INDUCTION AND ENHANCEMENT OF ANTITUMOR IMMUNITY INVOLVING SINDBIS VIRUS VECTORS EXPRESSING IMMUNE CHECKPOINT PROTEINS

Abstract

Provided are polynucleotides and viral vectors, particularly, Alphavirus vectors such as Sindbis viral vectors, which encode an immune checkpoint protein, or a ligand binding portion of the checkpoint protein, or an immune checkpoint protein or ligand binding portion thereof fused to one or more immunoglobulin (Ig) domains, e.g., an Ig hinge region and an Ig heavy chain constant domain. Methods of treating a mammalian subject having a cancer or tumor are provided, in which the viral vectors, e.g., a Sindbis virus vector, encoding the immune checkpoint protein, a ligand binding portion thereof, or a checkpoint protein fusion protein as described, are administered to the subject, resulting in an anti-cancer or anti-tumor immune response, significant reduction in tumor growth in the treated subject and increased survivability.

Claims

1. A therapeutic composition comprising a Sindbis virus encoding an immune checkpoint protein or a cognate ligand binding portion thereof.

2. The therapeutic composition of claim 1, wherein the immune checkpoint protein or the cognate ligand binding portion thereof is fused to an immunoglobulin hinge region and an immunoglobulin heavy chain constant domain.

3. The therapeutic composition of claim 2, wherein the Sindbis virus encoding a fusion polypeptide comprises a secretory signal sequence linked to the immunoglobulin heavy chain constant domain, which is linked to the immune checkpoint protein, or an extracellular domain thereof; and wherein the fusion protein comprises one or more linker sequences.

4. The therapeutic composition of claim 2, wherein the fusion protein comprises a linker sequence between the hinge region and the immunoglobulin heavy chain constant domain.

5. The therapeutic composition of claim 2, wherein the immunoglobulin is IgG, IgG1, or IgG2a.

6. The therapeutic composition of claim 5, wherein the heavy chain constant domain is the CH3 domain.

7. The therapeutic composition of claim 3, wherein the linker sequence comprises the sequence GGGSSGGGSGG (SEQ ID NO: 1) or GGGSSGGGSGS (SEQ ID NO: 2).

8. The therapeutic composition of claim 3, wherein the secretory signal sequence comprises the amino acid sequence METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 23).

9. The therapeutic composition of claim 1, wherein the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, OX40, OX40L, CTLA-4, 4-1BB, 4-1BBL, KIR, LAG-3, IDO1, TIM-3, A2AR, B7-H3, B7-H4, B7-1/B7-2, BTLA and VISTA, or a cognate ligand binding portion thereof.

10.-15. (canceled)

16. The therapeutic composition of claim 1, wherein the Sindbis virus encodes a tumor associated antigen (TAA) or an epitope of the TAA.

17. The therapeutic composition of claim 16, wherein the TAA is NY-ESO-1.

18. A method of increasing survival of a subject with cancer or a tumor, the method comprising administering to the subject an effective amount of the therapeutic composition of claim 1, thereby increasing the survival of the subject relative to a control subject.

19-28. (canceled)

29. A polynucleotide encoding an Alphavirus protein, or a fragment thereof, and an immune checkpoint molecule or a cognate ligand binding portion thereof.

30. A polynucleotide of claim 29 wherein the Alphavirus protein, or a fragment thereof, and an immune checkpoint protein or a ligand binding portion thereof is fused to an immunoglobulin hinge region and an immunoglobulin heavy chain constant domain.

31. (canceled)

32. A polynucleotide of claim 29 wherein the Alphavirus protein or a fragment thereof, and a fusion polypeptide comprises a secretory signal sequence linked to the immunoglobulin heavy chain constant domain, which is linked to the immune checkpoint protein, or an extracellular domain thereof; and wherein the fusion protein comprises one or more linker sequences.

33-48. (canceled)

49. A cell comprising the polynucleotide of claim 29.

50. A cell comprising the viral vector or viral particle of claim 42.

51. A pharmaceutical composition comprising the polynucleotide of claim 29. and a pharmaceutically acceptable vehicle, carrier, or diluent.

52. A method of inducing an immune response against a cancer or tumor cell, the method comprising contacting the cancer or tumor cell with an effective amount of the polynucleotide of claim 29 to induce the immune response against the cancer or tumor cell.

53. A method of treating cancer in a subject who has, or is at risk or having, cancer or tumorigenesis, the method comprising administering to the subject an effective amount of the polynucleotide of claim 29 to treat cancer in the subject.

54. A method of reducing tumor growth and/or increasing survivability in a subject who has cancer or a tumor, the method comprising administering to the subject an effective amount of the polynucleotide of claim 29 to reduce tumor growth and/or increase survivability in the subject.

55-60. (canceled)

61. A viral vector pseudotyped with one or more Alphavirus envelope proteins, wherein the viral vector comprises the polynucleotide of claim 29.

62. A Sindbis viral vector comprising the polynucleotide of claim 29.

63. A viral vector comprising the polynucleotide of claim 30.

64. A Sindbis viral vector comprising the polynucleotide of claim 30.

65. A Sindbis viral vector comprising the polynucleotide of claim 32.

66-73. (canceled)

74. The polynucleotide of claim 29, wherein the Alphavirus protein or a fragment thereof is derived from Barmah Forest virus, Barmah Forest virus complex, Eastern equine encephalitis virus (EEEV), Eastern equine encephalitis virus complex, Middelburg virus, Middelburg virus complex, Ndumu virus, Ndumu virus complex, Semliki Forest virus, Semliki Forest virus complex, Bebaru virus, Chikungunya virus, Mayaro virus, Subtype Una virus, O'Nyong Nyong virus, Subtype Igbo-Ora virus, Ross River virus, Subtype Getah virus, Subtype Bebaru virus, Subtype Sagiyama virus, Subtype Me Tri virus, Venezuelan equine encephalitis virus (VEEV), VEEV complex, Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Western equine encephalitis virus (WEEV), Rio Negro virus, Trocara virus, Subtype Bijou Bridge virus, Western equine encephalitis virus complex, Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Eilat virus, Salmon pancreatic disease virus (SPDV), Southern elephant seal virus (SESV), Tai Forest virus, or Tonate virus.

75. The therapeutic composition of claim 3, wherein the fusion polypeptide encoded by Sindbis virus comprises soluble 4-1BBL and a carboxy (C) terminal trimerization domain.

76. (canceled)

77. The therapeutic composition of claim 75, wherein the trimerization domain has the amino sequence IKQIEDKIEEILSKIYHIENEIARIKKL (SEQ ID NO: 24).

78. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0100] FIGS. 1A and 1B present polynucleotide sequences and amino acid sequences related to the PD-1 checkpoint protein. FIG. 1A presents the polynucleotide sequence (top, SEQ ID NO: 16) and the encoded amino acid (aa) sequence (bottom, SEQ ID NO: 17) of PD-1 (Programmed cell death protein 1) precursor [Homo sapiens]. The PD-1 polynucleotide sequence was cloned into the T7StuI-R vector (Accession No. NP_005009 VERSION NP_005009.2). The elements delineated in the sequences and depicted from the 5 end of the sequences are as follows: The soluble PD-1 amino acid (aa) sequence (1-169 aa) is shown in gray; the hinge region is shown by double underlining; the Linker sequence is shown in Italics; and the Ig CH3 domain is shown by single underlining. The CH3 and hinge domains are from human IgG1 (Accession No.: P01857.1). The Linker is synthetically produced. FIG. 1B shows an amino acid sequence comparison (alignment) of the human WT-PD-1 amino acid sequence (SEQ ID NO: 18) to PD-1 amino acid sequences of other species, namely, mouse (SEQ ID NO: 19) and monkey (SEQ ID NO: 20).

[0101] FIG. 2 presents Helper and Replicon DNA plasmids used to make the SV-PD-1WT vector for in vivo experiments as described herein.

[0102] FIG. 3: presents a schematic depiction of the design of the in vivo experiments described herein. For animals that received treatment with the immune checkpoint inhibitor, anti-PD-1 antibody, (aPD-1), the mice were dosed at days 4, 6, 8, 11, 13, 15; as shown the diagram. For animals that received treatment with the SV vector, SV_PD-1WT, Sindbis virus vector treatment was administered to the animals 4 times a week for 4 weeks, at days 5, 6, 7, 8 (week 1); 12, 13, 14, 15 (week 2); 20, 21, 22, 23 (week 3) and 27, 28, 29, 30 (week 4), as indicated. Tumor growth analysis was performed once a week bioluminescence was measured in the mice using IVIS at days 4, 11, 18, 25, 32, 39 and 46.

[0103] FIG. 4 presents graphs of tumor growth curves of mice treated with SV_PD-1WT Sindbis vector and of untreated (control) mice. Tumor growth is shown as fold changes relative to bioluminescence on the day before treatment of the same mouse with the Sindbis virus vector: (day1/day1); (day 6/day1); (day 13/day1) and (day 20/day1). Each time point shows the tumor growth average of the 5 mice in each group. Day 13 is the last day, with 5 mice/group for control (untreated) and PD1 antibody treated groups.

[0104] FIG. 5 shows representative bioluminescence images of control mice, anti-PD1 antibody-treated mice and SV_PD-1WT vector-treated mice bearing CT26.Fluc.NYESO1 tumors. Images correspond to days 4, 11 and 18 after tumor inoculation. Image scale min=50 Max=150 counts.

[0105] FIG. 6 shows survival curves of untreated Control animals (n=5), animals treated with anti-PD-1 antibody, (aPD1), (n=5) and animals treated with the Sindbis virus vector SV_PD-1WT, (n=5).

[0106] FIG. 7 shows a schematic depiction of the experimental design of the in vivo study described in Example 5. In accordance with the study, Sindbis virus vector treatments were administered to animals 4 times a week for 4 weeks at days: 6, 7, 8, 9 (week 1); days 13, 14, 15, 16 (week 2); days 21, 22, 23, 24 (week 3); and days 28, 29, 30, 31 (week 4) after tumor cell inoculation.

[0107] FIG. 8 presents a graph showing tumor growth curves over time (days) of treated and untreated (control) mice (n=5 for each group) as described in Example 5. Tumor growth curves are shown as fold changes relative to the bioluminescence on the first image taken of the same mouse before start of treatment.

[0108] FIGS. 9A and 9B present Kaplan-Meir survival plots of untreated control animals (n=5) and treated animal groups (n=5 per group). In FIG. 9A, four animal treatment groups are shown: (1) control untreated animals; (2) animals treated with a Sindbis virus (SV) vector encoding the tumor-associated antigen (TAA) NY-ESO-1. The designation of this SV vector is SV-NYESO1; (3) animals treated with an anti-OX40L antibody (commercially available from Bio-X-Cell, West Lebanon, N.H.; InVivoMAb anti-mouse OX40L (CD134L), Clone RM134L, Catalog #BE0033-1); and (4) animals treated with a combination of SV-NYESO1 and the anti-OX40L antibody. In FIG. 9B, four groups of treated animals are shown: (1) control untreated animals; (2) animals treated with SV-NYESO1; (3) animals treated with a Sindbis virus (SV) vector encoding OX40L. The designation of this SV vector is SV-OX40L; and (4) animals treated with a combination of SV-OX40L and SV-NYESO1. Comparing FIG. 9B with FIG. 9A, it can be seen that treatment with SV-OX40L is more efficacious than treatment with anti-OX40L antibody in promoting survival of the treated animals and that the combination of SV-NYESO-1 and SV-OX40L confers 100% survival of the treated animals, which is superior to the combination treatment of SV-NYESO1 and anti-OX40L antibody. The results suggest that armed Sindbis virus vectors (e.g., Sindbis virus vectors encoding a checkpoint inhibitor protein or a TAA as described herein) can provide tumor treatment that is superior to antibody-mediated therapy.

[0109] FIG. 10 presents a schematic depiction illustrating that a Sindbis virus vector is typically capable of expressing two full sized genes, one from each subgenomic promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0110] Provided by the present invention are polynucleotides and viral vectors, particularly, Alphavirus vectors, that encode an immune checkpoint protein, a costimulatory molecule, or a portion thereof that binds to the cognate ligand of the checkpoint protein or to the cognate ligand of the costimulatory molecule, which induce a potent immune response in a subject against the subject's cancer or tumor. An immune checkpoint molecule may also be referred to herein as a costimulatory molecule.

[0111] The present invention provides a polynucleotide that encodes an Alphavirus, lentivirus, or retrovirus protein or a fragment thereof, and an immune checkpoint molecule, or a cognate ligand binding portion or fragment thereof. In embodiments, the immune checkpoint molecule is, without limitation, PD-1, PD-L1, OX40, 4-1BB, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), or CTLA-4. In a particular embodiment, the immune checkpoint protein molecule is PD-1 or the extracellular domain of PD-1. In other particular embodiments, the immune checkpoint protein molecule is OX40L or 4-1BBL, or the extracellular domains thereof.

[0112] In an embodiment of the foregoing aspects, the polynucleotide encodes an Alphavirus (e.g., Sindbis virus protein or a fragment thereof) and an immune checkpoint molecule or a cognate ligand binding portion or fragment thereof. In an embodiment, the Alphavirus is Sindbis virus, a Sindbis virus vector, or Sindbis viral particle. In particular embodiments, the Sindbis virus vector contains a polynucleotide that encodes one or more immune checkpoint proteins, or a fragment or portion of the immune checkpoint protein that binds to its cognate ligand, for example and without limitation, the PD-1 immune checkpoint protein or a fragment or portion of PD-1 that binds to its cognate ligand PD-L1; the OX40L or a fragment or portion of OX40L that binds to its cognate receptor; or the 4-1BBL or a fragment or portion of 4-1BBL that binds to its cognate receptor. In a particular embodiment, the Sindbis virus vector contains a polynucleotide that encodes the PD-1 immune checkpoint protein, or an extracellular domain of PD-1, that binds to its cognate ligand PD-L1. In a particular embodiment, the Sindbis virus vector contains a polynucleotide that encodes the OX40L immune checkpoint protein, or an extracellular domain of OX40L, that binds to its cognate receptor. In a particular embodiment, the Sindbis virus vector contains a polynucleotide that encodes the 4-1BBL immune checkpoint protein, or an extracellular domain of 4-1BBL, that binds to its cognate receptor. In an embodiment, the checkpoint protein is a soluble form of the protein.

[0113] In an embodiment, the checkpoint protein encoded by the Sindbis virus vector is in the form of a minibody, as described herein, in which checkpoint protein or a portion of the checkpoint protein, e.g., the extracellular domain, is fused to portions of an immunoglobulin (Ig) molecule, thereby forming a fusion protein. In particular, checkpoint protein or a ligand binding portion of the checkpoint protein, e.g., the extracellular domain, is fused to an Ig hinge region, and an Ig heavy chain constant region domain, such as the CH1, CH2, or CH3 domain of an Ig heavy chain. In an embodiment, a spacer (or linker) sequence is inserted between the hinge region and the Ig heavy chain CH domain for flexibility. In an embodiment, the spacer (or linker) sequence is glycine-rich and is or comprises the amino acid sequence GGGSSGGGSGG (SEQ ID NO: 1) or the amino acid sequence GGGSSGGGSGS (SEQ ID NO: 2). In embodiments, the Ig is of the IgG (e.g., IgG1, IgG2a, IgG2b, IgG4 subtypes), IgM, IgA, IgD, or IgE type. In a specific embodiment, the Ig chain is the IgG1 heavy chain and the Ig constant region domain is the CH3 domain. In an embodiment, a glycine-rich spacer (or linker) sequence is inserted between the hinge region and the Ig heavy chain CH domain for flexibility. In an embodiment, the spacer (or linker) sequence is or comprises the sequence GGGSSGGGSGG (SEQ ID NO: 1).

[0114] In embodiments of the foregoing, the checkpoint protein is, without limitation, PD-1, PD-L1, OX40, OX40L, CTLA-4, 4-1BB, 4-1BBL, KIR, LAG-3, IDO1, TIM-3, A2AR, B7-H3, B7-H4, B7-1/B7-2, BTLA and VISTA, a cognate ligand binding portion thereof, or extracellular domain thereof. As will be appreciated by the skilled practitioner in the art, the following table categorizes checkpoint molecules (i.e., costimulatory molecules or receptors) as either activating molecules or receptors, which, upon being targeted and bound by agonistic antibodies, may enhance T cell stimulation to promote an immune response such as tumor destruction, or as blocking inhibitory molecules or receptors, which, upon being targeted and bound by blocking or inhibitory antibodies, may enhance T cell stimulation to promote an immune response such as tumor destruction. The molecules presented in the below table are illustrative and are not intended to be limiting.

Representative Checkpoint Molecules/Costimulatory Molecules/Receptors that may be Targeted and Bound by Antibodies

[0115]

TABLE-US-00011 Activating molecule/ Inhibitory molecule/ costimulatory costimulatory molecule/receptor molecule/receptor CD28 CTLA-4 OX40 PD-1 GITR TIM-3 CD137 BTLA CD37 VISTA HVEM LAG-3 (adapted from I. Mellman et al., 2011, Nature, 480(7378):480-489, which is incorporated herein by reference)

[0116] In a specific embodiment, the immune checkpoint proteins include PD-1, PD-L1, OX40, OX40L, 4-1BB, 4-1BBL, and CTLA-4. In an embodiment of the foregoing, the checkpoint protein is the extracellular domain of the protein. In a particular embodiment of the foregoing, the checkpoint protein is PD-1 or the extracellular domain of PD-1. In other particular embodiments of the foregoing, the checkpoint protein is OX40L, 4-1BBL or the extracellular domain thereof.

[0117] The invention is based, at least in part, on the discovery that a Sindbis virus vector encoding an immune checkpoint protein, such as the extracellular domain of a checkpoint protein, e.g., PD-1, OX40L, or 4-1BBL, resulted in a significant decrease in tumor growth and the long-term survival of tumor-bearing mice following treatment of the animals with a Sindbis virus vector encoding the checkpoint protein or a ligand binding portion thereof. In particular, treatment of animals with a Sindbis virus vector encoding the extracellular portion of wild-type PD-1 checkpoint protein or encoding OX40L, or 4-1BBL significantly reduced tumor growth in tumored animals relative to control animals, for example, for over 2 weeks, e.g., at least 20 days in the case of PD-1 or at least 18 days in the case of 4-1BBL. Treatment of tumored animals with this vector encoding PD-1 also resulted in a greater survival of animals following implantation of tumors. For example, by day 40 after tumor implantation, percent survival of tumored animals was approximately 3-times greater for animals that had been treated with the Sindbis virus vector encoding the PD-1 checkpoint protein compared with control animals. Treatment of tumored animals with an SV vector encoding OX40L (SV-OX40L) also resulted in a greater survival of animals following implantation of tumors compared with control animals (FIG. 9B). In addition, treatment of tumored animals with an SV vector encoding 4-1BBL (SV-4-1BB) resulted in a decrease in tumor growth in animals following implantation of tumors compared with control animals (FIG. 9A).

[0118] Surprisingly and unexpectedly, treatment of tumored animals with the Sindbis virus vector encoding the checkpoint protein (e.g., WT PD-1), as exemplified herein, resulted in a significant reduction in tumor growth compared with tumored animals that had been treated with an anti-PD-1 antibody, e.g., a more conventional checkpoint protein inhibitor treatment, and also compared with untreated control animals. In addition, and surprisingly, a significantly higher percentage of tumored animals survived following treatment with the Sindbis virus vector encoding the checkpoint protein (e.g., WT PD-1) compared with tumored animals that were treated with checkpoint inhibitor treatment with anti-PD-1 antibody.

[0119] Without wishing or intending to be bound by theory, following the administration of a Sindbis virus vector encoding an immune checkpoint protein, such as, e.g., PD-1, to a subject, large quantities of the checkpoint protein are expressed by the virus vector and soluble checkpoint protein is secreted systemically. Such large quantities of the checkpoint protein then circulate in a treated subject and are available to bind the cognate ligand, such as PD-L1, on tumor cells. The large amount of the checkpoint protein produced following administration of the Sindbis viral vector may thus directly compete with the binding of tumor cell-expressed cognate ligand (e.g., PD-L1) to T-cell expressed checkpoint protein (e.g., PD-1), thereby effectively blocking the binding of T-cell-expressed checkpoint protein to the tumor cell-expressed, interacting ligand. In such a system, the checkpoint protein encoded by the Sindbis virus vector, expressed in and produced from infected cells, may flood the tumor environment with soluble checkpoint protein that binds to the interacting ligand on tumor cells. Because of the occupation of the tumor-cell expressed ligand (e.g., cognate receptor protein, such as PD-L1) by the circulating checkpoint protein (e.g., PD-1), the tumor cell is unable to bind to cytotoxic T cell-expressed checkpoint protein. Consequently, cytotoxic T cells expressing checkpoint protein (e.g., PD-1) are not bound to and do not interact with cognate ligand on tumor cells (e.g., PD-L1), and the T cell cytotoxic activity is maintained and directed against the tumor cells, which are killed. Administration regimens for the checkpoint protein encoding viral vectors as described herein can be determined by a medical practitioner or clinician having skill in the art.

[0120] PD-1, the Programmed Death 1 (PD-1) protein, is a key immune checkpoint protein (receptor protein) that is expressed by activated T cells and mediates immunosuppression. PD-1 functions mainly in peripheral tissues where T cells may encounter the immunosuppressive PD-1 ligands PD-L1 (B7-H1) and PD-L2 that are expressed by tumor cells, stromal cells, or both. PD-1 produced in significant quantity by the Sindbis virus vector described herein serves to bind large quantities of PD-L1 on tumor cells, thus effectively inhibiting the normal interaction between cell-expressed PD-1 and PD-L1. Consequently, T-cell responses could be enhanced in vitro and could also mediate antitumor activity. Blockade of inhibitory receptors such as PD-L1 on tumors by the relatively large-scale, in vivo availability of Sindbis virus vector-produced, soluble checkpoint protein molecules encoded and expressed by the polynucleotides, Sindbis virus vectors and virus particles described herein offer a beneficial approach to prevent the inhibition of an anti-tumor immune response by T-cells and to augment the anti-tumor activity of T-cells whose inhibitory receptors are not blocked by binding to cognate ligand/receptors on tumor cells. The soluble checkpoint proteins expressed by the viral vectors as described herein may further act as decoys that bind ligand/receptors on tumor cells and block binding of the tumor cell ligand/receptor to the same checkpoint proteins that are expressed on the surfaces of effector T cells, such as cytotoxic T cells (CD8+ T cells). Such binding of the Sindbis virus vector-expressed checkpoint protein (or ligand binding portion thereof) to the cognate receptor protein expressed on tumor cells prevents a tumor cell from binding to the cytotoxic T cell that expresses the checkpoint protein, thereby preventing T cell anergy, which allows the cytotoxic T cell to kill the tumor.

[0121] In another embodiment, the treatment of tumored animals with the Sindbis virus vector encoding 4-1BBL, as exemplified herein, surprisingly resulted in a significant reduction in tumor growth compared with tumored animals that had been treated with an anti-4-1BB antibody, e.g., a more conventional checkpoint protein inhibitor treatment, compared with untreated control animals. (FIG. 8). In addition, and surprisingly, a significantly higher percentage of tumored animals survived following treatment with the Sindbis virus vector encoding the checkpoint protein OX40L compared with tumored animals that were treated with checkpoint inhibitor treatment with anti-OX40 antibody. (FIGS. 9A and 9B). Of note, combination treatment of tumored animals involving the administration of a Sindbis virus vector encoding the TAA, NY-ESO-1, plus a Sindbis virus vector encoding either 4-1BBL (SV4-1BBL) or OX40L (SV-OX40L) to the animals resulted in a significant reduction in tumor growth and a significantly higher percentage of survival of tumored animals, respectively, compared with antibodies directed against the respective checkpoint protein and with untreated controls.

[0122] In an embodiment, the immune response involves the activity of cytotoxic T cells which express checkpoint proteins on their surface, but are not made anergic by binding to cognate ligand expressed by tumor cells. In this embodiment, the checkpoint protein produced following administration of the Sindbis virus vector encoding the checkpoint protein binds to tumor cell-expressed ligand and prevents the tumor ligand from binding to and inactivating the anti-tumor activity that specifically kills the cancer or tumor cells. In an embodiment, the SV-encoded checkpoint protein-Ig fusion proteins as described and exemplified herein e.g., SV_PD-1, may facilitate binding to cells through the CH3 portion of the fusion protein, as well as trigger antibody dependent cell cytotoxicity (ADCC). Such checkpoint protein-Ig fusion proteins as described and exemplified herein may also be more stably expressed and have a longer half-life in vivo due to the Ig region components in the fusion protein.

[0123] The molecularly engineered viral vectors described herein provide an efficient and effective delivery system designed to harbor the genetic information of one or more checkpoint protein molecules and to promote a specific immune response, which ultimately allows cytotoxic T cells (e.g., effector CD8+ T cells) to remain activated to specifically kill the cancer or tumor.

[0124] The invention generally features virus vector-based compositions and methods that are useful for treating cancer and tumorigenesis and/or the symptoms thereof in a subject in need thereof, such as a patient having cancer. Methods utilizing viral vectors, which are designed to harbor polynucleotides encoding a checkpoint protein or a cognate binding portion thereof as described herein, involve administering a therapeutically effective amount of the viral vector, a viral particle, or a pharmaceutical composition comprising the viral vector or particle to a subject (e.g., a mammal such as a human), in particular, to elicit a T-cell-mediated immune response to the subject's cancer or tumor.

[0125] In an embodiment, particularly for the treatment and therapy of cancers, the polynucleotides, viral vectors and viral particles described herein may encode one or more checkpoint protein molecules, which following expression, bind to ligands with which they specifically interact.

[0126] In an embodiment, a wild-type (non-mutated) checkpoint protein is encoded by the Sindbis virus vector. In an embodiment, the wild-type checkpoint protein may bind more effectively to its cognate ligand than a checkpoint protein that has been genetically mutated or altered. In a particular embodiment, a wild-type PD-1 checkpoint protein is encoded by the Sindbis virus vector.

Tumor Associated Antigens (TAAs)

[0127] In some embodiments, the tumor associated antigens from which the epitopes that may be expressed by polynucleotides and viral vectors described herein are derived may be associated with, or expressed by, e.g., either extracellularly or intracellularly, a cancer or tumor, such as, without limitation, a/an ovarian cancer, breast cancer, testicular cancer, pancreatic cancer, liver cancer, colorectal cancer, thyroid cancer, lung cancer, prostate cancer, kidney cancer, melanoma, squamous cell carcinoma, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, promyelocytic leukemia, multiple myeloma, B-cell lymphoma, bladder carcinoma, head and neck cancer, esophageal cancer, brain cancer, pharynx cancer, tongue cancer, synovial cell carcinoma, neuroblastoma, uterine cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma. lymphangiosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'tumor, cervical cancer, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma. Hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroglioma, and retinoblastoma.

[0128] Additional examples of TAAs are known in the art and are described, for example, in Reuschenbach et al., Cancer Immunol. Immunother. 58:1535-1544 (2009); Parmiani et al., J. Nat. Cancer Inst. 94:805-818 (2002); Zarour et al., Cancer Medicine. (2003); Bright et al., Hum. Vaccin. Immunother. 10:3297-3305 (2014); Wurz et al., Ther. Adv. Med. Oncol. 8:4-31 (2016); Criscitiello, Breast Care 7:262-266 (2012); Chester et al., J. Immunother. Cancer 3:7 (2015); Li et al., Mol. Med. Report 1:589-594 (2008); Liu et al., J. Hematol. Oncol. 3:7 (2010); Bertino et al., Biomed. Res. Int. 731469 (2015); and Suri et al., World J. Gastrointest. Oncol. 7:492-502 (2015).

[0129] Any tumor associated antigen (TAA) having epitopes and expressed by a cancer cell or solid tumor can be utilized in conjunction with the compositions and methods described herein. However, it is expected that variability may exist in the efficacy of different TAAs and their associated epitopes to induce or increase an immune response in a subject, because some TAAs and/or their epitopes may potentially induce more robust responses (i.e., immunodominant TAAs). Relevant reports, e.g., preclinical and clinical study reports, can be used to guide the choice of TAAs or epitopes thereof to be incorporated into a polynucleotide (minigene), viral vector, viral particle, or pharmaceutical composition of the invention. In some embodiments, coding sequences of TAAs or the epitopes thereof that are capable of inducing a robust immune response, that bind MHC class I proteins with high affinity, or that bind MHC class II proteins with high affinity are incorporated into the polynucleotide, viral vector, viral particle, or pharmaceutical composition of the invention. By way of example, NY-ESO-1, the cancer-testis antigen, is desirable for use as a tumor associated antigen for cancer immunotherapy, because it is expressed in several different cancer and tumor types, e.g., breast cancer, lung cancer, melanoma, as well as in the testis and placenta; however, it is not expressed in other normal adult tissues.

[0130] Ways in which TAAs may be selected for inclusion in virus vectors are described in co-pending PCT Application No. PCT/US18/20985, the contents of which are incorporated by reference herein.

Sindbis Virus Vectors (T7Sindbis Vectors) Expressing Checkpoint Molecules/T-cell Costimulatory Molecules

[0131] Sindbis vectors were designed to express molecules that enhance the antitumor immune response. Optimal activation of T cells requires a strong T cell receptor-peptide antigen-MHC interaction, in addition to the ligation of co-receptors, on the surface of T cells, with cognate checkpoint molecules or costimulatory molecules expressed on antigen-presenting cells (APCs). Co-signaling molecules have been shown to control T-cell activation by regulating T-cell proliferation, cytokine production, cytotoxicity, T-cell apoptosis, and survival.

[0132] The co-signaling molecules can be grouped into two superfamilies based on their structure: the immunoglobulin (Ig) superfamily and the tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily. The Ig superfamily includes the costimulatory molecule, CD28 and ICOSL. The tumor necrosis factor (TNF) superfamily contains multiple receptor/ligands that play pivotal roles in the immune response. Members of the TNF superfamily all share a TNF homology domain that can form non-covalent homotrimers. While the TNF ligands are typically expressed as cell surface molecules, the extracellular domain can be proteolytically shed from the membrane. Representative and nonlimiting members of the TNF family of costimulatory molecules include 4-1BB/4-1BBL and OX40/OX40L.

[0133] As described herein the Sindbis virus vector platform can advantageously incorporate multiple checkpoint protein/immunomodulatory molecules and/or tumor associated antigens (TAAs) to achieve optimal anti-tumor immune responses in tumored subjects.

Viruses and Viral Vectors

Alphavirus, Sindbis Virus and Sindbis Virus Vectors

[0134] Alphaviruses belong to the group IV Togaviridae family of viruses that are small, spherical, enveloped, positive-sense, single-stranded RNA viruses. Most alphaviruses infect and replicate in vertebrate hosts and in hematophagous arthropods, such as mosquitoes. Alphavirus virions are spherical with an iscoahedral nucleocapsid enclosed in a lipid-protein envelope. Alphavirus RNA is a single 42S strand of approximately 410.sup.6 daltons that is capped and polyadenylated. The Alphavirus envelope comprises a lipid bilayer derived from the host cell plasma membrane and contains two viral glycoproteins, E1 (48,000 daltons) and E2 (52,000 daltons). A third, small E3 protein (10,000-12,000 daltons) is released from the virus as a soluble protein in alphaviruses other than Semliki Forest virus, where the E3 protein remains virus-associated.

[0135] As described herein, polynucleotides encoding an Alphavirus protein, or a fragment thereof, and a checkpoint protein or a ligand binding fragment thereof are embraced by the invention. In addition, the present invention encompasses viral vectors and particles that are pseudotyped with proteins, e.g., envelope proteins, from other virus types. The polynucleotides, viral vectors and viral particles described herein encompass nucleic acid sequences and polypeptide sequences of members of the Alphavirus genus, including various strains, antigenic complexes, species and subtypes. Encompassed by the invention are alphaviruses, phylogenetically related alphaviruses, Alphavirus complexes, and their structural components, such as envelope proteins, e.g., E1, as described, for example, in Powers, A. M. et al., 2011, J. Virol., 75 (21):10118-10131. Nonlimiting examples of alphaviruses, and polynucleotides and proteins thereof, as well as fragments of their polynucleotides and proteins, that may be used in the polynucleotides, viral vectors and viral particles as described herein include Barmah Forest virus, Barmah Forest virus complex, Eastern equine encephalitis virus (EEEV), Eastern equine encephalitis virus complex, Middelburg virus, Middelburg virus complex, Ndumu virus, Ndumu virus complex, Semliki Forest virus, Semliki Forest virus complex, Bebaru virus, Chikungunya virus, Mayaro virus, Subtype Una virus, O'Nyong Nyong virus, Subtype Igbo-Ora virus, Ross River virus, Subtype Getah virus, Subtype Bebaru virus, Subtype Sagiyama virus, Subtype Me Tri virus, Venezuelan equine encephalitis virus (VEEV), VEEV complex, Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Western equine encephalitis virus (WEEV), Rio Negro virus, Trocara virus, Subtype Bijou Bridge virus, Western equine encephalitis virus complex, Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Eilat virus, Salmon pancreatic disease virus (SPDV), Southern elephant seal virus (SESV), Tai Forest virus and Tonate virus.

[0136] As an Alphavirus, Sindbis virus is a small, enveloped, positive-sense, single strand RNA virus. Other members of the Alphavirus genus include, without limitation, Semliki Forest virus (SFV), Venezuelan equine encephalitis virus (VEEV) and Ross River Virus (RRV). Alphaviruses, including Sindbis virus, form spherical particles of 60-70 nm in diameter; the icosahedral structures of many alphaviruses have been defined to very high resolutions by cryo-electron microscopy (cryo-EM) and crystallographic studies, revealing details of the interactions between the structural proteins (Jose, J. et al., 2009, Future Microbiol., 4:837-856). The genome is composed of a single strand of positive-sense RNA that is approximately 11 to 12 kb in length and encodes four nonstructural proteins (nsP1-nsP4) involved in virus replication and pathogenesis, and five structural proteins that compose the virion particle, i.e., the nucleocapsid protein C and the envelope proteins, P62 (proteolytically cleaved into the mature envelope proteins E2 and E3) and the E1 protein. Alphaviruses exhibit efficient replication and have broad range of susceptible and permissive hosts; therefore, these viruses are highly suitable for heterologous gene expression and as gene therapy delivery vectors. Alphavirus vectors are suitable for use in encoding the polynucleotides (minigenes) for delivering the multi-epitopes of tumor associated antigens as described herein.

[0137] Any Sindbis viral vector is suitable for use in conjunction with the polynucleotides, virus vectors, compositions and methods of the present invention, including replication-competent vectors (see, e.g., U.S. Pat. No. 8,282,916) and replication-defective vectors (see, e.g., U.S. Pat. Nos. 7,303,898, 7,306,792, and 8,093,021). Replication-defective vectors are preferred for use in the present invention, as they offer another layer of protection against infection of healthy tissues. Sindbis vectors can also be constructed to contain more than one subgenomic promoter to express more than one gene using methods known in the art.

[0138] By way of example, to produce the pT7StuI-R/epitope vector, the replicon plasmid encoding the Sindbis replicase genes (nsP1-nsP4) and a helper plasmid, encoding the viral structural genes (capsid protein C, E1, E2, E3, and 6K), were transcribed in vitro. To limit viral replication in vivo, the replicon genes have been separated from the structural genes, which additionally contain a mutated packaging signal to prevent incorporation into virus particles (Bredenbeek, P. J. et al., 1993, J Virol 67: 6439-6446). Virus particles were produced by transient transfection of baby hamster kidney (BHK) cells with in vitro synthesized Sindbis replicon RNA and helper RNA transcripts. Within the cell, genomic RNA was replicated by the Sindbis replicase and expressed from the capped replicon RNA transcript. Structural proteins were expressed from the helper RNA transcript. Only the replicon RNA was packaged into the capsid to form the nucleocapsid, which then associates with the viral glycoproteins E1 and E2 and buds out of the cell. The resulting virion contained the capped SV single-stranded RNA message for nsP1-nsP4 genes, which encode the viral replicase, a subgenomic promoter (Psg) from which the replicase can transcribe an inserted gene of interest and a poly A tail. Example 2 herein describes methods to produce a Sindbis viral vector encoding a checkpoint protein or a ligand binding portion thereof.

Lentivirus

[0139] Lentiviral vectors are particularly useful for long-term expression of genes, as they have the ability to infect both dividing and non-dividing cells. Third generation lentiviral systems are preferred for increased safety (Breckpot, K., et al., 2007, Gene Ther, 14: 847-862). These include, e.g., a transfer plasmid into which nucleic acid sequences encoding two or more epitopes of a tumor associated antigen is inserted, a packaging plasmid for gag and pol genes and another packaging plasmid for the rev gene. For optimal expression, the transfer expression vectors contain a splice donor, a packaging signal (psi), a Rev-responsive element (RRE), splice acceptor, central poly-purine tract (cPPT), and Wood chuck hepatitis virus transcriptional response element (WPRE) (Shaw and Cornetta, 2014, Biomedicines, 2:14-35). Transfer vector constructs may also contain a promoter for expression in mammalian cells. Constitutive promoters, such as the cytomegalovirus (CMV), mammalian beta-actin, or ubiquitin promoters may be incorporated into a composition of the invention. In some embodiments, tissue-specific promoters are utilized, such as CD4+T cell-specific promoters.

[0140] Plasmids for generating lentiviral vectors can be obtained from Addgene (Cambridge, Mass., a non-profit plasmid repository) and modified, as necessary, using standard techniques in the art. Standard 3.sup.rd generation packaging plasmids can be used. Suitable transfer vectors include, for example, pLX301, pFUGW, and pWPXL. These vectors contain all of the requisite characteristics mentioned above. To increase safety, the lentivirus transfer vectors can be mutated to decrease integration and increase episomal replication in infected cells. For instance, using standard techniques known in the field, the following modifications can be performed: a deletion within the U3 region of the 3 LTR to create a self-inactivating LTR (SIN-LTR) is made; LTR att sites within the U3 and U5 LTR regions are deleted or mutated; the 3 LTR-proximal polypurine tract (PPT) are deleted or modified (Shaw and Cornetta, 2014).

[0141] Pseudotyped viral vectors and virions are also suitable for use in connection with the polynucleotides and compositions of the invention. Such virions contain a viral particle and one or more foreign virus envelope proteins. (D. A. Sanders, 2002, Curr. Opin. Biotechnol., 13:437-442). In some embodiments, a viral vector of the invention may be a lentivirus containing an Alphavirus protein or a fragment thereof, e.g., an envelope protein or a functional fragment thereof. In some embodiments, a viral vector of the invention may be a lentivirus containing a Sindbis virus envelope glycoprotein, or certain Sindbis virus envelope glycoproteins. By way of example, to produce a construct (e.g., a pseudotyped viral vector) comprising a lentivirus backbone pseudotyped with one or more Sindbis envelope proteins, a Sindbis envelope plasmid, e.g., T7 DM helper #101 (U.S. Pat. No. 8,093,021) is transfected into BHK or 293 cells along with the lentiviral plasmids resulting in pseudotyped virions.

Retrovirus

[0142] Retroviral vectors are also suitable for use according to the invention. In some embodiments, the retroviral vector is Moloney murine leukemia virus (Mo-MuLV) pseudotyped with Sindbis envelope proteins. Pseudotyping can be performed using methods known in the art (see, e.g., Sharkey et al., 2001, J. Virology, 75 (6):2653-2659). In some embodiments, the Mo-MuLV-based retrovirus particles are engineered to include and express the glycoproteins of the Alphavirus Ross River virus (RRV) using methods known and practiced in the art.

Sindbis Virus Envelope Pseudotyped Vectors

[0143] The Sindbis virus (SV) envelope is advantageous for use as a gene or polynucleotide delivery vector. SV is a blood-borne virus with a relatively long half-life. Stable virus is easily produced and can be concentrated for administration. Modification of the Sindbis E2 envelope protein, which binds to cell surface molecules, does not affect the E1 fusogenic envelope protein that is required for cell entry, thus allowing for engineered targeting of the virus. Sindbis virus specifically targets tumors by interacting with the high-affinity laminin receptor (LAMR) (U.S. Pat. No. 7,306,792), which is found in the 40S ribosome and is over-expressed by many tumors (e.g., breast, thyroid, colon, prostate, stomach, pancreas, ovary, melanocytes, lung, liver, uterus), but does not infect normal tissues. As a blood-borne virus, Sindbis virus is capable of contacting disseminated metastatic tumor cells via the bloodstream.

[0144] Sindbis viral envelope structural proteins can pseudotype other viral vectors, such as lentivirus, retrovirus and Vesicular Stomatitis virus (VSV) to improve their targeting capabilities and increase virion stability. In particular, the Sindbis-ZZ protein, designed to contain the Fc binding domain of S. aureus protein A inserted into the E2 envelope protein (U.S. Pat. No. 6,432,699), is useful in conjunction with cell surface specific antibodies for redirecting the targeting of SV and other vectors.

[0145] In certain embodiments in which long-term, stable expression of encoded protein is desired, retroviral or lentiviral vectors pseudotyped with wild type or engineered Sindbis virus envelope proteins are employed. Lentiviral vectors are advantageous for infection of both dividing and non-dividing cells. Like the Sindbis virus genome, the lentivirus genome can be split into two or three vectors, and genes can be modified or deleted to improve safety. A retrovirus subtype lentivirus naturally integrates into the host genome. However, vectors containing either long terminal repeats (LTR) or integrase enzyme mutations can exist as stable, non-integrating episomes in the cell nucleus (Breckpot, K., et al., 2007, Gene Ther., 14:847-862).

[0146] In particular embodiments, a therapeutic composition of the invention comprises a replication defective Sindbis virus described in U.S. Pat. Nos. 7,303,898, 9,423,401; 8,530,232; or 8,093,021.

Pharmaceutical Compositions

[0147] The present invention includes pharmaceutical compositions or formulations for treating subjects who are afflicted with cancer or a tumor, or who are at risk of developing cancer or a tumor. In an embodiment, the pharmaceutical composition includes viral vector, e.g., a Sindbis virus vector containing a polynucleotide encoding a checkpoint protein or a checkpoint protein minibody as described herein, or a cognate ligand binding portion thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. In an embodiment, the pharmaceutical composition includes a Sindbis viral vector or a pseudotyped viral vector as described herein and a pharmaceutically acceptable carrier, excipient, or diluent. When formulated in a pharmaceutical composition, a therapeutic compound or product of the present invention can be admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0148] The administration of a composition comprising the therapeutic Sindbis vectors described herein for the treatment of a cancer or tumor may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a cancer in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Routes of administration include, for example, subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (i.p.), intramuscular (i.m.), or intradermal administration, e.g., by injection, that optimally provide continuous, sustained levels of the agent in the patient. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age, physical condition and body weight of the patient, and with the clinical symptoms of the cancer or tumor. Generally, amounts will be in the range of those used for other viral vector-based agents employed in the treatment of a cancer or tumor, although in certain instances lower amounts will be needed if the agent exhibits increased specificity. A composition is administered at a dosage that shows a therapeutic effect, such as increasing immune cell (e.g., effector T cell; CD8+ T cell) levels, or that decreases cancer cell proliferation or reduces tumor size, as determined by methods known to one skilled in the art.

[0149] The therapeutic agent(s) may be contained in any appropriate amount in any suitable carrier substance, and is/are generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for a parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route, such that the agent, such as a viral vector described herein, is systemically delivered. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0150] Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the agent within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with a tumor; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a cancer using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., cancer or tumor cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level of the administered agent at a therapeutic level.

[0151] Methods by which to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question are not meant to be limiting. By way of example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic agent is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the agent in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0152] The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation, and can be found, for example, in Remington: The Science and Practice of Pharmacy, supra.

[0153] Compositions for parenteral delivery and administration may be provided in unit dosage forms (e.g., in single-dose ampules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent (e.g., a polynucleotide, viral vector or particle described herein), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

[0154] In some embodiments, the composition comprising the active therapeutic(s) (i.e., a polynucleotide, viral vector or particle described herein) is formulated for intravenous delivery. As noted above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be employed include water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Methods of Delivery

[0155] In embodiments, the viral vector, viral particle, or pharmaceutical composition of the invention may be delivered, such as to a cell (particularly a cancer or tumor cell) in any manner such that the viral vector, particle or composition is functional and active to express the encoded sequences. Illustratively, a Sindbis virus vector harboring a polynucleotide encoding a checkpoint protein or a ligand binding portion thereof may be delivered to cells for heterologous expression in the cells. Thus, the present invention features viral vectors, or viral particles delivered to a cell by contacting the cell with the Sindbis virus vector, or a composition comprising the vector, or viral particles, or by heterologously expressing the polynucleotides, viral vectors, or viral particles in the cell.

Polynucleotide Therapy

[0156] One therapeutic approach for treating a cancer or tumorigenesis is polynucleotide therapy using a polynucleotide encoding a checkpoint protein molecule as described herein. Expression of such polynucleotides or nucleic acid molecules in relevant cells and production of the protein is expected to stimulate an immune response, such as a cytotoxic T cell response, reduce survival of the cell and/or increase cell death. Such nucleic acid molecules can be delivered to cells of a subject having a cancer or tumor. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the encoded products can be produced.

[0157] Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for delivering encoded proteins and peptide products to cells, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy, 8:423-430, 1997; Kido et al., Current Eye Research, 15:833-844, 1996; Bloomer et al., Journal of Virology, 71:6641-6649, 1997; Naldini et al., Science, 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A., 94:10319, 1997).

[0158] For example, a polynucleotide encoding a checkpoint protein or a ligand binding portion thereof, as well as a checkpoint protein minibody as described herein, can be cloned into a vector, e.g., a Sindbis virus vector or a pseudotyped virus vector, as described herein, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus (see, for example, the vectors of Miller, Human Gene Therapy, 15-14, 1990; Friedman, Science, 244:1275-1281, 1989; Eglitis et al., BioTechniques, 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology, 1:55-61, 1990; Sharp, The Lancet, 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology, 36:311-322, 1987; Anderson, Science, 226:401-409, 1984; Moen, Blood Cells, 17:407-416, 1991; Miller et al., Biotechnology, 7:980-990, 1989; Le Gal La Salle et al., Science, 259:988-990, 1993; and Johnson, Chest, 107:77S-83S, 1995). Retroviral vectors are well developed and have been used, for example, as described in Rosenberg et al., NEJM, 323:370, 1990; Anderson et al., and U.S. Pat. No. 5,399,346. In some embodiments, the viral vector containing a polynucleotide encoding a checkpoint protein, a ligand binding portion thereof, or a checkpoint protein minibody is administered systemically. In an embodiment, administration is performed intravenously or intraperitoneally.

[0159] As will be appreciated by the skilled practitioner, non-viral approaches can also be employed for the introduction of a therapeutic polypeptide to a cell of a subject requiring induction of a T cell immune response to inhibit growth of a cancer or tumor or to induce cancer or tumor cell death. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters, 17:259, 1990; Brigham et al., Am. J. Med. Sci., 298:278, 1989; Staubinger et al., Methods in Enzymology, 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry, 263:14621, 1988; Wu et al., Journal of Biological Chemistry, 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science, 247:1465, 1990). In addition, the nucleic acids can be administered in combination with a liposome and protamine.

[0160] Gene transfer can also be achieved using in vitro transfection methods. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell.

[0161] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the Sindbis virus promoter, the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

Methods of Administration and Treatment Protocols

[0162] Provided are methods of administering a therapeutic agent to a subject in need, such as a subject having cancer or a tumor, or identified as needing such treatment), in which an effective amount of a polynucleotide, viral vector, or viral particle as described herein, or a composition described herein, is administered to a subject to produce a therapeutic effect. According to the present invention, a therapeutic effect includes, without limitation, an immune response against cancer and tumor cells expressing checkpoint protein binding molecules (e.g., receptors that bind checkpoint protein) on their surface, e.g., by effector T cells (e.g., CD8+ T cells). Identifying a subject in need of such treatment can be achieved based on the judgment of a subject or a health or medical care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

[0163] The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the agents described herein, such as a polynucleotide, a viral vector, a viral particle, or composition containing the aforementioned agents, to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for cancer or a tumor. Determination of those subjects at risk can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker or biomarker, family history, and the like). The polynucleotide and viral vector agents described herein may be also used in the treatment of any other diseases or disorders in which checkpoint proteins and their interacting binding molecules (protein receptors) may be implicated.

[0164] In preclinical studies using mice, intraperitoneal (i.p.) injections of a therapeutically effective amount of the Sindbis viral vector encoding the checkpoint protein (e.g., a checkpoint protein minibody), (10.sup.5 virus particle transforming units), resulted in an immune response directed against the tumor and a reduction in tumor growth and increased survival of treated animals (Example 2, infra). It will be appreciated by the skilled practitioner that other regimens may be necessary for achieving a maximal response in human subjects. For example, in human patients, therapeutically effective amounts of the vectors described herein can broadly range between about 6 and about 12 Log.sub.10 vector particles/kg per treatment administered over time, e.g., between about 1 and about 8 i.p. (intraperitoneal) injections over a time period of between about 1 week and many weeks, with the possibility of injecting one or more booster injections, week, months, or years, e.g., 1 or more years, later.

[0165] Viral vectors, polynucleotides and pharmaceutical compositions of the present invention can be used therapeutically to treat patients suffering from cancer or tumors, or prophylactically to vaccinate patients at risk for certain cancers or tumors, such as a prophylactic vaccine for cancer in the general population. A prophylactically effective amount of the vectors of the present invention may range between about 10.sup.2 TU (transducing units) per kilogram body weight of the recipient and about 10.sup.8 TU kilogram body weight of the recipient. Mouse models of relevant cancers can be used to optimize dosages and regimens. To promote an effective, persistent immune response that includes both effector and memory CD8+ T cells, optimal dosage and immunization intervals are established. A CD8+ T cell response to an initial Alphavirus vaccine quickly contracts, allowing development of memory T cells. Prior to this contraction, additional administration of the viral vector does not increase the immune response (Knudsen, M. L. et al., 2014, J Virol., 88:12438-12451). The strong type I interferon (IFN) response to Alphavirus RNA amplification stimulates the generation of memory T cells by activating dendritic cells to promote cross-priming (Fuertes, M. B. et al., J Exp Med, 208: 2005-2016).

[0166] A typical treatment regimen using a vector or composition as described herein may include SV_checkpoint protein viral vector administration followed by monitoring of lymphocytes, several times per week, using flow cytometry to determine the peak and decline of effector CD8+ T cells (CD62L.sup.CD127.sup. phenotype). In an embodiment, a boost of vector can be administered allowing an increase in effector memory T cells (CD62L.sup.CD127+), central memory T cells (CD62L.sup.+CD127.sup.+) and T cells with persistent high recall capacity (CD27.sup.+CD43.sup.). Efficacy is determined by positive immune response and low tumor recurrence.

[0167] The vectors used for immunization boost(s) are not limiting. The distribution of T cell subpopulations induced by a DNA-launched Alphavirus replicon can be altered by heterologous boost (Knudsen, M. L. et al., 2-14, J. Virology, 88:12438-12451). For example, boosting with a poxvirus vector (Modified Vaccinia Ankara or MVA) can boost the expansion of T cell compartments that can greatly augment efficacy. In this embodiment, the viral vector employed in the booster administration encodes multiple (e.g., two or more) epitopes of one or more tumor associated antigens. Any antigen delivery system can be used to boost the immune response induced by the vectors of the present invention. Non-limiting examples include replication-defective adenoviruses, fowl pox viruses, vaccinia virus, influenza virus, Sendai virus, naked DNA, plasmids and peptides (Woodland, D. L., 2004, TRENDS in Immunology, Vol. 25 (2):98-104).

[0168] Exemplary routes of vector administration include, without limitation, parenteral administration, such as by intraperitoneal, intravenous, subcutaneous, stereotactic, intramuscular, intranasal, intradermal, intraorbital, intranodular and intratumoral injection. Other modes of administration may include oral, intracranial, ocular, intraorbital, intra-aural, rectal, intravaginal, suppositories, intrathecal, inhalation, aerosol, and the like.

[0169] In a certain embodiment, the vector used for treatment is a defective Sindbis viral vector, the tumor is a cancer or tumor, such as colon cancer or ovarian cancer, and the checkpoint protein encoded by the viral vector is PD-1. In other embodiments, one or more checkpoint proteins selected from PD-L1, OX40, OX40L, CTLA-4, 4-1BB, 4-1BBL, KIR, LAG-3, IDO1, TIM-3, A2AR, B7-H3, B7-H4, B7-1/B7-2, BTLA, VISTA, or a cognate ligand binding portion thereof may be used.

[0170] Patients to whom the viral vectors of the present invention are administered may also benefit from adjunct or additional treatments, such as an anti-cancer or tumor agent, chemotherapy and/or radiation treatments, as are well known to the skilled practitioner in the art. In particular, the Sindbis viral vector encoding a checkpoint protein (SV/checkpoint protein) can be combined with chemotherapy treatment. In certain cases, SV and chemotherapy synergize (e.g., U.S. Patent Application Publication No. 2016/0008431), thus providing the potential for an improved treatment effect and/or outcome. Suitable chemotherapy includes, without limitation, chemotherapy treatment that stimulates the immune system, or that inhibits suppressor elements in the immune system, or that affects tumor cells and makes them more susceptible to T cell (or other immune cell) cytotoxicity. For example, there are certain chemotherapies that can facilitate treatment and therapy with the Sindbis viral vectors described herein, because they attenuate the activity of immunosuppressive cells, thereby enhancing immunostimulation by the viral vector. In addition, chemotherapy may enhance tumor cell susceptibility to T cell mediated cytotoxicity.

Kits

[0171] Provided are kits for the treatment or prevention of cancer or tumors. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a polynucleotide, viral vector, or viral particle as described herein, which comprises a polynucleotide that encodes a checkpoint protein, a ligand binding portion of the checkpoint protein (e.g., an extracellular domain of the checkpoint protein), or a minibody checkpoint protein fusion protein. In an embodiment, the polynucleotide encodes an Alphavirus protein or a fragment thereof. In an embodiment, the Alphavirus protein or a fragment thereof is a Sindbis virus protein or a fragment thereof. In an embodiment, the encoded checkpoint protein is PD-1. In other embodiments, the checkpoint protein is one or more of PD-L1, OX40, OX40L, CTLA-4, 4-1BB, 4-1BBL, KIR, LAG-3, IDO1, TIM-3, A2AR, B7-H3, B7-H4, B7-1/B7-2, BTLA, VISTA, or a cognate ligand binding portion thereof. In some embodiments, the kit comprises a sterile container which contains the therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. The containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0172] If desired, a composition comprising one or more checkpoint protein-encoding viral vector agents of the invention is provided together with instructions for administering the agent to a subject having or at risk of developing cancer or a tumor. The instructions will generally include information about the use of the composition for the treatment or prevention of the cancer or tumor. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0173] One having skill in the art will appreciate that the practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides, viral vectors and viral particles of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0174] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the virus products, compositions and therapeutic methods as described, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1Methods

[0175] Vector preparation: Construction of recombinant viral vectors was performed using standard techniques well known to those of ordinary skill in the field of molecular biology, including, but not limited to, plasmid purification, restriction endonuclease digestion, ligation, transformation, polymerase chain reaction and DNA sequencing (e.g., Current Protocols in Molecular Biology, E M. Ausubel et al. (Eds), John Wiley and Sons, Inc., NY, USA. (1998) and Molecular Cloning: A Laboratory Manual (2nd Ed.), J. Sambrook, E. F. Fritsch and T. Maniatis (Eds), Cold Spring Harbor Laboratory Press, NY, USA. (1989)).

[0176] For the experiments using Sindbis viral vector encoding PD-1 (SV/PD-1) and SV/Fluc and SV/GFP as control vectors, the vectors were produced as previously described. (Tseng J. C. et al, 2004, Nat. Biotechnol., 22:70-77). Briefly, plasmids carrying the replicon (SinRep5-LacZ, SinRep5-GFP, or SinRep5-Fluc) or DHBB helper RNAs (SinRep5-tBB) were linearized with XhoI (for SinRep5-LacZ, SinRep5-GFP, and SinRep5-tBB) or PacI (for SinRep5-Fluc). In vitro transcription was performed using the mMessage mMachine RNA transcription kit (Ambion, Austin, Tex.). Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37 C. in -MEM supplemented with 10% FBS. After 12 hours, the medium was replaced with OPTI-MEM I (Invitrogen, Carlsbad, Calif.), supplemented with CaCl.sub.2 (100 g/ml), and cells were incubated at 37 C. After 24 hours, the supernatant was collected, centrifuged to remove cellular debris, and frozen at 80 C. Vector titers were determined as known in the art (Tseng J. C., et al., 2002, J Natl Cancer Inst., 94:1790-1802) and were similar in all three vectors (SV/LacZ, SV/Fluc, and SV/GFP).

[0177] Cell lines and Cell Culture: Baby hamster kidney (BHK), CT26.WT cells were obtained from the American Type Culture Collection (ATCC), (Manassas, Va.). BHK cells were maintained in minimum essential -modified media (-MEM) (Mediatech, Va.) with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.). CT26.WT cells were maintained in Dulbecco modified essential media (DMEM) containing 4.5 g/L glucose (Mediatech) supplemented with 10% FBS. All basal medium was supplemented with 100 mg/mL of penicillin-streptomycin (Mediatech) and 0.5 mg/mL of amphotericin B (Mediatech).

[0178] Virion Production: Sindbis virus vectors were produced as described in U.S. Pat. Nos. 7,303,898, 7,306,792, and 8,093,021. Briefly, plasmids carrying the replicon pT7StuI-R or DHBB helper RNAs (SinRep5-tBB) were linearized with appropriate restriction enzymes. In vitro transcription was performed using the mMessage RNA transcription kit (Ambion, TX) according to the manufacturer's instructions. Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37 C. in MEM supplemented with 10% FBS. After 12 hours, the medium was replaced with OPTIMEM I (Life Sciences, CA) supplemented with CaCl.sub.2 (100 g/mL) and cells were incubated at 37 C. After 24 hours, the supernatant was collected, centrifuged to remove cellular debris, and frozen at 80 C. Titers of the vectors were determined using RT-qPCR as practiced in the art.

[0179] Therapeutic Efficacy: Therapeutic efficacy was monitored in three ways: tumor volume (for subcutaneous tumors, measured with mechanical calipers), tumor luminescence and survival. Noninvasive bioluminescent imaging was performed using the IVIS Spectrum imaging system (Caliper Life Sciences, Inc., MA), and tumor growth was quantified using the Living Image 3.0 software (Caliper Life Sciences). Survival of the animals was monitored and recorded daily.

[0180] Flow cytometry: Flow cytometry was used to analyze lymphocytes extracted from organs, peritoneum or peripheral blood. Cells were treated with 1 RBC lysis buffer (eBioscience) to eliminate red blood cells. Peritoneal cells were collected and stained with various Abs, washed twice with HBSS buffer (Mediatech), and analyzed using an LSR II machine (BD Biosciences, San Jose, Calif.). Data were analyzed using FlowJo (Tree Star, San Carlos, Calif.).

[0181] Bioluminescent imaging of SV/Fluc: Tumor-bearing and tumor-free mice were injected with SV/Fluc (10.sup.7 plaque-forming units in 0.5 ml of OPTI-MEM I 0.5ml) i.p. After the treatment, bioluminescence signal was detected by IVIS at the indicated time points (Tseng, J. C. et al., 2004).

Example 2Sindbis Virus Vector Encoding the Immune Checkpoint Protein PD-1 Provided Anti-tumor Efficacy In Vivo

[0182] This Example describes studies conducted utilizing a Sindbis virus vector which contained a polynucleotide encoding the extracellular portion of PD-1, a checkpoint protein (receptor protein) expressed by T cells, which plays a role in downregulating the immune response.

Materials and Methods

Cell Lines

[0183] Baby hamster kidney cells (BHK-21; ATTC CCL-10) were maintained in minimum essential -modified medium (-MEM) (Corning CellGro) supplemented to contain 5% fetal bovine serum (FCS, Gibco) and 100 mg/mL penicillin-streptomycin (Corning CellGro). BHKSINLuc2 cells (ATCC CRL12071) were cultured in a manner similar to that of BHK cells, and 400 g/mL Geneticin was included in the culture medium.

[0184] The BALB/c colon carcinoma (CT26) cell line was obtained from the American Type Culture Collection (ATCC: CRL 2638). Firefly luciferase (Fluc)-expressing CT26 cells (CT26.Fluc) were generated by stable transfection of the pGL4.20_Fluc plasmid that expresses luciferase from an SV40 promoter and has puromycin as a selection marker. The CT26 cell line expressing both Firefly luciferase and NYESO1 (CT26.Fluc.NYESO1) was generated by stably transfecting the CT26.Fluc cell line with the expression plasmid pReceiver-M02 (GeneCopoeia) that contains the polynucleotide encoding NYESO1 (NM_001327.1) under the control of the CMV promoter and that contains neomycin as a selection marker. The CT26.Fluc.NYESO1 cell line was maintained in Dulbecco's modified Eagles medium (DMEM) containing 4.5 g/L Glucose (Corning CellGro) supplemented to contain 10% FCS, 100 mg/mL penicillin-streptomycin, 7.5 g/mL Puromycin and 800 g/mL Geneticin. All cell lines were cultured at 37 C. and 5% CO.sub.2.

Preparation of pT7StuIR-WT PD-1 Minibody Vector

[0185] The extracellular domain of the human PD-1 protein is encoded by nucleotides 69-576 of the GenBank-NCBI sequence, Ref. Seq. NM_005018.2 (FIG. 1A). The encoded human polypeptide is 59% identical and 69% similar to the mouse PD-1 sequence. To produce the Sindbis virus vector encoding PD-1 protein, the wild-type human PD-1 sequence was fused to the hinge region and the CH3 heavy chain constant region domain of human immunoglobulin (Ig) G isotype 1, IgG1 (GenBank, P01857.1). (FIG. 1A). A glycine-rich, artificial spacer or linker sequence was added between the hinge and CH3 domains to provide greater flexibility between the protein domains. Amino acid sequences were optimized for expression and function in the mouse. The sequence was synthesized by GenArt (Lifetechnologies). The PD-1 sequence fused to the IgG1 hinge region and the heavy chain constant region CH3 domain is termed a minibody or minibody fragment herein. An XbaI restriction enzyme site was included to facilitate subcloning from the GenArt pMK-RQ-Bb vector. The synthesized sequence was excised from the pMK-RQ-Bb plasmid using the restriction enzymes XbaI and PmeI. The Sindbis virus plasmid, pT7StuIR, was also digested with the XbaI/PmeI enzymes and was ligated with the PD-1 minibody fragment. Plasmid DNAs, isolated from bacterial colonies obtained following transformation with the ligation reactions, were analyzed by restriction enzyme digestion and positive plasmids were sequenced. FIG. 1B shows an amino acid sequence comparison (alignment) of the human WT-PD-1 amino acid sequence to PD-1 amino acid sequences of other species, e.g., mouse and monkey.

[0186] To produce the Sindbis virus_PD-1WT minibody viral vector (SV_PD-1WT), the DNA plasmids pT7StuIR1-PD-1 WT Minibody and T7DM-Helper (maps in FIG. 2) were linearized with PacI and XhoI restriction enzymes, respectively, before performing in vitro transcription using the mMACHINE RNA transcription kit (Ambion, Austin, Tex.) following the manufacturer's protocol. Helper and replicon RNAs were mixed at a 1:1 ratio and then were electroporated into BHK cells. After 8 to 10 hours, the cell culture medium was replaced with OPTI-MEM (Invitrogen), supplemented to contain 100 g/mL CaCl.sub.2. After 24 hours, the supernatant was collected, centrifuged to remove cellular debris and stored at 80 C.

[0187] The vector titer was determined by infecting BHKSINLuc2 cells that expressed Firefly luciferase under the Sindbis promoter, which produced Luciferase signal only in infected cells in which the Sindbis replicase wa expressed. Briefly, 10.sup.5 BHKSINLUC2 cells in 12 well plates were infected with serial dilutions of vector (250 L/well) in Optimem-CaCl.sub.2 for an hour at room temperature (RT). Cells were washed with -MEM medium and were incubated overnight (O/N) at 37 C. and in 5% CO.sub.2. Thereafter, the medium was removed and the cells were lysed using M-PER Mammalian Protein Extraction Reagent (100 L/well) for 10 min at RT. Thereafter, 100 L of SteadyGlo Reagent (Promega E2520) was added. Following shaking at RT for 10 min, bioluminescence was measured in a Glomax Biorad luminometer. The SV_PD1WT vector was titered in parallel to Sindbis virus vector expressing GFP (Sindbis-GFP) to establish a correlation between the visual titer (GFP positive cells) and the Luminescent signal. Vector titers refer to the number of infectious particles, transducing units (TU), per milliliter of supernatant (TU/mL). In this study the SV_PD-1WT vector was used at titer of 5-10.sup.5 TU/ml.

In Vivo Studies Using the SV PD-1WT Vector

[0188] All experiments were performed in accordance with the Institute of Animal Care and Use Committee at New York University Health.

[0189] Four to eight week old female BALB/c mice were purchased from Taconic (Germantown, N.Y.). For the animal tumor model, 710.sup.4 CT26.Fluc.NYESO1 cells in 500 L OPTI-MEM medium were injected (i.p. administration) into animals 5 days before treatment with the Sindbis vector (SV_PD-1WT), (day 0). Four days after the cells were injected, tumor implantation in mice was assessed by IVIS imaging, and mice in the group receiving anti-PD-1 antibody received a first dose (250 g/mouse) of anti-PD-1 antibody (clone RPMI-14, BioXCell) via i.p. injection. Anti-PD-1 antibody was administrated 3 days a week for a total of 2 weeks: days 4, 6, 8 and 11, 13 15 after tumor cell implantation. For treatments, 10.sup.5 TU of SV_PD-1WT vector in a total volume of 500 L was injected into mice (i.p.) 4 days a week for a total of 4 weeks. Days after cells inoculation: 5, 6, 7, 8 (week one); 12, 13, 14, 15 (week 2); 20, 21, 22, 23 (week 3); and 27, 28, 29, 30 (week 4). The schematic diagram of the experiment design is shown in FIG. 3. The therapeutic efficacy of the treatment was monitored in two ways: by tumor luminescence and by animal survival.

[0190] Noninvasive bioluminescent imaging was performed using the IVIS Spectrum imaging system (Caliper Life Science) and tumor growth was quantified using the Living Image 3.0 software (Caliper Life Science). The first tumor bioluminescent image was collected on day 4 after tumor cell inoculation, and then imaging was continued weekly for 6 weeks. Relative tumor growth for each mouse was calculated by dividing total body counts on a given day by total body counts on the first day of IVIS imaging (at day 4). Graphs showing relative tumor growth (fold change) at different days after treatment are shown in FIG. 4. Representative bioluminescence images of control, anti-PD1 antibody treated and SV_PD-1WT vector-treated mice bearing established CT26.Fluc.NYESO1 tumors are shown in FIG. 5. Animal survival was monitored and recorded daily; survival plots of untreated and SV_PD-1WT treated mice are shown in FIG. 6.

Example 3Sindbis Virus Vector Encoding the Immune Checkpoint Protein 4-1BB Ligand (4-1BBL)

[0191] (4-1BB ligand) is a transmembrane cytokine that is part of the tumor necrosis factor (TNF) ligand superfamily. 4-1BBL is a bidirectional signal transduction molecule that serves as a ligand for 4-1BB (alternatively called tumor necrosis factor receptor superfamily member 9 (TNFRSF9), CD137, and induced by lymphocyte activation (ILA)), which is a costimulatory receptor/immune checkpoint molecule expressed by T cells. 4-1BBL and its receptor, 4-1BB (TNFRSF9), play a role in antigen presentation by cells of the immune system, e.g., dendritic cells, macrophages, APC, and in the generation of cytotoxic T cells. While the 4-1BB receptor (4-1BBR) is absent from resting T cells, its expression is rapidly induced in T cells upon antigenic stimulation. 4-1BB reactivates anergic T cells and promotes T cell proliferation. 4-1BBL is involved in generating an optimal response in CD8+ T cells. 4-1BBL is also expressed by carcinoma cell lines and is thought to be involved in T cell-tumor cell interaction. 4-1BBL is expressed as a transmembrane surface protein on activated B cells, macrophages, dendritic cells, activated T cells, neurons and astrocytes.

[0192] The interaction of 4-1BBL with its receptor on activated T cells and natural killer (NK) cells promotes the upregulation of anti-apoptotic molecules, proliferation and IL-2 production. Both 4-1BB ligand and agonist 4-1BB receptor antibodies have been shown to have anti-tumor effects in preclinical mouse models (Melero, I. et al., 1997, Nature Medicine, 3:682-685).

[0193] The full-length murine 4-1BBL cDNA sequence (shown supra) was excised from MG50067-UT plasmid DNA (Sino Biological Inc., Wayne, Pa.) using HindIII and XbaI restriction enzymes. The cDNA fragment was purified by agarose gel electrophoresis. An XbaI-HindIII linker adaptor was ligated to the 5 end of the agarose gel purified fragment, and an XbaI-ApaI linker adapter was ligated to the 3 end. The fragment containing the 5 and 3 end linkers was then ligated into pT7-StuIR Sindbis virus vector (SV) digested with XbaI/ApaI, e.g., as described in Example 6 infra.

[0194] A cDNA polynucleotide encoding a soluble form of the 4-1BBL (s4-1BBL) polypeptide, corresponding to amino acids 106-314 of the 4-1BBL amino acid sequence identified as NCBI Ref Seq NP_033430.1, presented supra, was cloned into the pT7-StuIR SV vector. The s4-1BBL sequence lacked the transmembrane and cytoplasmic domains. The s4-1BBL amino acid sequence is as shown below:

TABLE-US-00012 (SEQIDNO:21) MRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLL AKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFL ELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKL VDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLV KPDNPWE.
The cDNA polynucleotide sequence encoding s4-1BBL is as shown below:

TABLE-US-00013 (SEQIDNO:22) gccaccatgcgcaccgagcctcggccagcgctcacaatcaccacctcgcc caacctgggtacccgagagaataatgcagaccaggtcacccctgtttccc acattggctgccccaacactacacaacagggctctcctgtgttcgccaag ctactggctaaaaaccaagcatcgttgtgcaatacaactctgaactggca cagccaagatggagctgggagctcatacctatctcaaggtctgaggtacg aagaagacaaaaaggagttggtggtagacagtcccgggctctactacgta tttttggaactgaagctcagtccaacattcacaaacacaggccacaaggt gcagggctgggtctctcttgttttgcaagcaaagcctcaggtagatgact ttgacaacttggccctgacagtggaactgttcccttgctccatggagaac aagttagtggaccgttcctggagtcaactgttgctcctgaaggctggcca ccgcctcagtgtgggtctgagggcttatctgcatggagcccaggatgcat acagagactgggagctgtcttatcccaacaccaccagctttggactattc ttgtgaaacccgacaacccatgggaatga.

[0195] The s4-1BBL sequence was synthesized by GenArt (Invitrogen GenArt Gene Synthesis, Lifetechnologies.com, ThermoFisher Scientific, Waltham, Mass.). A 5 XbaI site and a 3 ApaI were included to facilitate subcloning from the GenArt pMK vector into the SV vector (PT7-StuIR SV vector). The synthesized sequence was excised from the pMK plasmid using the restriction enzymes XbaI and ApaI.

[0196] In some cases, a secretory signal sequence was ligated to the amino (N) terminus of the polypeptide, e.g., the 4-1BBL, to optimize the synthesis of the soluble ligand. A non-limiting secretory signal sequence that is suitable for use can be obtained from Ig and has the amino acid sequence METDTLLLWVLLLWVPGSTGD (NCBI Accession No. NCBI:AAH80787.1), (SEQ ID NO: 23)

[0197] In some cases, a trimerization domain was also added to the carboxy (C) terminus of the polypeptide to increase the affinity of the soluble ligand for the 4-1BB receptor. An example of a trimerization domain that is suitable for use has the amino sequence IKQIEDKIEEILSKIYHIENEIARIKKL (SEQ ID NO: 24). This sequence is an isoleucine zipper from the yeast protein GCN4 (Morris, N. P. et al., 2007, Mol. Immunol., 44:3112-3121).

Example 4Sindbis Virus Vector Encoding the Immune Checkpoint Protein OX40 Ligand (OX40L)

[0198] OX40 ligands (OX40Ls) are expressed on activated antigen presenting cells. The OX40 receptor is transiently expressed after antigen recognition by T cells. The interaction between OX40L and its receptor OX40 is important for survival of effector T cells and for the generation of memory T cells. In preclinical tumor models, OX40 agonists were shown to be effective in eradicating immunogenic tumors, though they were less effective in poorly immunogenic tumors (Sanmamed, M. F., 2015, Seminars in Oncology, 42:640-655).

[0199] Sindbis virus vectors were designed to contain a polynucleotide encoding the complete OX40L polypeptide; a soluble form of the OX40 ligand that contained an immunoglobulin Fc region, and an OX40 ligand coexpressed with a TAA.

[0200] The full-length murine OX40L cDNA sequence was excised from plasmid MG53582-UT (Sino Biological Inc.) using the restriction enzymes KpnI and XbaI. The cDNA fragment was purified using agarose gel electrophoresis. An XbaI-KpnI linker adaptor was ligated to the 5 end of the agarose gel purified fragment and an XbaI-ApaI linker adapter was ligated to the 3 end. The fragment containing the 5 and 3 end linkers was then ligated into pT7StuIR SV digested with XbaI/ApaI.

OX40L Polypeptide

[0201] A cDNA polynucleotide encoding the mouse OX40 ligand (OX40L) polypeptide amino acid sequence was cloned into the SV vector. The OX40L amino acid sequence identified by Accession No. NCBI P43488 is as shown below:

OX40L: NCBI P43488 Mouse Amino Acid Sequence

[0202]

TABLE-US-00014 (SEQIDNO:25) MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLLCFIYVCLQL SSSPAKDPPIQRLRGAVTRCEDGQLFSSYKNEYQTMEVQNNSVVIKCDGL YIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDK VYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL.
The cDNA polynucleotide sequence encoding OX40L identified by Accession No. NM_009452.2 is as shown below:

OX40L: NM_009452.2 Mouse cDNA Sequence

[0203]

TABLE-US-00015 (SEQIDNO:26) atggaaggggaaggggttcaacccctggatgagaatctggaaaacggatc aaggccaagattcaagtggaagaagacgctaaggctggtggtctctggga tcaagggagcagggatgcttctgtgcttcatctatgtctgcctgcaactc tcttcctctccggcaaaggaccctccaatccaaagactcagaggagcagt taccagatgtgaggatgggcaactattcatcagctcatacaagaatgagt atcaaactatggaggtgcagaacaattcggttgtcatcaagtgcgatggg ctttatatcatctacctgaagggctcctttttccaggaggtcaagattga ccttcatttccgggaggatcataatcccatctctattccaatgctgaacg atggtcgaaggattgtcttcactgtggtggcctctttggctttcaaagat aaagtttacctgactgtaaatgctcctgatactctctgcgaacacctcca gataaatgatggggagctgattgttgtccagctaacgcctggatactgtg ctcctgaaggatcttaccacagcactgtgaaccaagtaccactgtga.

Soluble Form of OX40L Containing an Immunoglobulin Fc Region (FcOX40L)

[0204] A DNA sequence encoding encodes a soluble form of the OX40L polypeptide was synthesized by GenArt (Lifetechnologies.com). More specifically, the sequence encodes a polypeptide (called FcOX40L herein) that comprises a secretory signal sequence (amino acids 1-18, from murine Ig heavy chain gamma-2A (NCBI: CAA49868.1), followed by the heavy chain constant (C.sub.H) region of IgG2a (amino acids 19-250), a flexible spacer or linker amino acid sequence (amino acids 250-260) and the external (extracellular) region of the OX40L polypeptide (amino acids 260-405) of the NCBI sequence P43488, shown supra.

[0205] A 5 XbaI restriction enzyme site and a 3 ApaI restriction enzyme site were included to facilitate subcloning from the GenArt pMK vector into SV. The synthesized sequence was excised from the pMK plasmid using the restriction enzymes XbaI and ApaI. Shown below are the amino acid sequence and the cDNA polynucleotide sequence of FcOX40L. In the FcOX40L amino acid sequence shown below, the secretory signal sequence at the N-terminus of the amino acid sequence is in bold font; the IgG2a C.sub.H region is underlined; the spacer sequence is in italic font; and the C-terminal OX40L external sequence is in regular font following the spacer sequence.

FcOX40L Amino Acid Sequence

[0206]

TABLE-US-00016 (SEQIDNO:27) MGWSWIFLFLLSGTAGVHPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKI KDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQ VYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPV LDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK GGGSSGGGSGSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNN SVVIKCDGLYITYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTV VASLAFKDKVYLTVNAPDTLCEHLQNDGELIVVQLTPGYCAPEGSYHSTV NQVPL.

[0207] In the FcOX40L-encoding cDNA sequence shown below, the secretory signal encoding polynucleotide sequence at the N-terminus is in bold font; the IgG2a C.sub.H region-encoding sequence is underlined; the spacer/linker-encoding sequence is in italic font; and the C-terminal OX40L external region encoding sequence is in regular font following the sequence encoding the spacer/linker.

FcOX40L cDNA Sequence

[0208]

TABLE-US-00017 (SEQIDNO:28) gccaccatgggctggtcctggatcttcctgttcctgctgtccggcaccgc cggcgtgcaccctcggggacccaccatcaagccctgccctccctgcaagt gtcccgctcccaacctgctgggcggcccctccgtgttcatctttccaccc aagatcaaggacgtgctgatgatctccctgtctcccatcgtgacctgcgt ggtggtggacgtgtccgaggacgaccccgacgtgcagatctcctggttcg tgaacaacgtggaggtgcacaccgcccagacccagacccaccgggaggac tacaactccaccctgcgggtggtgtccgccctgcccatccagcaccagga ctggatgtccggcaaggagttcaagtgcaaggtgaacaacaaggacctgc ccgcccccatcgagcggaccatctccaagcccaagggctccgtgcgggct ccccaggtgtacgtgctgcctcctcctgaggaggagatgaccaagaagca ggtgaccctgacctgcatggtgaccgacttcatgcccgaggacatctacg tggagtggaccaacaacggcaagaccgagctgaactacaagaacaccgag cccgtgctggactccgacggctcctacttcatgtactccaagctgcgggt ggagaagaagaactgggtggagcggaactcctactcctgctccgtggtgc acgagggcctgcacaaccaccacaccaccaagtccttctcccggacccct ggcaagggaggaggctctagcggaggagggtctggatcccctgccaagga ccctcccatccagcggctgcggggcgccgtgacccggtgcgaggacggcc agctgttcatctcctcctacaagaacgagtaccagaccatggaggtgcag aacaactccgtggtgatcaagtgcgacggcctgtacatcatctacctgaa gggctccttcttccaggaggtgaagatcgacctgcacttccgggaggacc acaaccccatctccatccccatgctgaacgacggccggcggatcgtgttc accgtggtggcctccctggccttcaaggacaaggtgtacctgaccgtgaa cgctcccgacaccctgtgcgagcacctgcagaacgacggcgagctgatcg tggtgcagctgacacccggctactgcgctcccgagggctcctaccactcc accgtgaaccaggtgcccctgtga.

Example 5Sindbis Virus Vector Encoding the Immune Checkpoint Protein 4-1BB Ligand (4-1BBL) or OX40 Ligand (OX40L) Reduced Tumor Size in In Vivo Mouse Models

SV Vector Titers

[0209] SV vector titers are determined by infecting BHKSINLuc2 cells that express the Firefly luciferase under control of the Sindbis virus promoter, which allows a luciferase signal only in infected cells where Sindbis replicase is expressed. Briefly, 10.sup.5 BHKSINLuc2 cells in 12 well tissue culture plates were infected with 250 l/well of the vector serial dilutions in Optimem-CaCl.sub.2 for one hour at room temperature (RT). Cells were washed with -MEM medium and were incubated overnight (O/N) at 37 C. and 5% CO.sub.2. The medium was then removed, and the cells were lysed with M-PER Mammalian Protein Extraction Reagent (100 l/well) for 10 minutes at RT. Thereafter, 100 l of SteadyGlo Reagent (Promega E2520) was added; the culture plates were shaken for 10 minutes at RT; and bioluminescence was measured in a Glomax Biorad luminometer. SV vectors containing polynucleotides encoding the multimer polypeptides, e.g., checkpoint protein ligand and TAA, were titered in parallel to the SV vectors encoding GFP to establish a correlation between the visual titer (GFP positives cells) and the luminescent signal. SV vector titers refer to the number of infectious virus particles, transducing units, per milliliter of supernatant (TU/ml).

In Vivo Studies in Mice

[0210] All experiments were performed in accordance with the Institute of Animal Care and Use Committee, New York University Langone Health System.

[0211] Four-to-eight week old female BALB/c mice were purchased from Taconic Biosciences (Germantown, N.Y.). For the CT26 mouse solid tumor model (Lechner, M. et al., 2013, J. Immunother., 36 (9):477-489), CT26.Fluc.NY-ESO1 cells (710.sup.4 cells in 500 l OPTI-MEM medium) were injected into mice intraperitoneally (i.p.) 6 days before Sindbis virus vector treatment (day 0). Four days after cell injection, tumor implantation in mice was assessed by IVIS imaging. Six days after tumor inoculation, the first dose of 10.sup.7 TU of the appropriate Sindbis vector in a total volume of 500 l was administered to the mice via i.p. injection. The treatment continued 4 days a week for a total of 4 weeks; days after cells inoculation: 6, 7, 8, 9 (Week one); 13, 14, 15, 16 (week 2); 21, 22, 23, 24 (week 3); and 28, 29, 30, 31 (Week 4). The experimental design of this study is shown in FIG. 7. The therapeutic efficacy of the treatment was monitored in two ways: tumor luminescence and survival.

[0212] Noninvasive bioluminescent imaging was performed using the IVIS Spectrum imaging system (Caliper Life Sciences/PerkinElmer, Hopkinton, Mass.), and tumor growth was quantified using the Living Image 3.0 software (Caliper Life Sciences/PerkinElmer). The first tumor bioluminescent image was obtained day 4 after inoculation of tumor cells, and then weekly thereafter for 6 weeks. Relative tumor growth for each mouse was calculated by dividing total body counts on a given day by total body counts on the first IVIS image at day 4.

[0213] The relative tumor growth curves at different days after treatment of animals with an anti-4-1BB antibody or with vector SV-4-1BBL (SV vector expressing 4-1BBL) alone or in combination with SV-NYESO1 (SV vector expressing NY-ESO-1) are shown in FIG. 8, which demonstrates an example of the antitumor efficacy of Sindbis virus vectors encoding checkpoint protein ligands and/or TAAs (called armed SV vectors herein) compared with an antibody directed against 4-1BB (an anti-4-1BB antibody), (Bio-X-Cell, West Lebanon, N.H.; InVivoMAb anti-mouse 4-1BB (CD137), Clone 3H3, Catalog #BE0239). As observed in FIG. 8, used alone, the anti-4-1BB antibody was not effective in reducing tumor growth in these in vivo mouse model studies. By contrast, the SV-4-1BBL vector expressing 4-1BBL demonstrated effectiveness in reducing tumor growth. Of note, use of the anti-4-1BB antibody and the SV-NY-ESO-1 vector expressing NY-ESO-1 TAA in combination, as well as use of the SV vector expressing NY-ESO-1 TAA (SV-NYESO1) and the SV vector expressing 4-1BBL (SV-4-1BBL) in combination were highly effective in reducing tumor growth over time.

[0214] In another study, treatment of animals with an anti-OX40L antibody (aOX40L) alone was compared with treatments using (i) Sindbis virus vector harboring NY-ESO-1 TAA encoding polynucleotide (SV-NYESO1) alone and (ii) a combination of anti-OX40L antibody and (SV-NYESO1) together, versus controls (FIG. 9A). In another study, treatment of animals with (i) Sindbis virus vector harboring NY-ESO-1 TAA encoding polynucleotide (SV-NYESO1) alone; (ii) Sindbis virus vector harboring OX40L encoding polynucleotide (SV-OX40L) alone; and (iii) a combination of SV-NYESO1 and SV-OX40L together were compared versus controls (FIG. 9B). As is observed by comparing the results shown in FIG. 9B and FIG. 9A, treatment with SV-OX40L was more efficacious than treatment with anti-OX40L antibody (i.e., a higher percentage of animals survived longer following treatment with SV-OX40L versus treatment with anti-OX40L antibody (aOX40L). In addition, the combination treatment using SV-NYESO1 and SVOX40L conferred 100% survival to animals (FIG. 9B), a result which is superior to the combination treatment using anti-OX40L antibody and SV-NYESO1 together (FIG. 9A). These results support the use of armed Sindbis virus vectors, which provide superior and more effective treatment compared with the use of antibody-mediated therapy.

Example 6General Protocols for Sindbis Virus Vector Preparation

[0215] Construction of recombinant vectors, particularly for the studies described in Example 5 supra, were performed using standard molecular biology techniques, including plasmid growth and purification, restriction endonuclease digestion, agarose gel electrophoresis and fragment extraction, ligation, transformation, polymerase chain reaction (PCR) methods and DNA sequencing, as described in Current Protocols in Molecular Biology, E M. Ausubel et al. (Eds), John Wiley and Sons, Inc., NY, USA. (1998) and Molecular Cloning: A Laboratory Manual (2nd Ed.), J. Sambrook, E. F. Fritsch and T. Maniatis (Eds), Cold Spring Harbor Laboratory Press, NY, USA. (1989).

[0216] Unless otherwise described, sequences were ligated into the 5 XbaI site and the 3 ApaI site of the pT7StuIR-LacZ vector, in which the LacZ sequence was removed. If cDNA sequences contained XbaI or ApaI restriction sites, these sites were mutated to remove them, while maintaining the native amino acid sequence. A schematic depiction of a Sindbis virus vector capable of expressing heterologous gene, e.g., a checkpoint molecule-encoding gene or a TAA-encoding gene, from each of its two subgenomic promoters is shown in FIG. 10. Plasmid DNAs, isolated from bacterial colonies obtained following transformation with the ligation reactions, were analyzed by restriction digestion and positive plasmids were sequenced.

Other Embodiments

[0217] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0218] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0219] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.