Immune Assay for Monitoring Response to Oncolytic Vaccinia Virus and Uses Thereof

Abstract

Provided herein are methods and compositions for assaying vaccinia virus-specific T cell responses in a sample from a subject undergoing treatment with an oncolytic vaccinia virus. The compositions comprise custom peptide pools from known immunogenic vaccinia virus epitopes in an HLA-agnostic format to profile peripheral CD8+T cell responses.

Claims

1. A composition for use in measuring an immune response against an oncolytic vaccinia virus, the composition comprising a population of peptides comprising at least 5 different peptides, wherein each peptide in the population consists essentially of an amino acid sequence selected from those set forth in SEQ ID NOs:1-70.

2. A composition for use according to claim 1, comprising a population of peptides, each peptide consisting essentially of an amino acid sequence forth in SEQ ID NOs: 1-18.

3. A composition for use according to claim 1, comprising a population of peptides, each peptide consisting essentially an amino acid sequence forth in SEQ ID NOs: 19-34.

4. A composition for use according to claim 1, comprising a population of peptides, each peptide consisting essentially of an amino acid sequence forth in SEQ ID NOs: 35-49.

5. A composition for use according to claim 1, comprising a population of peptides, each peptide consisting essentially of an amino acid sequence forth in SEQ ID NOs: 50-70.

6. A method for monitoring the number and/or status of vaccinia virus-reactive T cells in a patient that has received one or more oncolytic vaccinia virus treatments, the method comprising determining the patient's immune reactivity to a composition according to claim 1.

7. A method for determining the prognosis of a cancer patient that has received one or more oncolytic vaccinia virus treatments, the method comprising determining the patient's immune reactivity to a composition according to claim 1.

8. A method for monitoring the efficacy of vaccinia virus treatment in a cancer patient that has been administered one or more doses of oncolytic vaccinia virus, comprising determining the patient's immune reactivity to a composition according to claim 1.

9. A method for monitoring the efficacy of a combination therapy comprising co-administration of an oncolytic vaccinia virus and one or more checkpoint inhibitors in a cancer patient that been administered one or more doses of oncolytic vaccinia virus and one or more doses of a checkpoint inhibitor, comprising determining the patient's immune reactivity to a composition according to claim 1.

10. The method of claim 6, comprising contacting a PBMC or whole blood sample from the patient with one or more compositions according to any one of claims 2-5.

11. The method of claim 6, wherein the patient's immune reactivity to the one or more compositions is assessed by ELISPOT assay.

12. The method of claim 11, wherein the ELISPOT assay quantifies the level of IFN-γ produced by CD8+T cells in response to the one or more compositions.

13. The method of claim 6, wherein the sample obtained from the patient is incubated with the one or more compositions for a period of at least 12, at least 24, at least 36 or at least 48 hours.

14. The method of claim 6, wherein T cells in the sample are expanded ex vivo in the presence of(i) the one or more compositions (ii) autologous antigen presenting cells, preferably dendritic cells, and (iii) one or more T cell supportive cytokines.

15. The method of claim 14, wherein unfractionated PBMCs are directly stimulated in culture with the one or more compositions, IL-4, IL-7, IL-15 and GM-CSF and wherein the culture does not comprise isolated antigen presenting cells pre-stimulated with the one or more compositions.

16. The method according to claim 6, wherein the patient has a cancer selected from brain cancer, renal cancer, liver cancer, lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, bone cancer, testicular cancer, cervical cancer, ovarian cancer, uterine cancer, rectal cancer, gastrointestinal cancer, lymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.

17. The method according to claim 16, wherein the patient has a cancer selected from renal cell carcinoma, hepatocellular carcinoma and melanoma.

18. The method according to claim 6, wherein the patient received one or more treatments of a wild type or genetically modified Wyeth or Western Reserve strain vaccinia virus.

19. The method according to claim 18, wherein the patient received one or more treatments of a genetically modified Wyeth or Western Reserve strain vaccinia virus lacking a functional thymidine kinase gene and/or lacking a functional vaccinia growth factor gene.

20. (canceled)

21. The method according to claim 19, wherein the Wyeth or Western Reserve strain vaccinia virus contained a transgene encoding a cytokine, preferably a transgene encoding GM-CSF.

22. The method according to claim 22, wherein the patient received one or more intratumoral and/or intravenous treatments of JX-594.

23. (canceled)

24. The method according to claim 6, wherein the vaccinia virus was administered to the patient at a dose of 10.sup.8 to 10.sup.9 pfu.

25. The method according to claim 6, wherein the patient was co-administered one or more immune checkpoint inhibitors.

26. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0125] FIG. 1 illustrates the genetic modifications made to Wyeth strain vaccinia virus to create PEXA-VEC (JX-594). Briefly, lacZ and GM-CSF genes were inserted into the native thymidine kinase gene. The resulting virus expresses GM-CSF to drive active immunity and is thymidine kinase deficient, increasing selectivity for cancer cells.

[0126] FIGS. 2A-2B illustrate the administration scheme of JX-594 to patients with renal cell carcinoma and collection of peripheral blood mononuclear cells (FIG. 2A) and the clinical response of these patients (FIG. 2B).

[0127] FIG. 3 compares traditional overlapping peptide pools to the epitope-based peptide pools of the present invention. By using only peptides found in the Immune Epitope Database, the present invention ensures that each peptide included in the pools has been validated as immune-reactive in humans and enables screening of the full set with each blood collection. In contrast, 1000s of peptides would need to be screened, covering the whole proteome of the virus by the use of traditional overlapping peptide pools, which in turn requires a significant (and infeasible) volume of blood to be drawn from each patient to successfully screen the entire peptide set. Additionally, traditional overlapping peptide pools does not allow for differentiation of CD8+ versus CD4+ responses.

[0128] FIG. 4 illustrates ELISPOT IFNγ response for PBMCs collected from patients with progressive disease (n=4) (left panel), patients with stable disease (n=12) (middle panel) and a patient with complete response (n=1) (right panel) at baseline, six weeks and 12 weeks, incubated with overlapping 15mer amino-acid peptides overlapping by 11 amino acid for coverage of the second half of major core Vaccinia protein P4a.

[0129] FIGS. 5A-5B FIG. 5A illustrates the procedure for measuring ELISPOT IFNγ response for PBMCs incubated with peptide pools. FIG. 5B illustrates the clinical response of each patient from whom PBMCs were collected and assigns a symbol to each patient/outcome corresponding to those used in FIGS. 6A and 6B.

[0130] FIGS. 6A-6B compares illustrates ELISPOT IFNγ response for PBMCs collected at the indicated time points from patients with progressive disease (n=4), patients with stable disease (n=12) and a patient with complete response (n=1) to overlapping versus epitope-based peptide pools (FIG. 6A) and illustrates ELISPOT IFNγ response for PBMCs collected at the indicated time points from patients with progressive disease (n=4), patients with stable disease (n=12) and a patient with complete response (n=1), incubated with epitope-based peptide pools according to Table 3 (FIG. 6B).

[0131] FIG. 7 illustrates ELISPOT IFNγ response for PBMCs

[0132] FIGS. 8A-8B FIG. 8A illustrates the fold-change over time in IFNγ response of PBMCs to the epitope-based peptide pools according to Table 3 compared to baseline. Three out of four patients with progressive disease exhibited a fold-change below baseline whereas 8/11 patients with stable or complete response exhibited a fold-change above baseline. FIG. 8B illustrates that the same analysis performed on the response to the overlapping peptide pool did not show the same grouping of inductions as the epitope-based pools. Rather, the change in response did not trend with any specific clinical response.

[0133] FIGS. 9A-9B illustrate correlation of the fold-change in peripheral T cell response at 6 weeks to overall survival of RCC patients treated with PEXA-VEC. The change in response at week 6 for all patients in the study was analyzed in comparison to clinical response. Radiographic change was determined by calculating percent change in tumor size (mm) of the primary lesion at week 6 compared to the size at baseline. FIG. 9A illustrates overall survival correlation to T cell response to all epitope-based peptide pools. FIG. 9B illustrates the same correlation with only A*02 supertype pool.

[0134] FIG. 10 illustrates the clinical response (tumor response and % change in tumor burden) of patients with advanced RCC who received combination therapy with JX-594 and Cemiplimab (the patients had received 6 to 15 rounds of Cemiplimab at the time of data collection).

[0135] FIG. 11 illustrates the percentage of tumor change (volume) in individual patients treated with JX-594+Cemiplimab over the course of treatment.

[0136] FIG. 12 illustrates correlation between T cell response (ELISPOT) and tumor volume change in patients treated with JX-594+Cemiplimab. The left panel illustrates the results of direct ex vivo stimulation of PBMCs from the patients with the OV peptides (without expansion) and the right panel illustrates the results of expansion of PBMCs from the patients with OV peptides in the presence of autologous dendritic cells and T cell supportive cytokines for 10 days.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0137] An “immune response” refers to a change in immunity, for example, a response of a cell of the immune system, such as a B cell, T cell or monocyte, to a stimulus (e.g. a response specific for a vaccinia virus antigen). In one example, an immune response is a T cell response, such as a CD4.sup.+ response or a CD8.sup.+ response.

[0138] “Measuring an immune response” refers to any measurement or determination of the level, presence or absence, reduction, or increase in an immune response in vitro or in vivo.

[0139] “Epitope”, also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by, for example, antibodies, B cells, or T cells. As used herein, “MHC Class I-restricted epitopes” are epitopes that are presented to immune cells by MHC class I molecules found on nucleated cells. In some embodiments, the epitope itself is an antigen. The T-cell epitopes presented by major histocompatibility complex class I (MHC I) molecules are CD8.sup.+T-cell epitopes, which are typically peptides 8-11 amino acids in length.

[0140] “Interferon-gamma (IFN- or IFNγ)” refers to a protein produced by T lymphocytes in response to a specific antigen or mitogenic stimulation. Sequences for IFN-γ are publicly available (exemplary IFN-γ protein sequences are available from GenBank Accession Nos: CAA00226; AAA72254; and 0809316A).

[0141] The term “subject” or “patient” refers to either a human or non-human, such as primates, mammals and vertebrates. In particular embodiments, the subject or patient is a human. In some embodiments, the subject or patient is a human with a cancer that is refractory to one or more standard treatments.

EXAMPLES

[0142] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.

Example 1

[0143] Clinical Trial Design

[0144] A phase 2 clinical trial in the clear cell variant of metastatic renal cell carcinoma was conducted. It was a single-center/investigator-initiated trial at Pusan National University Yangsan Hospital in South Korea. 17 patients (median age 62, range 37-73; 12 male and 5 female) were enrolled, all of which were refractory to standard therapy. All patients had received at least one tyrosine kinase inhibitor or anti-angiogenic and most had also received at least one mTOR inhibitor (n=14) or at least one immunotherapeutic (n=10).

[0145] Patients were given Pexa-Vec (JX-594; a Wyeth vaccinia virus vaccine-derived oncolytic with disruption of the viral thymidine kinase gene and expression of the human granulocyte-monocyte colony stimulating factor (hGM-CSF) and β-galactosidase transgenes under control of the synthetic early-late and p7.5 promoters, respectively, see FIG. 1) intravenously at 10.sup.9 plaque-forming units (pfu) once a week for five weeks and every three weeks thereafter. Blood was collected for PBMC isolation at Baseline, Week 6, Week 12 and upon study completion. See FIG. 2A.

[0146] Stimulation of PBMCs with Peptides Corresponding to Renal Cell Carcinoma Antigens

[0147] Peripheral blood mononuclear cells (PBMC) were isolated from 16 renal cell carcinoma patients enrolled in the clinical trial at baseline (15/16 patients, prior to treatment) and at weeks 6 (16/16 patients) and 12 (12/16 patients) of a treatment protocol with the oncolytic vaccinia virus JX-594 (5 weekly intravenous infusions of 1×10.sup.9 pfu and every three weeks thereafter) and tested for immunoreactivity against a selection of 8 renal cell carcinoma-related antigens by ELISPOT: (i) RGS5=Regulator of G-protein signaling 5 (ii) MMP7=Matrix metallopeptidase 7 (iii) Survivin=BIRC5=baculoviral inhibitor of apoptosis repeat-containing 5 (iv) IGF-BP3=Insulin-like growth factor-binding protein 3 (v) MAGE-A3=Melanoma Antigen family A3 (Cancer/Testis Antigen Family 1, Member 3) (vi) TYMS=Thymidylate Synthetase (vii) HIG2=Hypoxia-inducible protein 2=Hypoxia Inducible Lipid Droplet Associated and (viii) PRUNE2=Prune Homolog 2. The 16 RCC patients were refractory to prior systemic treatments (mean (SD) of 2.9 lines of systemic therapy per patient including TKI/antioangiogenic, mTOR inhibitor, immunotherapeutic and/or chemotherapeutic treatments).

[0148] Seven of the eight antigens were used for stimulation as a peptide pool of 15mers overlapping by 11 amino acids spanning the entire protein. This format allows the efficient presentation of all potential class I- and class II-related epitopes to the patients' PBMC without the need to know patient HLA composition. In the case of PRUNE2, a single HLA-A2.01-restricted peptide was used for stimulation instead, which has been shown to elicit responses in RCC patients. The use of a peptide pool in this case was prohibitive due to the length of the protein (3088 amino acids=770 peptides).

[0149] A control peptide pool (CEFT) was included as an internal control for consistency of response status over time. The CEFT peptide pool consists of defined peptides from CMV, EBV, Influenza virus and Tetanus, which are known to elicit CD8 (CMV, EBV, Flu) and CD4 (Tetanus) responses in a consistent manner over time. For the assessment of background reactivity of each sample, PBMC were tested with medium plus DMSO alone (negative control). All samples were also assessed for overall functional status with non-specific anti-CD3 stimulation (positive control). Performance consistency (=Trending control) was assessed by simultaneous testing of the same External Reference PBMC sample against medium, CEFT and anti-CD3 in all experiments. Based on the trending control, the inter-assay variability (% CV) was determined to be <10.

[0150] PBMC from all time points of each patient were tested in the same experiment and an external reference sample included as a trending control between experiments. Each antigen condition was plated in triplicates, with 250,000 cells per well plated. The negative control (PBMC plus medium only) was plated in six replicates of 250,000 cells per well, to allow for proper statistical testing. As a positive control, 50,000 PBMC were stimulated with anti-CD3.

[0151] Elispot plates were read using a KS Elispot reader (Carl Zeiss Inc., Thornwood, N.Y.) equipped with KS Elispot Version 4.9.16 software. Spot parameters and evaluation algorithms were adapted to the specific occurrence of spots and background signals in each sample. All responses are based on Distribution-free Resampling (DFR)(eq) and DFR(2×) testing using the raw spot counts, none extrapolated. The cut-off for responses was 6 spots.

[0152] Results

[0153] Four patients (25%) were found to have developed responses against one or more RCC-related antigens after vaccination, which were not detectable at Baseline. Four patients (25%) exhibited one or more responses against RCC-related antigens already at Baseline, one of whom developed responses to other antigens after vaccination. All-together, responses against RCC-related antigens were detectable in 7 out of 16 patients (44%) at least one time point (BL, wk6. Wk12), including 4 patients with new responses after JX-594 treatment. Patients with T cell responses to the antigens all had stable disease, not progressive disease (i.e. all patients with T cell responses to the antigens were responders).

[0154] The response rate stratified by the clinical response (SD=stable disease; PD=progressive disease) at week 6 is shown at Table 1:

TABLE-US-00001 TABLE 1 SD at 6 weeks PD at 6 weeks Response(DFR) Response(DFR) Antigen Time Point N (%) N (%) Survivin Baseline 0 (0%) 0 (0%) Survivin Week 6 1 (8%) 0 (0%) Survivin Week 12/ 1 (9%) 0 (0%) Complete MAGEA3 Baseline 0 (0%) 0 (0%) MAGEA3 Week 6 1 (8%) 0 (0%) MAGEA3 Week 12/ 0 (0%) 0 (0%) Complete PRUNE2 Baseline 0 (0%) 0 (0%) PRUNE2 Week 6 1 (8%) 0 (0%) PRUNE2 Week 12/ 0 (0%) 0 (0%) Complete Vaccinia Baseline 1 (9%) 2 (50%) Vaccinia Week 6 5 (42%) 2 (50%) Vaccinia Week 12/ 6 (55%) 0 (0%) Complete CEFT Baseline 9 (82%) 2 (50%) CEFT Week 6 7 (58%) 1 (25%) CEFT Week 12/ 7 (64%) 1 (100%) Complete HIG2 Baseline 0 (0%) 0 (0%) HIG2 Week 6 1 (8%) 0 (0%) HIG2 Week 12/ 1 (9%) 0 (0%) Complete TYMS Baseline 2 (18%) 0 (0%) TYMS Week 6 2 (17%) 0 (0%) TYMS Week 12/ 2 (18%) 0 (0%) Complete RGS5 Baseline 2 (18%) 0 (0%) RGS5 Week 6 2 (17%) 0 (0%) RGS5 Week 12/ 2 (18%) 0 (0%) Complete MMP7 Baseline 1 (9%) 0 (0%) MMP7 Week 6 1 (8%) 0 (0%) MMP7 Week 12/ 1 (9%) 0 (0%) Complete IGF-BP3 Baseline 3 (27%) 0 (0%) IGF-BP3 Week 6 4 (33%) 0 (0%) IGF-BP3 Week 12/ 3 (27%) 0 (0%) Complete

[0155] Response rate to ay tumor antigen are summarized at Table 2 (response rates by antigen and time point stratified by clinical response at 6 weeks):

TABLE-US-00002 TABLE 2 SD at 6 weeks PD at 6 weeks Response(DFR) Response(DFR) Antigen Time Point N (%) N (%) Any Tumor* Baseline 4 (36%) 0 (0%) Week 6 4 (33%) 0 (0%) Week 12/Complete 4 (36%) 0 (0%) Vaccinia Baseline 1 (9%) 2 (50%) Week 6 5 (42%) 2 (50%) Week 12/Complete 6 (55%) 0 (0%) CEFT Baseline 9 (82%) 2 (50%) Week 6 7 (58%) 1 (25%) Week 12/Complete 7 (64%) 1 (100%) *A patient had a response to at least one of the 8 tumor antigens. At each time point there was a different combination of 4 patients that had a response to at least one antigen.

[0156] Six patients (38%) had a response to IGF-BP3 at least one of the three assessed time points (BL, wk6, wk12). Three out of the four patients who developed new responses against RCC-related antigens developed a response against IGF-BP3.

[0157] The results illustrate that a baseline (pretreatment) T lymphocyte response to certain renal cell carcinoma antigens, particularly IGF-BP3 (and to a lesser extent RGS5 and TYMS), in RCC patients is useful for identifying an RCC patient who will respond favorably to treatment with oncolytic vaccinia virus (exemplified here by JX-594).

Example 2

[0158] A critical component to delineating anti-tumor activity of oncolytic viruses is to monitor peripheral immune response to the virus. Assays to monitor functional CD8+T cell responses in the blood patients treated with oncolytic vaccinia virus JX-594 were developed.

[0159] Peripheral blood mononuclear cells (PBMC) from 17 patients enrolled in the clinical trial described at Example 1 were collected by Ficoll separation and cryopreserved prior to treatment, at six and twelve weeks post-Pexa-Vec initiation, and upon completion of study. Clinical response was determined for all patients according to mRECIST1.0 criteria at weeks six, twelve, and upon study completion: Stable Disease (<20% increase and <20% decrease in lesion size), Progressive Disease (>20% increase), or Response (>20% decrease). See FIG. 2B.

[0160] This example compares the use of an overlapping vaccina peptide pool (covering the second half of Major core protein P4a of vaccinia virus) to the use of epitope-based peptide pools to track peripheral CD8+T cell response to vaccinia virus in patients enrolled in the clinical trial.

[0161] As described in detail below, the custom peptide pools were designed from known immunogenic vaccinia virus epitopes in an HLA-agnostic format to profile peripheral CD8+T cell responses. All curated MHC class I-restricted epitopes to vaccinia virus were retrieved from the Immune Epitope Database and duplicates were removed. The remaining epitopes (n=70) were synthesized and pooled based on known HLA restriction. See FIG. 3. Individual pools comprised no more than 20 peptides. PBMCs were stimulated for 48 hours and response to peptide pools were measured by dual IFNγ/granzyme B ELISPOT. Additionally, patient HLA haplotypes were determined by sequencing and all samples were evaluated by flow cytometry to assess T cell phenotypes.

[0162] Overlapping Vaccinia Peptide Pool ELISPOT Testing

[0163] Briefly, patient PBMCs were assayed by IFNγELISPOT (R&D Systems, Minneapolis, Minn.) for response to a Vaccinia peptide pool containing 104 peptides of 15 residues in length overlapping by 10 covering the second half of Major core protein P4a of vaccinia virus (JPT Peptide Technologies, Acton, Mass.).

[0164] Vaccinia Epitope-Based Peptide Pool Design

[0165] All peptides included in the study had been identified as human HLA class I epitopes for Vaccinia and curated by the Immune Epitope Database. Search criteria for peptides were as follows: Linear Epitope; Vaccinia virus (ID:10245); Positive Assays Only; T Cell Assays; MHC Class I; Humans; Any Disease; Any Reference Type. From the resulting 212 epitopes, specific HLA class I restriction was noted, and only epitopes with positive reactivity in at least two human donors confirmed through a literature search were included. Additionally, overlapping epitope sequences with identical HLA class I restriction were combined provided the resulting sequence was under eleven residues in length. The 70 remaining epitopes (Table 3) were synthesized at Genscript (Piscataway, N.J.) to >90% purity, reconstituted in DMSO, and pooled based on reported HLA class I restriction as noted on Table 3. Individual pools were comprised of no more than 20 peptides.

TABLE-US-00003 TABLE 3 HLA class I Pool Restriction Parent Protein Sequence Start End A*02 A*02 DNA polymerase FLNISWFYI (SEQ ID 107 115 supertype NO: 1) A*02 A*02 Early transcription factor 82 kDa FLVIAINAM (SEQ ID 342 350 supertype subunit NO: 2) A*02 A*02 Intermediate transcription factor 3 YLFRCVDAV (SEQ ID 72 80 supertype small subunit NO: 3) A*02 A*02 Kelch repeat and BTB domain- YIYGIPLSL (SEQ ID 78 86 supertype containing protein A55 NO: 4) A*02 A*02 mRNA-capping enzyme regulatory SLFKNVRLL (SEQ ID 174 182 supertype subunit NO: 5) A*02 A*02 Plaque-size/host range protein SVVTLLCVLPAVVYS 5 19 supertype (SEQ ID NO: 6) A*02 A*02 Poly(A) polymerase catalytic FLIDLAFLI (SEQ ID 213 221 supertype subunit NO: 7) A*02 A*02 Profilin LMDENTYAM (SEQ 74 82 supertype ID NO: 8) A*02 A*02 Protein A36 MMLVPLITV(SEQ ID 1 9 supertype NO: 9) A*02 A*02 Protein A6 ILSDENYLL (SEQ ID 172 180 supertype NO: 10) A*02 A*02 Protein E2 KIDYYIPYV (SEQ ID 249 257 supertype NO: 11) A*02 A*02 Protein F12 NLFDIPLLTV(SEQ ID 286 295 supertype NO: 12) A*02 A*02 Protein F12 FLTSVINRV (SEQ ID 404 412 supertype NO: 13) A*02 A*02 Protein N1 RMIAISAKV(SEQ ID 71 79 supertype NO: 14) A*02 A*02 Protein N2 YVNAILYQI (SEQ ID 93 101 supertype NO: 15) A*02 A*02 Protein O1 GLNDYLHSV (SEQ ID 247 255 supertype NO: 16) A*02 A*02 Ribonucleoside-diphosphate SMHFYGWSL (SEQ 720 728 supertype reductase large subunit ID NO: 17) A*02 A*02 RNA helicase NPH-II KLLLWFNYL (SEQ ID 197 205 supertype NO: 18) A*02: 01 pool A*02: 01 A-type inclusion protein A25 YLYTEYFLFL (SEQ ID 177 186 NO: 19) A*02: 01 pool A*02: 01 DNA-directed RNA polymerase 7 SLKDVLVSV (SEQ ID 27 35 kDa subunit NO: 20) A*02: 01 pool A*02: 01 Envelope protein H3 SLSAYIIRV (SEQ ID 184 192 NO: 21) A*02: 01 pool A*02: 01 Interferon antagonist C7 KVDDTFYYV (SEQ ID 74 82 NO: 22) A*02: 01 pool A*02: 01 Intermediate transcription factor 3 ALDEKLFLI (SEQ ID 273 281 large subunit NO: 23) A*02: 01 pool A*02: 01 Kelch repeat and BTB domain- AMLNGLIYV (SEQ ID 391 399 containing protein A55 NO: 24) A*02: 01 pool A*02: 01 mRNA-capping enzyme regulatory KLFTHDIML (SEQ ID 62 70 subunit NO: 25) A*02: 01 pool A*02: 01 mRNA-capping enzyme regulatory RVYEALYYV (SEQ ID 251 259 subunit NO: 26) A*02: 01 pool A*02: 01 Protein A47 LLYAHINAL (SEQ ID 155 163 NO: 27) A*02: 01 pool A*02: 01 Protein B14 CLTEYILWV (SEQ ID 79 87 NO: 28) A*02: 01 pool A*02: 01 Protein B14 TLLDHIRTA (SEQ ID 137 145 NO: 29) A*02: 01 pool A*02: 01 Protein E5 KLFSDISAI (SEQ ID 107 115 NO: 30) A*02: 01 pool A*02: 01 Putative nuclease G5 ILDDNLYKV (SEQ ID 18 26 NO: 31) A*02: 01 pool A*02: 01 Soluble interferon alpha/beta KLIIHNPEL (SEQ ID 207 215 receptor B18 NO: 32) A*02: 01 pool A*02: 01 Soluble interferon gamma receptor KITSYKFESV (SEQ ID 18 27 B8 NO: 33) A*02: 01 pool A*02: 01 Thymidylate kinase IVIEAIHTV (SEQ ID 187 195 NO: 34) Other A* pool A*01 mRNA-capping enzyme regulatory GTHVLLPFY (SEQ ID 11 19 subunit NO: 35) Other A* pool A*01 Protein A27 LRAAMISLAKKIDVQ 89 103 (SEQ ID NO: 36) Other A* pool A*01 Protein A38 VSEHFSLLF (SEQ ID 257 265 NO: 37) Other A* pool A*01 Protein C10 QSDTVFDYY(SEQ ID 298 306 NO: 38) Other A* pool A*01 Soluble interferon gamma receptor DMCDIYLLY(SEQ ID 139 147 B8 NO: 39) Other A* pool A*01 Soluble interferon gamma receptor FGDSKEPVPY (SEQ 153 162 B8 ID NO: 40) Other A* pool A*01 Transcript termination protein A18 LSDLKKTIY (SEQ ID 255 263 NO: 41) Other A* pool A*01: 01 DNA-directed RNA polymerase 133 ITDFNIDTY(SEQ ID 278 286 kDa polypeptide NO: 42) Other A* pool A*03 Intermediate transcription factor 3 AVKDVTITKK (SEQ 79 88 small subunit ID NO: 43) Other A* pool A*03 Protein C5 KVMFVIRFK(SEQ ID 158 166 NO: 44) Other A* pool A*23: 01 Thymidylate kinase TYNDHIVNL (SEQ ID 58 66 NO: 45) Other A* pool A*23: 01 Primase D5 VWINNSWKF (SEQ 349 357 ID NO: 46) Other A* pool A*24 DNA polymerase processivity factor KYQSPVNIF (SEQ ID 144 152 component A20 NO: 47) Other A* pool A*26 Major core protein 4a precursor DTRGIFSAY (SEQ ID 636 644 NO: 48) Other A* pool A*29: 03/02 Serine proteinase inhibitor 1 VYINHPFMY (SEQ ID 326 334 NO: 49) B* pool B*07 Protein B14 TVADVRHCL (SEQ ID 53 61 NO: 50) B* pool B*07: 02 DNA-directed RNA polymerase 147 MPAYIRNTL (SEQ ID 303 311 kDa polypeptide NO: 51) B* pool B*07: 02 mRNA-capping enzyme catalytic HPRHYATVM (SEQ 686 694 subunit ID NO: 52) B* pool B*07: 02 Protein C1 KPKPAVRFAI (SEQ 102 111 ID NO: 53) B* pool B*07: 02 Protein O1 RPMSLRSTII (SEQ ID 335 344 NO: 54) B* pool B*07: 02 Ribonucleoside-diphosphate APNPNRFVI (SEQ ID 6 14 reductase small chain NO: 55) B* pool B*08: 01 DNA ligase WLKIKRDYL (SEQ ID 395 403 NO: 56) B* pool B*15: 01 Protein K7 SIIDLIDEY (SEQ ID 25 33 NO: 57) B* pool B*35 Plaque-size/host range protein CIDGKWNPILPTCVR 225 239 (SEQ ID NO: 58) B* pool B*44 Dual specificity protein SEVKFKYVL (SEQ ID 48 56 phosphatase H1 NO: 59) B* pool B*44 Plaque-size/host range protein TKYFRCEEKNGNTSW 105 119 (SEQ ID NO: 60) B* pool B*44: 03 lnterleukin-18-binding protein DEIKCPNLN (SEQ ID 21 29 NO: 61) B* pool B*44: 03 Intermediate transcription factor 3 HDVYGVSNF (SEQ 287 295 large subunit ID NO: 62) B* pool B*44: 03 Major core protein 4b DEVASTHDW (SEQ 90 98 ID NO: 63) B* pool B*44: 03 Major core protein 4b YEFRKVKSY (SEQ ID 264 272 NO: 64) B* pool B*44: 03 mRNA-capping enzyme catalytic EERHIFLDY (SEQ ID 126 134 subunit NO: 65) B* pool B*44: 03 Primase D5 LENGAIRIY (SEQ ID 298 306 NO: 66) B* pool B*44: 03 Primase D5 EEIPDFAFY (SEQ ID 691 699 NO: 67) B* pool B*44: 03 Protein E3 DDVSREKSM (SEQ 86 94 ID NO: 68) B* pool B*44: 03 Protein I3 IEGELESLS (SEQ ID 173 181 NO: 69) B* pool B*44: 03 Protein M2 AELTIGVNY (SEQ ID 38 46 NO: 70)

[0166] ELISPOT Assay

[0167] All samples were assayed for their response to the Vaccinia overlapping peptide pool or to the novel epitope-based peptide pools by ELISPOT as follows. Cryopreserved PBMCs were thawed and allowed to rest for 16 hours in media with human serum at 37° C. Subsequently, the PBMCs were harvested and stimulated at 200,000 cells per well with the peptides pools at 5 μg/ml per individual peptide in 200 ul using the Human IFNγ/GranzymeB Double-Color FluoroSpot (ImmunoSpot, Cleveland, Ohio). Stimulations were incubated for 48 hours at 37° C. and developed using the manufacturer's protocol. Fluorescent spot forming cells (SFC) were imaged and counted on an ImmunoSpot S6 Universal. Subsequent analyses of the data were done on Microsoft Excel and Graphpad Prism.

[0168] Overlapping Peptide Pool Results

[0169] Initially, anti-Vaccinia T cell responses were determined to an overlapping peptide pool covering the second half of the Vaccinia protein P4a. PBMCs from patients were stimulated with the peptide pool for 24 hours and responding cells were quantified by IFNγELISPOT. Of note, for the majority of samples, the ELISPOT assay had a particularly high background (30-60 SFC) and/or a large variability between the triplicate wells for each stimulation. To normalize analysis, the background spot detection was subtracted from each sample. Due to the high variation of replicates, only responses greater than 10 SFC were considered above the level of detection. The results are illustrated at FIG. 4.

[0170] For the majority of samples, the assay with the overlapping peptide pool had a particularly high background (30-60 SFC) and/or a large variability between the triplicate wells for each stimulation. Interestingly, in patients with Stable Disease, an increase in the response to the overlapping peptide pool was observed (*p<0.05, paired non-parametric Student's t-test). Previous studies have found strong peripheral T cell responses to Vaccinia for many years after vaccination, so it was likely that the chosen overlapping pool did not contain sufficient epitope specific to accurately measure the response. Unfortunately, a full screen of all potential Vaccinia epitopes would consist of thousands of peptides and require larger amounts of PBMC from patients than what could be safely collected. Thus, we concluded that this format of the assay is insufficient to accurately track peripheral responses to Pexa-Vec.

[0171] Epitope-Based Peptide Pool Results

[0172] Increased Detection of Responses to Vaccinia Virus

[0173] As the detected response to the overlapping vaccinia peptide pool was below what was expected, custom peptide pools from known immunogenic Vaccinia epitopes in an HLA-agnostic format to profile peripheral CD8.sup.+T cell responses as described above in order to accurately track anti-Vaccinia responses in these patients and efficiently use small volumes of collected blood.

[0174] Upon screening PBMC from patients included in the study with the new peptide pools in the 48-hour ELISPOT as described above, we found that the epitope-based, HLA-agnostic peptide pools were successful in tracking CD8.sup.+T cell responses in RCC patients treated with the oncolytic virus Pexa-Vec, with increased response detected for almost every sample with epitope-based pools in comparison to the overlapping pool. See FIG. 6A. At Baseline and Week 6, this difference between the two stimuli was significantly different (*p<0.05, paired non-parametric Student's t-test) and the trend was still evident at Baseline and Week 12. Additionally, the responses to each of the two pool formats were compared using specific thresholds of detection. Using response thresholds of 10 SFC (p<0.05) or 20 SFC (p<0.005) per 2×10.sup.5 PBMC, the anti-vaccinia response to the epitope-based pools were statistically higher than the overlapping peptide pool, with there being statistically more patients with responses higher than 10 SFC (p<0.05) and 20 SFC (p<0.005) with the epitope-based pools (Table 4, see also FIG. 6B):

TABLE-US-00004 TABLE 4 >10 <10 >20 <20 SFC SFC SFC SFC Fisher’s exact test Overlapping  6 11  3 14 <0.05 Epitope-Based 13  4 12  5 <0.005

[0175] Overall, these data show that the epitope-based pools are a more accurate method to detect peripheral responses to vaccinia in these patients than the overlapping peptide pool.

[0176] Response to Epitope-Based Peptide Pools at Week 6 Correlates with Clinical Response

[0177] Consistent with the HLA allele frequencies of the South Korean population, most of the induction was specific to the A*01,03,24 pool, but there was also a large amount of induction in the A*02:01 pool (see FIG. 7). To determine whether there is a correlation between induction of T-cell response and clinical response, the SFC measured on treatment was normalized to the response at Baseline for each patient. To calculate the “fold change” of each patient longitudinally as compared to the Baseline response, the absolute SFC value at Week 6, Week 12, or additional time-points were divided by the absolute value of the Baseline response. Thus, a fold change of greater than 1 is an increase in response, and less than 1 is a decrease. See FIG. 8A. Interestingly, the majority (8/11) of patients with Stable Disease had a measurable increase in T cell response to epitope-based pools at Week 6 and beyond. Additionally, no Stable Disease patients displayed a decreased overall response. In contrast, three of four patients with Progressive Disease decreased their response to the pools at Week 6 (p=0.0517; Table 5):

TABLE-US-00005 TABLE 5 Increased No or Response Decreased post vaccination Response Fisher’s exact test Stable Disease 8 1 0.0517 Progressive Disease 1 3

[0178] Further, the same analysis performed on the response to the overlapping pool did not show the same grouping of inductions as the epitope-based pools. Rather, the change in response did not trend with any specific clinical response. See FIG. 8B.

[0179] Correlations with Clinical and Peripheral Epitope-Based Responses

[0180] Finally, we investigated the correlations between the fold change in peripheral T cell response to clinical observations. The change in response at week 6 for all patients was analyzed in comparison to clinical response, excluding patients for which a baseline or week 6 PBMC sample was not available to assay or for which no response to the vaccinia pools were detected. Correlation was determined by linear regression of all patients.

[0181] A trend of correlation was observed between overall survival and the combined response to all of the vaccinia epitope-based pools. See FIG. 9A. A strong correlation between overall survival and the response to the A*02 supertype pool was observed. See FIG. 9B.

CONCLUSIONS

[0182] The results demonstrate that the HLA-agnostic peptide pools were successful in tracking CD8+T cell responses in RCC patients treated with JX-594. Interestingly, 8/11 patients with Stable Disease at week six had increased responses to the peptide pools relative to baseline, whereas three out of four Progressive Disease patients displayed decreased responses (p<0.005, Fisher t test). Additionally, an increase in PBMC response at week six to the A*02 supertype pool directly correlated to overall survival of the patients, whereas no correlation was detected with the other peptide pools.

[0183] Thus, profiling of the peripheral CD8+T cell responses to vaccinia virus (exemplified here by JX-594) can be achieved with a high degree of accuracy and minimal sample requirements in an HLA-agnostic manner using custom peptide pools based on known Vaccinia epitopes. This method allows for screening with small peptide pools numbers (10s rather than 100-1000s) and thus requires significantly fewer valuable clinical trial samples. By reducing and improving the number of candidate epitopes, the assay can track systemic patient responses while using less of the valuable clinical samples.

[0184] The CD8.sup.+T cell response to Pexa-Vec at Week 6 after treatment initiation suggests that patients with Stable disease had a higher induction of T cell response to epitope-based Vaccinia peptide pools than those with Progressive disease.

[0185] The peptide pools can be used to predict clinical responses to oncolytic vaccinia virus at early time points in a variety of cancers. Paired tumor biopsies allow the correlation with vaccinia-specific TCRs identified in the periphery with TCRs on intra-tumoral CD8+T cells.

Example 3

[0186] The epitope-based peptide pools described above in Example 2 were used to evaluate the circulating vaccinia virus-specific CD8+T cell response in patients with advanced renal cell carcinoma (naive or refractory to prior systemic treatment and who had no prior treatment with immune checkpoint inhibitors) during the course of treatment with 4 weekly intravenous infusions of JX-594 (Pexa-Vec) at 10.sup.9 plaque forming units (pfu) starting at Day-7 plus the monoclonal anti-PD-1 antibody Cemiplimab (350 mg every 3 weeks) from Day 1. Radiographic assessments per RECIST 1.1 were performed centrally every 9 weeks from Day 1. Peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved at baseline and at 29 days post initial JX-594 treatment.

[0187] IFNγELISPOT analysis was performed on longitudinal PMBC samples using the epitope-based peptide pools described above in Example 2 (OV peptides) and culture conditions designed to measure existing oncolytic virus (OV)-specific memory T cell cytolytic activity. PBMC samples from patients were tested for IFNγ release following stimulation with OV peptides using two different assay conditions: (1) measurement following direct ex vivo stimulation with OV peptides alone and (2) measurement following 10 days of T cell expansion in the presence of OV peptides, the T cell supportive cytokines GM-CSF, IL-4, IL-7 and IL-15 and autologous dendritic cells. The number of OV-specific IFNγ spots was correlated with the clinical response and tumor regression.

[0188] Remarkably, 8 of the 11 (72.7%) patients showed tumor burden reduction, 4 of whom had ≥30% confirmed reduction (FIGS. 10 and 11). OV-specific IFNγ-producing T cells were detected in only 3 of 11 patients in the nonexpanded ELISPOT culture conditions (FIG. 12, left panel), but in 8 out of 11 patients when T cells were first expanded for 10 days in the presence of OV peptides prior to ELISPOT, which trended toward a correlation with the preliminary clinical response assessment (FIG. 12, right panel). Prolonged stimulation with CMV, EBV and Influenza peptides did not show any correlation (R.sup.2=0.005), suggesting that the treatment and culture expansion influenced relevant OV-specific memory T cell proliferation. There was a 9-fold increase in the detection range of IFNγ producing cells up to 1000 SFC/2×10.sup.5 cells (FIG. 12, right panel), from a detection range of fewer than 110 SFC/2×10.sup.5 cells (FIG. 12, left panel). Pre-expanded ELISPOT revealed a trend of correlation between CD8+T cell response and tumor volume reduction even at an early sample collection time point (Day 29) with R2=0.3031 and p-value=0.0793.

[0189] Conclusion: These results demonstrate that OV-specific T cell responses can be induced by OV therapy. In addition, 10-day expansion of low levels of OV-specific circulating T cells can amplify signals in ELISPOT analysis and might enable systemic tracking of patient responses in blood samples collected at early time points. The observed CD8+T cell response to oncolytic vaccinia virus in patients supports the rationale for combination treatment with OV (e.g. Pexa-Vec) and immune checkpoint inhibitors (e.g. Cemiplimab).

[0190] While the materials and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention