccaaagc 2400 ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac ttctggcctg gcaggagaaa 2460 ctgcatcagc cgattatcat caccgaatac ggcgtggata cgttagccgg gctgcactca 2520 atgtacaccg acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc 2580 gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc cgattttgcg 2640 acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac tcgcgaccgc 2700 aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga ctggcatgaa cttcggtgaa 2760 aaaccgcagc agggaggcaa acaatgagag ctcggttgtt gatggatctg tgatgcatgc 2820 aatagctgat aatagaactt acgcaaatat tagcaaaaat atattagaca atactacaat 2880 taacgatgag tgtagatgct gttattttga accacagatt aggattcttg atagagatga 2940 gatgctcaat ggatcatcgt gtgatatgaa cagacattgt attatgatga atttacctga 3000 tgtaggcgaa tttggatcta gtatgttggg gaaatatgaa cctgacatga ttaagattgc 3060 tctttcggtg gctggcggcc cgctcgagaa aaattgaaaa taaatacaaa ggttcttgag 3120 ggttgtgtta aattgaaagc gagaaataat cataaataag ccaccaccgt ttaaacagtc 3180 gacggtatcg ataagcttga tatcgaattc ctgcagcccg tacgcgcagg cagcatggag 3240 tcgcgcggtc gccgttgtcc cgaaatgata tccgtactgg gtcccatttc ggggcacgtg 3300 ctgaaagccg tgtttagtcg cggcgacacg ccggtgctgc cgcacgagac gcgactcctg 3360 cagacgggta tccacgtgcg cgtgagccag ccctcgctga tcctggtgtc gcagtacacg 3420 cccgactcga cgccatgcca ccgcggcgac aatcagctgc aggtgcagca cacgtacttt 3480 acgggcagcg aggtggagaa cgtgtcggtc aacgtgcaca accccacggg ccggagcatc 3540 tgccccagcc aagagcccat gtcgatctat gtgtacgcgc tgccgctcaa gatgctgaac 3600 atccccagca tcaacgtgca ccactacccg tcggcggccg agcgcaaaca ccgacacctg 3660 cccgtagctg acgctgtgat tcacgcgtcg ggcaagcaga tgtggcaggc gcgtctcacg 3720 gtctcgggac tggcctggac gcgtcagcag aaccagtgga aagagcccga cgtctactac 3780 acgtcagcgt tcgtgtttcc caccaaggac gtggcactgc ggcacgtggt gtgcgcgcac 3840 gagctggttt gctccatgga gaacacgcgc gcaaccaaga tgcaggtgat aggtgaccag 3900 tacgtcaagg tgtacctgga gtccttctgc gaggacgtgc cctccggcaa gctctttatg 3960 cacgtcacgc tgggctctga cgtggaagag gacctgacga tgacccgcaa cccgcaaccc 4020 ttcatgcgcc cccacgagcg caacggcttt acggtgttgt gtcccaaaaa tatgataatc 4080 aaaccgggca agatctcgca catcatgctg gatgtggctt ttacctcaca cgagcatttt 4140 gggctgctgt gtcccaagag catcccgggc ctgagcatct caggtaacct attgatgaac 4200 gggcagcaga tcttcctgga ggtgcaagcg atacgcgaga ccgtggaact gcgtcagtac 4260 gatcccgtgg ctgcgctctt ctttttcgat atcgacttgc tgctgcagcg cgggcctcag 4320 tacagcgaac accccacctt caccagccag tatcgcatcc agggcaagct tgagtaccga 4380 cacacctggg accggcacga cgagggtgcc gcccagggcg acgacgacgt ctggaccagc 4440 ggatcggact ccgacgagga actcgtaacc accgagcgca agacgccccg cgttaccggc 4500 ggcggcgcca tggcgggcgc ctccacttcc gcgggccgca aacgcaaatc agcatcctcg 4560 gcgacggcgt gcacggcggg cgttatgaca cgcggccgcc ttaaggccga gtccaccgtc 4620 gcgcccgaag aggacaccga cgaggattcc gacaacgaaa tccacaatcc ggccgtgttc 4680 acctggccgc cctggcaggc cggcatcctg gcccgcaacc tggtgcccat ggtggctacg 4740 gttcagggtc agaatctgaa gtaccaggag ttcttctggg acgccaacga catctaccgc 4800 atcttcgccg aattggaagg cgtatggcag cccgctgcgc aacccaaacg tcgccgccac 4860 cggcaagacg ccttgcccgg gccatgcatc gcctcgacgc ccaaaaagca ccgaggttga 4920 tttttatggc gcgccctgca gggaaagttt tataggtagt tgatagaaca aaatacataa 4980 ttttgtaaaa ataaatcact ttttatacta atatgacacg attaccaata cttttgttac 5040 taatatcatt agtatacgct acaccttttc ctcagacatc taaaaaaata ggtgatgatg 5100 caactttatc atgtaatcga aataatacaa atgactacgt tgttatgagt gcttggtata 5160 aggagcccaa ttccattatt cttttagctg ctaaaagcga cgtcttgtat tttgataatt 5220 ataccaagga taaaatatct tacgactctc catacgatga tctagttaca actatcacaa 5280 ttaaatcatt gactgctaga gatgccggta cttatgtatg tgcattcttt atgacatcgc 5340 ctacaaatga cactgataaa gtagattatg aagaatactc cacagagttg attgtaaata 5400 cagatagtga atcgactata gacataatac tatctggatc tacacattca ccagaaacta 5460 gttaagcttg tctccctata gtgagtcgta ttagagcttg gcgtaatcat ggtcatagct 5520 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 5580 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 5640 actgcccgct ttcgagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 5700 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 5760 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5820 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 5880 caggaaccgt aaaaaggccg cgttgctggc gtttttcgat aggctccgcc cccctgacga 5940 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 6000 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 6060 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 6120 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 6180 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 6240 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 6300 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 6360 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 6420 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 6480 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 6540 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 6600 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 6660 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 6720 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 6780 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 6840 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 6900 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 6960 tagtttgcgc aacgttgttg gcattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 7020 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 7080 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 7140 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 7200 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 7260 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 7320 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 7380 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 7440 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 7500 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 7560 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 7620 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 7680 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 7740 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 7800 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 7860 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat 7920 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc 7980 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 8040 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 8100 gtcacgacgt tgtaaaacga cggccagtga attggattta ggtgacacta ta 8152 <210> SEQ ID NO 31 <211> LENGTH: 2709 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IE1 antigen and the IE2 antigen fusion protein ((SEQ ID NO:C) <400> SEQUENCE: 31 atggtcaaac agattaaggt tcgagtggac atggtgcggc atagaatcaa ggagcacatg 60 ctgaaaaaat atacccagac ggaagagaaa ttcactggcg cctttaatat gatgggagga 120 tgtttgcaga atgccttaga tatcttagat aaggttcatg agcctttcga ggagatgaag 180 tgtattgggc taactatgca gagcatgtat gagaactaca ttgtacctga ggataagcgg 240 gagatgtgga tggcttgtat taaggagctg catgatgtga gcaagggcgc cgctaacaag 300 ttggggggtg cactgcaggc taaggcccgt gctaaaaagg atgaacttag gagaaagatg 360 atgtatatgt gctacaggaa tatagagttc tttaccaaga actcagcctt ccctaagacc 420 accaatggct gcagtcaggc catggcggca ctgcagaact tgcctcagtg ctcccctgat 480 gagattatgg cttatgccca gaaaatattt aagattttgg atgaggagag agacaaggtg 540 ctcacgcaca ttgatcacat atttatggat atcctcacta catgtgtgga aacaatgtgt 600 aatgagtaca aggtcactag tgacgcttgt atgatgacca tgtacggggc atctctctct 660 taagtgagtt ctgtcgggtg ctgtgctgct atgtcttaga ggagactagt gtgatgctgg 720 ccaagcggcc tctgataacc aagcctgagg ttatcagtgt aatgaagcgc cgcattgagg 780 agatctgcat gaaggtcttt gcccagtaca ttctgggggc cgatcctctg agagtctgct 840 ctcctagtgt ggatgaccta cgggccatcg ccgaggagtc agatgaggaa gaggctattg 900 tagcctacac tttggccacc gctggtgtca gctcctctga ttctctggtg tcacccccag 960 agtcccctgt acccgcgact atccctctgt cctcagtaat tgtggctgag aacagtgatc 1020 aggaagaaag tgagcagagt gatgaggaag aggaggaggg tgctcaggag gagcgggagg 1080 acactgtgtc tgtcaagtct gagccagtgt ctgagataga ggaagttgcc ccagaggaag 1140 aggaggatgg tgctgaggaa cccaccgcct ctggaggcaa gagcacccac cctatggtga 1200 ctagaagcaa ggctgaccag ggtgacatcc tcgcccaggc tgtcaatcat gccggtatcg 1260 attccagtag caccggcccc acgctgacaa cccactcttg cagcgttagc agcgcccctc 1320 ttaacaagcc gacccccacc agcgtcgcgg ttactaacac tcctctcccc ggggcatccg 1380 ctactcccga gctcagcccg cgtaagaaac cgcgcaaaac cacgcgtcct ttcaaggtga 1440 ttattaaacc gcccgtgcct cccgcgccta tcatgctgcc cctcatcaaa caggaagaca 1500 tcaagcccga gcccgacttt accatccagt accgcaacaa gattatcgat accgccggct 1560 gtatcgtgat ctctgatagc gaggaagaac agggtgaaga agtcgaaacc cgcggtgcta 1620 ccgcgtcttc cccttccacc ggcagcggca cgccgcgagt gacctctccc acgcacccgc 1680 tctcccagat gaaccaccct cctcttcccg atcccttggg ccggcccgat gaagatagtt 1740 cctcttcgtc ttcctcctcc tgcagttcgg cttcggactc ggagagtgag tccgaggaga 1800 tgaaatgcag cagtggcgga ggagcatccg tgacctcgag ccaccatggg cgcggcggtt 1860 ttggtggcgc ggcctcctcc tctctgctga gctgcggcca tcagagcagc ggcggggcga 1920 gcaccggacc ccgcaagaag aagagcaaac gcatctccga gttggacaac gagaaggtgc 1980 gcaatatcat gaaagataag aacaccccct tctgcacacc caacgtgcag actcggcggg 2040 gtccgcgtca agattgacga ggtgagccgc atgttccgca acaccaatcg ctctcttgag 2100 tacaagaacc tgcccttcac gattcccagt atgcaccagg tgttagatga ggccatcaaa 2160 gcctgcaaaa ccatgcaggt gaacaacaag ggcatccaga ttatctacac ccgcaatcat 2220 gaggtgaaga gtgaggtgga tgcggtgcgg tgtcgcctgg gcaccatgtg caacctggcc 2280 ctctccactc ccttcctcat ggagcacacc atgcccgtga cacatccacc cgaagtggcg 2340 cagcgaacag ccgatgcttg taacgaaggc gtcaaggccg cgtggagcct caaagaattg 2400 cacacccacc aattatgccc ccgttcctcc gattaccgca acatgatcat ccacgctgcc 2460 acccccgtgg acctgttggg cgctctcaac ctgtgcctgc ccctgatgca aaagtttccc 2520 aaacaggtca tggtgcgcat cttctccacc aaccagggtg ggttcatgct gcctatctac 2580 gagacggccg cgaaggccta cgccgtgggg cagtttgagc agcccaccga gacccctccc 2640 gaagacctgg acaccctgag cctggccatc gaggcagcca tccaggacct gaggaacaag 2700 tctcagtaa 2709

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 31 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker <400> SEQUENCE: 2 Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly 1 5 10 <210> SEQ ID NO 3 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4 hinge with mutation (S228P) <400> SEQUENCE: 3 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> SEQ ID NO 4 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4 hinge with mutation (S228P) + linker <400> SEQUENCE: 5 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly 20 <210> SEQ ID NO 6 <211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 hinge <400> SEQUENCE: 6 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> SEQ ID NO 7 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8 hinge <400> SEQUENCE: 7 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> SEQ ID NO 8 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8 hinge <400> SEQUENCE: 8 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> SEQ ID NO 9 <211> LENGTH: 129 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(HL-CH3)(includes S228P in hinge) <400> SEQUENCE: 9 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gly Gly Ser 1 5 10 15 Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 20 25 30 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 35 40 45 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 50 55 60 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 65 70 75 80 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 85 90 95 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 100 105 110 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 115 120 125 Lys <210> SEQ ID NO 10 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(L235E,N297Q) <400> SEQUENCE: 10 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> SEQ ID NO 11 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(S228P, L235E,N297Q) <400> SEQUENCE: 11 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Gln Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> SEQ ID NO 12 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IgG4(CH3) <400> SEQUENCE: 12 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100 105 <210> SEQ ID NO 13 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD3z J04132.1 <400> SEQUENCE: 13 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu 20 <210> SEQ ID NO 14 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 NM_006139 <400> SEQUENCE: 14 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> SEQ ID NO 15 <211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28(M) NM_006139 <400> SEQUENCE: 15 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> SEQ ID NO 16 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD4 M35160 <400> SEQUENCE: 16 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 1 5 10 15 Gly Leu Gly Ile Phe Phe 20 <210> SEQ ID NO 17 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm NM_001768 <400> SEQUENCE: 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> SEQ ID NO 18 <211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm2 - NM_001768 <400> SEQUENCE: 18 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr 20 <210> SEQ ID NO 19 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD8tm3 - NM_001768 <400> SEQUENCE: 19 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> SEQ ID NO 20 <211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 41BB - NM_001561 <400> SEQUENCE: 20 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu 1 5 10 15 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val 20 25 <210> SEQ ID NO 21 <211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD3 J04132.1 <400> SEQUENCE: 21 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> SEQ ID NO 22 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28 NM_006139 <400> SEQUENCE: 22 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> SEQ ID NO 23 <211> LENGTH: 41 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CD28gg* NM_006139 <400> SEQUENCE: 23 Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> SEQ ID NO 24 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence

<220> FEATURE: <223> OTHER INFORMATION: 41BB NM_001561 <400> SEQUENCE: 24 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> SEQ ID NO 25 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: OX40 <400> SEQUENCE: 25 Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His 1 5 10 15 Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln 20 25 30 Ala Asp Ala His Ser Thr Leu Ala Lys Ile 35 40 <210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: ribosomal skip sequence <400> SEQUENCE: 27 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro Arg 20 <210> SEQ ID NO 28 <211> LENGTH: 354 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: truncated EGFR <400> SEQUENCE: 28 Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu 1 5 10 15 Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys 20 25 30 Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys 35 40 45 Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly 50 55 60 Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile 65 70 75 80 Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp 85 90 95 Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile 100 105 110 Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser 115 120 125 Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp 130 135 140 Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr 145 150 155 160 Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile 165 170 175 Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys 180 185 190 His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp 195 200 205 Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys 210 215 220 Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu 225 230 235 240 Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr 245 250 255 Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile 260 265 270 Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu 275 280 285 Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 290 295 300 Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu 305 310 315 320 Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met 325 330 335 Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu 340 345 350 Phe Met <210> SEQ ID NO 29 <211> LENGTH: 9388 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: mH5-IEfusion-pZWIIA (GUS) plasmid (SEQ ID NO:A) <400> SEQUENCE: 29 cctcctgaaa aactggaatt taatacacca tttgtgttca tcatcagaca tgatattact 60 ggatttatat tgtttatggg taaggtagaa tctccttaat atgggtacgg tgtaaggaat 120 cattatttta tttatattga tgggtacgtg aaatctgaat tttcttaata aatattattt 180 ttattaaatg tgtatatgtt gttttgcgat agccatgtat ctactaatca gatctattag 240 agatattatt aattctggtg caatatgaca aaaattatac actaattagc gtctcgtttc 300 agacatggat ctgtcacgaa ttaatacttg gaagtctaag cagctgaaaa gctttctctc 360 tagcaaagat gcatttaagg cggatgtcca tggacatagt gccttgtatt atgcaatagc 420 tgataataac gtgcgtctag tatgtacgtt gttgaacgct ggagcattga aaaatcttct 480 agagaatgaa tttccattac atcaggcagc cacattggaa gataccaaaa tagtaaagat 540 tttggctatt cagtggactg gatgattcga ggtaccgact attgttctat attatatatg 600 gttgttgatg gatctgtgat gcatgcaata gctgataata gaacttacgc aaatattagc 660 aaaaatatat tagacaatac tacaattaac gatgagtgta gatgctgtta ttttgaacca 720 cagattagga ttcttgatag agatgagatg ctcaatggat catcgtgtga tatgaacaga 780 cattgtatta tgatgaattt acctgatgta ggcgaatttg gatctagtat gttggggaaa 840 tatgaacctg acatgattaa gattgctctt tcggtggctg ggtaccaggc gcgcatttca 900 ttttgttttt ttctatgcta taaatggtac gtcctgtaga aaccccaacc cgtgaaatca 960 aaaaactcga cggcctgtgg gcattcagtc tggatcgcga aaactgtgga attgatcagc 1020 gttggtggga aagcgcgtta caagaaagcc gggcaattgc tgtgccaggc agttttaacg 1080 atcagttcgc cgatgcagat attcgtaatt atgcgggcaa cgtctggtat cagcgcgaag 1140 tctttatacc gaaaggttgg gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc 1200 attacggcaa agtgtgggtc aataatcagg aagtgatgga gcatcagggc ggctatacgc 1260 catttgaagc cgatgtcacg ccgtatgtta ttgccgggaa aagtgtacgt atcaccgttt 1320 gtgtgaacaa cgaactgaac tggcagacta tcccgccggg aatggtgatt accgacgaaa 1380 acggcaagaa aaagcagtct tacttccatg atttctttaa ctatgccgga atccatcgca 1440 gcgtaatgct ctacaccacg ccgaacacct gggtggacga tatcaccgtg gtgacgcatg 1500 tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt ggtggccaat ggtgatgtca 1560 gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc actagcggga 1620 ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc tatgaactgt 1680 gcgtcacagc caaaagccag acagagtgtg atatctaccc gcttcgcgtc ggcatccggt 1740 cagtggcagt gaagggcgaa cagttcctga ttaaccacaa accgttctac tttactggct 1800 ttggtcgtca tgaagatgcg gacttgcgtg gcaaaggatt cgataacgtg ctgatggtgc 1860 acgaccacgc attaatggac tggattgggg ccaactccta ccgtacctcg cattaccctt 1920 acgctgaaga gatgctcgac tgggcagatg aacatggcat cgtggtgatt gatgaaactg 1980 ctgctgtcgg ctttaacctc tctttaggca ttggtttcga agcgggcaac aagccgaaag 2040 aactgtacag cgaagaggca gtcaacgggg aaactcagca agcgcactta caggcgatta 2100 aagagctgat agcgcgtgac aaaaaccacc caagcgtggt gatgtggagt attgccaacg 2160 aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc gccactggcg gaagcaacgc 2220 gtaaactcga cccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 2280 ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggatggtatg 2340 tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 2400 aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 2460 tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 2520 atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 2580 attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaaaggg atcttcactc 2640 gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 2700 tcggtgaaaa accgcagcag ggaggcaaac aatgagagct cggttgttga tggatctgtg 2760 atgcatgcaa tagctgataa tagaacttac gcaaatatta gcaaaaatat attagacaat 2820 actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag gattcttgat 2880 agagatgaga tgctcaatgg atcatcgtgt gatatgaaca gacattgtat tatgatgaat 2940 ttacctgatg taggcgaatt tggatctagt atgttgggga aatatgaacc tgacatgatt 3000 aagattgctc tttcggtggc tggcggcccg ctcgagaaaa attgaaaata aatacaaagg 3060 ttcttgaggg ttgtgttaaa ttgaaagcga gaaataatca taaataagcc accaccgttt 3120 aaacgccacc acaatggtca aacagattaa ggttcgagtg gacatggtgc ggcatagaat 3180

caaggagcac atgctgaaaa aatataccca gacggaagag aaattcactg gcgcctttaa 3240 tatgatggga ggatgtttgc agaatgcctt agatatctta gataaggttc atgagccttt 3300 cgaggagatg aagtgtattg ggctaactat gcagagcatg tatgagaact acattgtacc 3360 tgaggataag cgggagatgt ggatggcttg tattaaggag ctgcatgatg tgagcaaggg 3420 cgccgctaac aagttggggg gtgcactgca ggctaaggcc cgtgctaaaa aggatgaact 3480 taggagaaag atgatgtata tgtgctacag gaatatagag ttctttacca agaactcagc 3540 cttccctaag accaccaatg gctgcagtca ggccatggcg gcactgcaga acttgcctca 3600 gtgctcccct gatgagatta tggcttatgc ccagaaaata tttaagattt tggatgagga 3660 gagagacaag gtgctcacgc acattgatca catatttatg gatatcctca ctacatgtgt 3720 ggaaacaatg tgtaatgagt acaaggtcac tagtgacgct tgtatgatga ccatgtacgg 3780 gggcatctct ctcttaagtg agttctgtcg ggtgctgtgc tgctatgtct tagaggagac 3840 tagtgtgatg ctggccaagc ggcctctgat aaccaagcct gaggttatca gtgtaatgaa 3900 gcgccgcatt gaggagatct gcatgaaggt ctttgcccag tacattctgg gggccgatcc 3960 tctgagagtc tgctctccta gtgtggatga cctacgggcc atcgccgagg agtcagatga 4020 ggaagaggct attgtagcct acactttggc caccgctggt gtcagctcct ctgattctct 4080 ggtgtcaccc ccagagtccc ctgtacccgc gactatccct ctgtcctcag taattgtggc 4140 tgagaacagt gatcaggaag aaagtgagca gagtgatgag gaagaggagg agggtgctca 4200 ggaggagcgg gaggacactg tgtctgtcaa gtctgagcca gtgtctgaga tagaggaagt 4260 tgccccagag gaagaggagg atggtgctga ggaacccacc gcctctggag gcaagagcac 4320 ccaccctatg gtgactagaa gcaaggctga ccagggtgac atcctcgccc aggctgtcaa 4380 tcatgccggt atcgattcca gtagcaccgg ccccacgctg acaacccact cttgcagcgt 4440 tagcagcgcc cctcttaaca agccgacccc caccagcgtc gcggttacta acactcctct 4500 ccccggggca tccgctactc ccgagctcag cccgcgtaag aaaccgcgca aaaccacgcg 4560 tcctttcaag gtgattatta aaccgcccgt gcctcccgcg cctatcatgc tgcccctcat 4620 caaacaggaa gacatcaagc ccgagcccga ctttaccatc cagtaccgca acaagattat 4680 cgataccgcc ggctgtatcg tgatctctga tagcgaggaa gaacagggtg aagaagtcga 4740 aacccgcggt gctaccgcgt cttccccttc caccggcagc ggcacgccgc gagtgacctc 4800 tcccacgcac ccgctctccc agatgaacca ccctcctctt cccgatccct tgggccggcc 4860 cgatgaagat agttcctctt cgtcttcctc ctcctgcagt tcggcttcgg actcggagag 4920 tgagtccgag gagatgaaat gcagcagtgg cggaggagca tccgtgacct cgagccacca 4980 tgggcgcggc ggttttggtg gcgcggcctc ctcctctctg ctgagctgcg gccatcagag 5040 cagcggcggg gcgagcaccg gaccccgcaa gaagaagagc aaacgcatct ccgagttgga 5100 caacgagaag gtgcgcaata tcatgaaaga taagaacacc cccttctgca cacccaacgt 5160 gcagactcgg cggggtcgcg tcaagattga cgaggtgagc cgcatgttcc gcaacaccaa 5220 tcgctctctt gagtacaaga acctgccctt cacgattccc agtatgcacc aggtgttaga 5280 tgaggccatc aaagcctgca aaaccatgca ggtgaacaac aagggcatcc agattatcta 5340 cacccgcaat catgaggtga agagtgaggt ggatgcggtg cggtgtcgcc tgggcaccat 5400 gtgcaacctg gccctctcca ctcccttcct catggagcac accatgcccg tgacacatcc 5460 acccgaagtg gcgcagcgca cagccgatgc ttgtaacgaa ggcgtcaagg ccgcgtggag 5520 cctcaaagaa ttgcacaccc accaattatg cccccgttcc tccgattacc gcaacatgat 5580 catccacgct gccacccccg tggacctgtt gggcgctctc aacctgtgcc tgcccctgat 5640 gcaaaagttt cccaaacagg tcatggtgcg catcttctcc accaaccagg gtgggttcat 5700 gctgcctatc tacgagacgg ccgcgaaggc ctacgccgtg gggcagtttg agcagcccac 5760 cgagacccct cccgaagacc tggacaccct gagcctggcc atcgaggcag ccatccagga 5820 cctgaggaac aagtctcagt aaaataaagg cgcgccataa aaatttttat actagtgtac 5880 cgcggtcgaa tcgatttaat taacgatgct agcattgtcg acggtggtgg cgcggccgcc 5940 agtgtgatgg atatctgcag aattcggctt ggggggctgc aggtggatgc gatcatgacg 6000 tcctctgcaa tggataacaa tgaacctaaa gtactagaaa tggtatatga tgctacaatt 6060 ttacccgaag gtagtagcat ggattgtata aacagacaca tcaatatgtg tatacaacgc 6120 acctatagtt ctagtataat tgccatattg gatagattcc taatgatgaa caaggatgaa 6180 ctaaataata cacagtgtca tataattaaa gaatttatga catacgaaca aatggcgatt 6240 gaccattatg gagaatatgt aaacgctatt ctatatcaaa ttcgtaaaag acctaatcaa 6300 catcacacca ttaatctgtt taaaaaaata aaaagaaccc ggtatgacac ttttaaagtg 6360 gatcccgtag aattcgtaaa aaaagttatc ggatttgtat ctatcttgaa caaatataaa 6420 ccggtttata gttacgtcct gtacgagaac gtcctgtacg atgagttcaa atgtttcatt 6480 gactacgtgg aaactaagta tttctaaaat taatgatgca ttaatttttg tattgattct 6540 caatcctaaa aactaaaata tgaataagta ttaaacatag cggtgtacta attgatttaa 6600 cataaaaaat agttgttaac taatcatgag gactctactt attagatata ttctttggag 6660 aaatgacaac gatcaaaccg ggcatgcaag cttgtctccc tatagtgagt cgtattagag 6720 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 6780 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 6840 actcacatta attgcgttgc gctcactgcc cgctttcgag tcgggaaacc tgtcgtgcca 6900 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6960 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 7020 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7080 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7140 cgataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7200 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7260 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7320 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7380 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7440 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7500 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7560 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7620 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7680 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7740 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7800 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 7860 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7920 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7980 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 8040 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 8100 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 8160 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttggcattg ctacaggcat 8220 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 8280 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 8340 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 8400 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 8460 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 8520 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 8580 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 8640 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 8700 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 8760 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 8820 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 8880 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 8940 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 9000 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 9060 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 9120 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 9180 ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 9240 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 9300 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgga 9360 tttaggtgac actatagaat acgaattc 9388 <210> SEQ ID NO 30 <211> LENGTH: 8152 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: mH5-pp65-pLW51(GUS) plasmid (SEQ ID NO:B) <400> SEQUENCE: 30 gaattcgttg gtggtcgcca tggatggtgt tattgtatac tgtctaaacg cgttagtaaa 60 acatggcgag gaaataaatc atataaaaaa tgatttcatg attaaaccat gttgtgaaaa 120 agtcaagaac gttcacattg gcggacaatc taaaaacaat acagtgattg cagatttgcc 180 atatatggat aatgcggtat ccgatgtatg caattcactg tataaaaaga atgtatcaag 240 aatatccaga tttgctaatt tgataaagat agatgacgat gacaagactc ctactggtgt 300 atataattat tttaaaccta aagatgccat tcctgttatt atatccatag gaaaggatag 360 agatgtttgt gaactattaa tctcatctga taaagcgtgt gcgtgtatag agttaaattc 420 atataaagta gccattcttc ccatggatgt ttcctttttt accaaaggaa atgcatcatt 480 gattattctc ctgtttgatt tctctatcga tgcggcacct ctcttaagaa gtgtaaccga 540 taataatgtt attatatcta gacaccagcg tctacatgac gagcttccga gttccaattg 600 gttcaagttt tacataagta taaagtccga ctattgttct atattatata tggttgttga 660 tggatctgtg atgcatgcaa tagctgataa tagaacttac gcaaatatta gcaaaaatat 720 attagacaat actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag 780 gattcttgat agagatgaga tgctcaatgg atcatcgtgt gatatgaaca gacattgtat 840 tatgatgaat ttacctgatg taggcgaatt tggatctagt atgttgggga aatatgaacc 900 tgacatgatt aagattgctc tttcggtggc tgggtaccag gcgcgcattt cattttgttt 960 ttttctatgc tataaatggt acgtcctgta gaaaccccaa cccgtgaaat caaaaaactc 1020

gacggcctgt gggcattcag tctggatcgc gaaaactgtg gaattgatca gcgttggtgg 1080 gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc 1140 gccgatgcag atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata 1200 ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac tcattacggc 1260 aaagtgtggg tcaataatca ggaagtgatg gagcatcagg gcggctatac gccatttgaa 1320 gccgatgtca cgccgtatgt tattgccggg aaaagtgtac gtatcaccgt ttgtgtgaac 1380 aacgaactga actggcagac tatcccgccg ggaatggtga ttaccgacga aaacggcaag 1440 aaaaagcagt cttacttcca tgatttcttt aactatgccg gaatccatcg cagcgtaatg 1500 ctctacacca cgccgaacac ctgggtggac gatatcaccg tggtgacgca tgtcgcgcaa 1560 gactgtaacc acgcgtctgt tgactggcag gtggtggcca atggtgatgt cagcgttgaa 1620 ctgcgtgatg cggatcaaca ggtggttgca actggacaag gcactagcgg gactttgcaa 1680 gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc tctatgaact gtgcgtcaca 1740 gccaaaagcc agacagagtg tgatatctac ccgcttcgcg tcggcatccg gtcagtggca 1800 gtgaagggcg aacagttcct gattaaccac aaaccgttct actttactgg ctttggtcgt 1860 catgaagatg cggacttgcg tggcaaagga ttcgataacg tgctgatggt gcacgaccac 1920 gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa 1980 gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac tgctgctgtc 2040 ggctttaacc tctctttagg cattggtttc gaagcgggca acaagccgaa agaactgtac 2100 agcgaagagg cagtcaacgg ggaaactcag caagcgcact tacaggcgat taaagagctg 2160 atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 2220 acccgtccgc aaggtgcacg ggaatatttc gcgccactgg cggaagcaac gcgtaaactc 2280 gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca caccgatacc 2340 atcagcgatc tctttgatgt gctgtgcctg aaccgttatt acggatggta tgtccaaagc 2400 ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac ttctggcctg gcaggagaaa 2460 ctgcatcagc cgattatcat caccgaatac ggcgtggata cgttagccgg gctgcactca 2520 atgtacaccg acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc 2580 gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc cgattttgcg 2640 acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac tcgcgaccgc 2700 aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga ctggcatgaa cttcggtgaa 2760 aaaccgcagc agggaggcaa acaatgagag ctcggttgtt gatggatctg tgatgcatgc 2820 aatagctgat aatagaactt acgcaaatat tagcaaaaat atattagaca atactacaat 2880 taacgatgag tgtagatgct gttattttga accacagatt aggattcttg atagagatga 2940 gatgctcaat ggatcatcgt gtgatatgaa cagacattgt attatgatga atttacctga 3000 tgtaggcgaa tttggatcta gtatgttggg gaaatatgaa cctgacatga ttaagattgc 3060 tctttcggtg gctggcggcc cgctcgagaa aaattgaaaa taaatacaaa ggttcttgag 3120 ggttgtgtta aattgaaagc gagaaataat cataaataag ccaccaccgt ttaaacagtc 3180 gacggtatcg ataagcttga tatcgaattc ctgcagcccg tacgcgcagg cagcatggag 3240 tcgcgcggtc gccgttgtcc cgaaatgata tccgtactgg gtcccatttc ggggcacgtg 3300 ctgaaagccg tgtttagtcg cggcgacacg ccggtgctgc cgcacgagac gcgactcctg 3360 cagacgggta tccacgtgcg cgtgagccag ccctcgctga tcctggtgtc gcagtacacg 3420 cccgactcga cgccatgcca ccgcggcgac aatcagctgc aggtgcagca cacgtacttt 3480 acgggcagcg aggtggagaa cgtgtcggtc aacgtgcaca accccacggg ccggagcatc 3540 tgccccagcc aagagcccat gtcgatctat gtgtacgcgc tgccgctcaa gatgctgaac 3600 atccccagca tcaacgtgca ccactacccg tcggcggccg agcgcaaaca ccgacacctg 3660 cccgtagctg acgctgtgat tcacgcgtcg ggcaagcaga tgtggcaggc gcgtctcacg 3720 gtctcgggac tggcctggac gcgtcagcag aaccagtgga aagagcccga cgtctactac 3780 acgtcagcgt tcgtgtttcc caccaaggac gtggcactgc ggcacgtggt gtgcgcgcac 3840 gagctggttt gctccatgga gaacacgcgc gcaaccaaga tgcaggtgat aggtgaccag 3900 tacgtcaagg tgtacctgga gtccttctgc gaggacgtgc cctccggcaa gctctttatg 3960 cacgtcacgc tgggctctga cgtggaagag gacctgacga tgacccgcaa cccgcaaccc 4020 ttcatgcgcc cccacgagcg caacggcttt acggtgttgt gtcccaaaaa tatgataatc 4080 aaaccgggca agatctcgca catcatgctg gatgtggctt ttacctcaca cgagcatttt 4140 gggctgctgt gtcccaagag catcccgggc ctgagcatct caggtaacct attgatgaac 4200 gggcagcaga tcttcctgga ggtgcaagcg atacgcgaga ccgtggaact gcgtcagtac 4260 gatcccgtgg ctgcgctctt ctttttcgat atcgacttgc tgctgcagcg cgggcctcag 4320 tacagcgaac accccacctt caccagccag tatcgcatcc agggcaagct tgagtaccga 4380 cacacctggg accggcacga cgagggtgcc gcccagggcg acgacgacgt ctggaccagc 4440 ggatcggact ccgacgagga actcgtaacc accgagcgca agacgccccg cgttaccggc 4500 ggcggcgcca tggcgggcgc ctccacttcc gcgggccgca aacgcaaatc agcatcctcg 4560 gcgacggcgt gcacggcggg cgttatgaca cgcggccgcc ttaaggccga gtccaccgtc 4620 gcgcccgaag aggacaccga cgaggattcc gacaacgaaa tccacaatcc ggccgtgttc 4680 acctggccgc cctggcaggc cggcatcctg gcccgcaacc tggtgcccat ggtggctacg 4740 gttcagggtc agaatctgaa gtaccaggag ttcttctggg acgccaacga catctaccgc 4800 atcttcgccg aattggaagg cgtatggcag cccgctgcgc aacccaaacg tcgccgccac 4860 cggcaagacg ccttgcccgg gccatgcatc gcctcgacgc ccaaaaagca ccgaggttga 4920 tttttatggc gcgccctgca gggaaagttt tataggtagt tgatagaaca aaatacataa 4980 ttttgtaaaa ataaatcact ttttatacta atatgacacg attaccaata cttttgttac 5040 taatatcatt agtatacgct acaccttttc ctcagacatc taaaaaaata ggtgatgatg 5100 caactttatc atgtaatcga aataatacaa atgactacgt tgttatgagt gcttggtata 5160 aggagcccaa ttccattatt cttttagctg ctaaaagcga cgtcttgtat tttgataatt 5220 ataccaagga taaaatatct tacgactctc catacgatga tctagttaca actatcacaa 5280 ttaaatcatt gactgctaga gatgccggta cttatgtatg tgcattcttt atgacatcgc 5340 ctacaaatga cactgataaa gtagattatg aagaatactc cacagagttg attgtaaata 5400 cagatagtga atcgactata gacataatac tatctggatc tacacattca ccagaaacta 5460 gttaagcttg tctccctata gtgagtcgta ttagagcttg gcgtaatcat ggtcatagct 5520 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 5580 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 5640 actgcccgct ttcgagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 5700 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 5760 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5820 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 5880 caggaaccgt aaaaaggccg cgttgctggc gtttttcgat aggctccgcc cccctgacga 5940 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 6000 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 6060 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 6120 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 6180 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 6240 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 6300 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 6360 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 6420 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 6480 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 6540 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 6600 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 6660 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 6720 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 6780 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 6840 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 6900 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 6960 tagtttgcgc aacgttgttg gcattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 7020 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 7080 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 7140 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 7200 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 7260 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 7320 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 7380 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 7440 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 7500 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 7560 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 7620 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 7680 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 7740 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 7800 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 7860 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat 7920 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc 7980 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 8040 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 8100 gtcacgacgt tgtaaaacga cggccagtga attggattta ggtgacacta ta 8152 <210> SEQ ID NO 31 <211> LENGTH: 2709 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: IE1 antigen and the IE2 antigen fusion protein ((SEQ ID NO:C) <400> SEQUENCE: 31

atggtcaaac agattaaggt tcgagtggac atggtgcggc atagaatcaa ggagcacatg 60 ctgaaaaaat atacccagac ggaagagaaa ttcactggcg cctttaatat gatgggagga 120 tgtttgcaga atgccttaga tatcttagat aaggttcatg agcctttcga ggagatgaag 180 tgtattgggc taactatgca gagcatgtat gagaactaca ttgtacctga ggataagcgg 240 gagatgtgga tggcttgtat taaggagctg catgatgtga gcaagggcgc cgctaacaag 300 ttggggggtg cactgcaggc taaggcccgt gctaaaaagg atgaacttag gagaaagatg 360 atgtatatgt gctacaggaa tatagagttc tttaccaaga actcagcctt ccctaagacc 420 accaatggct gcagtcaggc catggcggca ctgcagaact tgcctcagtg ctcccctgat 480 gagattatgg cttatgccca gaaaatattt aagattttgg atgaggagag agacaaggtg 540 ctcacgcaca ttgatcacat atttatggat atcctcacta catgtgtgga aacaatgtgt 600 aatgagtaca aggtcactag tgacgcttgt atgatgacca tgtacggggc atctctctct 660 taagtgagtt ctgtcgggtg ctgtgctgct atgtcttaga ggagactagt gtgatgctgg 720 ccaagcggcc tctgataacc aagcctgagg ttatcagtgt aatgaagcgc cgcattgagg 780 agatctgcat gaaggtcttt gcccagtaca ttctgggggc cgatcctctg agagtctgct 840 ctcctagtgt ggatgaccta cgggccatcg ccgaggagtc agatgaggaa gaggctattg 900 tagcctacac tttggccacc gctggtgtca gctcctctga ttctctggtg tcacccccag 960 agtcccctgt acccgcgact atccctctgt cctcagtaat tgtggctgag aacagtgatc 1020 aggaagaaag tgagcagagt gatgaggaag aggaggaggg tgctcaggag gagcgggagg 1080 acactgtgtc tgtcaagtct gagccagtgt ctgagataga ggaagttgcc ccagaggaag 1140 aggaggatgg tgctgaggaa cccaccgcct ctggaggcaa gagcacccac cctatggtga 1200 ctagaagcaa ggctgaccag ggtgacatcc tcgcccaggc tgtcaatcat gccggtatcg 1260 attccagtag caccggcccc acgctgacaa cccactcttg cagcgttagc agcgcccctc 1320 ttaacaagcc gacccccacc agcgtcgcgg ttactaacac tcctctcccc ggggcatccg 1380 ctactcccga gctcagcccg cgtaagaaac cgcgcaaaac cacgcgtcct ttcaaggtga 1440 ttattaaacc gcccgtgcct cccgcgccta tcatgctgcc cctcatcaaa caggaagaca 1500 tcaagcccga gcccgacttt accatccagt accgcaacaa gattatcgat accgccggct 1560 gtatcgtgat ctctgatagc gaggaagaac agggtgaaga agtcgaaacc cgcggtgcta 1620 ccgcgtcttc cccttccacc ggcagcggca cgccgcgagt gacctctccc acgcacccgc 1680 tctcccagat gaaccaccct cctcttcccg atcccttggg ccggcccgat gaagatagtt 1740 cctcttcgtc ttcctcctcc tgcagttcgg cttcggactc ggagagtgag tccgaggaga 1800 tgaaatgcag cagtggcgga ggagcatccg tgacctcgag ccaccatggg cgcggcggtt 1860 ttggtggcgc ggcctcctcc tctctgctga gctgcggcca tcagagcagc ggcggggcga 1920 gcaccggacc ccgcaagaag aagagcaaac gcatctccga gttggacaac gagaaggtgc 1980 gcaatatcat gaaagataag aacaccccct tctgcacacc caacgtgcag actcggcggg 2040 gtccgcgtca agattgacga ggtgagccgc atgttccgca acaccaatcg ctctcttgag 2100 tacaagaacc tgcccttcac gattcccagt atgcaccagg tgttagatga ggccatcaaa 2160 gcctgcaaaa ccatgcaggt gaacaacaag ggcatccaga ttatctacac ccgcaatcat 2220 gaggtgaaga gtgaggtgga tgcggtgcgg tgtcgcctgg gcaccatgtg caacctggcc 2280 ctctccactc ccttcctcat ggagcacacc atgcccgtga cacatccacc cgaagtggcg 2340 cagcgaacag ccgatgcttg taacgaaggc gtcaaggccg cgtggagcct caaagaattg 2400 cacacccacc aattatgccc ccgttcctcc gattaccgca acatgatcat ccacgctgcc 2460 acccccgtgg acctgttggg cgctctcaac ctgtgcctgc ccctgatgca aaagtttccc 2520 aaacaggtca tggtgcgcat cttctccacc aaccagggtg ggttcatgct gcctatctac 2580 gagacggccg cgaaggccta cgccgtgggg cagtttgagc agcccaccga gacccctccc 2640 gaagacctgg acaccctgag cctggccatc gaggcagcca tccaggacct gaggaacaag 2700 tctcagtaa 2709

Claims

1-50. (canceled)

51. A method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition of part (a) to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5).

52. The method of claim 51, wherein the viral vector of part (a) is a MVA virus.

53. The method of claim 52, wherein expression of (i) CMV pp65 and (ii) the fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) is under the control of mH5 promoter.

54. The method of claim 51, wherein the patient is CMV-seronegative prior to treatment.

55. The method of claim 51, wherein the patient is CMV-seropositive prior to treatment.

56. The method of claim 51, wherein the CAR is targeted to CD19.

57. The method of claim 51, wherein the viral vector is administered to the patient both prior to and subsequent to the administration of the composition comprising a population of T cells.

58. The method of claim 51, wherein the step of providing a population of T cells expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a1) providing PBMC or a T cell subpopulation from a CMV-seropositive human donor; (a2) exposing the PBMC or the T cell subpopulation of part (a1) to at least one CMV antigen; (a3) treating the exposed cells of part (a2) to produce a population of cells enriched for cells specific for CMV; and (a4) transducing at least a portion of the enriched population of cells of part (a3) with a vector expressing a CAR.

59. The method of claim 58, wherein the step of treating the exposed cells to produce a population of cells enriched for cells specific for CMV comprises treating the cells to produce a population of cells enriched for cells expressing an activation marker.

60. The method of claim 51, wherein the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen further comprises: (a1) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a human donor to convert a CMV-seronegative human donor to one containing T cells responsive to CMV antigens pp65, IE1 and IE2; (a2) obtaining PBMC from the CMV-seropositive human donor of (a1); (a3) exposing the PBMC of (a2) to at least one CMV antigen; (a4) treating the exposed cells of (a3) to produce a population of cells enriched for stimulated cells specific for CMV; and (a5) transducing at least a portion of the enriched population of cells of (a4) with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

61. The method of claim 51, wherein the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a1) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (a2) obtaining PBMC from the CMV-seropositive human donor of (a1); (a3) exposing the PBMC of (a2) to at least one CMV antigen; (a4) treating the exposed cells of (a3) to produce a population of cells enriched for stimulated cells specific for CMV; (a5) transducing at least a portion of the enriched population of cells of (a4) with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

62. A method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen, the method comprising: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (c) exposing the PBMC to at least one CMV antigen; (d) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; and (e) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

63. A method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen, the method comprising: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (c) (b) exposing the PBMC to at least one CMV antigen; (d) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; and (e) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.

64. The method of claim 51, wherein the CAR is selective for an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13.alpha.2, PSA, HER-2, EGFRv3, CEA, and C7R.

65. The method of claim 51, wherein the CAR comprises: a scFv selective for a non-CMV antigen; a hinge/linker region; a transmembrane domain; a co-signaling domain; and CD3 .xi. signaling domain.

66. The method of claim 65, wherein the co-signaling domain is selected from a CD28 co-signaling domain and a 4-IBB co-signaling domain.

67. The method of claim 65, wherein the transmembrane domain is selected from a CD28 transmembrane domain and a CD4 transmembrane