Sachin Puri

Signaling Through OX40 Enhances Antitumor Immunity

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

Cancer Immunotherapy: The Role Regulatory T Cells Play and What Can be Done to Overcome their Inhibitory Effects

Since multiple lines of experimental and clinical data clearly identified regulatory T cells as an integral part of the immune response, these cells have become a major focus of investigation in tumor immunology. Regulatory T cells are in place to dampen ongoing immune responses and to prevent autoimmunity, but they also have profound effects in blocking therapeutic anti-tumor activity. Therefore regulatory T cells are seen as a major hurdle that must be overcome in order for cancer immunotherapy to reach its therapeutic potential. Regulatory T cells are heterogeneous with sub-populations that exhibit distinct functional features. Here we will review the individual sub-populations in regards to their mode of action and their potential impact on blocking anti-tumor immunity. Approaches to measure function and frequency of regulatory T cells in model systems and clinical trails will be discussed. Finally, we will describe possible ways to interfere with regulatory T cell-mediated immune suppression with the focus on recent pre-clinical and clinical findings.

Sustained complete response to CTLA-4 blockade in a patient with metastatic, castration-resistant prostate cancer

Graff and colleagues analyzed sera and biopsies from a prostate cancer patient, which showed amplification of the 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) gene with strong protein expression, suggesting that the augmented antibody response to HIBCH contributed to the durable immune response in prostate cancer. We present the case of a man with metastatic, castration-resistant prostate cancer, who had a complete prostate-specific antigen (PSA) response after 2½ doses of ipilimumab. His treatment course was complicated by diarrhea and autoimmune hepatitis, both of which resolved within 4 months. Sera and biopsy specimens were accessed, and sera from pretreatment and day 113 were analyzed. Augmented antibody responses were detected against 11 potential tumor antigens, with responses ranging from 5- to 20-fold in day 113 sera compared with baseline. Genes that were targets of a strong antibody response (arbitrarily set at 10-fold or greater increase) were analyzed by real-time PCR for expression in the tumor biopsy cDNA. Of the top 5 genes, only 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) could be identified in the amplified tumor biopsy cDNA. Using an antibody to HIBCH, immunohistochemical analysis documented strong expression of the protein. Together, these data suggest that an augmented antibody response to HIBCH, an antigen that was expressed by the patients prostate cancer, could have contributed to the clinical response. After 16 months of PSA stability, he discontinued his androgen-suppression therapy. With the return of his testosterone, his PSA increased slightly, likely originating from his intact prostate. He has been disease free for the past 6 years without any additional therapy. Cancer Immunol Res; 2(5); 399–403. ©2014 AACR.

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer

ABSTRACT The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous – single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85–22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.

Pyruvate kinase M2 may be a therapeutic target in a mouse model of human non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common cause of cancer related mortality worldwide and in need of new treatment options. Previously, we have shown that 97 genes are overexpressed in 9 primary NSCLC cell lines by DNA microarray technology. Pyruvate kinase M2 (PKM2), an allosteric isoform of pyruvate kinase and metabolic enzyme necessary for aerobic glycolysis and cell proliferation, was identified to be over-expressed in the NSCLC cell lines. Real-time qRT-PCR and immunohistochemistry (IHC) techniques confirmed the microarrays results. Consistent with the over-expression of PKM2, all 9 NSCLC cell lines showed higher PKM2 enzyme activity than normal lung cell lines. Since a chemical inhibitor of PKM2 decreased PKM2 enzyme activity and proliferation of NSCLC cell lines, we hypothesized that PKM2 inhibitors may have a role in antitumor effects in vivo. Therefore, we evaluated antitumor effects of a PKM2 inhibitor in murine models of NSCLC. Mice tolerated intra-tumoral administration of the PKM2 inhibitor (Small molecule inhibitor for PKM2) up to 500 µg/kg/day without any evidence of visible toxicity or weight loss. Despite its anti-proliferative activities in vitro, the PKM2 inhibitor did not mediate significant antitumor effects in NSCLC tumor xenografts. The mechanism of lack of activity in vivo is not clear. It is possible that PMK2 inhibitor levels were not sufficient or sustained in vivo for a measurable antitumor effect. To further study whether PKM2 was a target for therapy, we silenced the PKM2 gene in the H1299 and H358 NSCLC cell lines using RNAi and proliferative activity of tumors was assessed in vitro and in vivo. Gene silencing in cells transfected with plasmids encoding shRNAs was confirmed by IHC, which showed >80% silencing measured by NIKON-S-Element software at the protein level. In clonogenic assays, we observed >50% fewer colonies in two PKM2 silenced NSCLC cell lines compared to mock transfected controls. In subcutaneous xenograft tumor models in athymic nude mice, preliminary results showed that PKM2-silenced tumors grew significantly slower compared to mock transfected tumors (p < 0.05). These results suggest that PKM2 may be a target for cancer therapy. Additionally, given its over-expression in NSCLC, it may also serve as a target for immunotherapy. Studies are ongoing to confirm these results, understand a mechanism of tumor response, and determine whether patients generate immune responses to PKM2.

Adoptive TIL in HPV-negative oral scca

Meeting abstracts A 22 year old male was diagnosed with clinical T2 squamous cell carcinoma of the lateral oral tongue. He underwent partial glossectomy with elective neck dissection of ipsilateral cervical nodes levels I-IV. Pathology showed a 2.1cm primary tumor with clear margins to 5mm, 1.6cm

Evaluation of circulating tumor cell gene expression profiles before and following combination immunotherapy for advanced prostate cancer

The Phase I/II clinical trial randomized patients to GVAX immunotherapy (two prostate cancer cell lines that secrete GM-CSF, Cell Genesys Inc.) alone or in combination with non-myeloablative chemotherapy and adoptive transfer of PBMC. Pre and week 11 aphereses were obtained to elutriate monocytes and cryopreserve PBMC for immune monitoring. We used ProtoArrays (8,217 proteins spotted arrays, Invitrogen) to evaluate the week 11 post treatment antibody responses of 11 patients who completed this IRB-approved DAMD-funded clinical trial for men with androgen-independent prostate cancer. Analysis of pre and week 11 sera identified increased antibody responses to a number of proteins and the patients with an increase in PSA-doubling time (PSA-DT) exhibited a strong response to a significantly (p 1.5 fold compared to a normal prostate tissue control. Current efforts are aimed at evaluating whether targets of the antibody response were contained in the oligonucleotide array and whether proteins for genes that decreased in intensity were contained on the protein array. The methods we describe may help assess whether a given treatment is inducing strong immune response and whether this immune response is sculpting the repertoire of antigens expressed by the tumor. Such tests are currently not available and development of such a test may aid the development and refinement of the novel combination immunotherapy strategies.

An image analysis algorithm based on the hue saturation density transformation, an important tool for melanoma immunotherapy research

Melanoma is the most serious form of skin cancer, resulting in nearly 10,000 deaths per year in the US alone. Approximately 76,000 people are diagnosed with melanoma each year, and that number has been growing steadily over the last 30 years. Tragically, melanoma affects younger as well as older people. In fact, it is one of the most common forms of cancer in people younger than 30 [1]. A significant challenge impeding melanoma research is that the very cells that cause the cancer, the melanocytes, can confound measurements made using traditional immunohistochemical (IHC) techniques. This is because the brown melanin in the cells creates a high background for bright-field IHC stains, which are often brown or red.

Novel strategy to monitor apparent tumor-specific T cells in patients with cancer

We hypothesize that immune responses that develop following immunotherapy, particularly in patients with objective responses, are directed against a wide range of antigenic determinants. However, in the absence of autologous tumor cells, monitoring is frequently limited to a small number of well-defined tumor-associated antigens (TAA) and underestimates the immune response. Further, we hypothesize that the majority of antigens expressed by HLA on tumor cells are short-lived proteins (SLiPs and DRiPs), which are rapidly degraded by the proteasome, bound to HLA and transported to the tumor’s surface. To develop an antigen source to evaluate this hypothesis in men with prostate cancer, gene expression datasets for prostate cancer (n=85) were compared with several tumor cell lines. One cell line, UbiLT3, had at least 1 overexpressed gene in common (avg 37.1 genes: range 0-200, Z-score 2) with 98.9% of the prostate cancers, suggesting that UbiLT3 had TAA in common with prostate cancer. Next UbiLT3 were cultured with proteosomal and lysosomal inhibitors to accumulate SLiPs and DRiPs in autophagosomes (DRibbles/DRb), which were harvested and used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX (PC3/ LNCaP). After one week of IVS with UbiLT3-DRb, we observed UbiLT3-DRb-specific CD8 T-cell responses in post-vaccine PBMC of 6/12 patients (range 2-22% of CD8). No responses were seen in pre PBMC or healthy donor controls. We could not induce T cells recognizing normal kidney (NK) control DRb after 1-week IVS nor could UbiLT3-DRb stimulated T cells recognize NK-DRb. The difference between pre vs. post DRibble-specific responses was highly significant (p=0.007). To rule out qualitative differences between pre- and post-vaccine antigen-presenting cells, UbiLT3-DRb stimulated CD11c+ cells were isolated from pre and post-vaccine PBMC and were cultured with isolated non-stimulated pre- or post vaccine T cells. Pre and post CD11c+ cells were equally able to induce CD8 responses in post T cells but not pre T cells (n=2). Our data show that vaccination with prostate GVAX induced T cell responses that recognized DRibbles from a cancer cell line that shared common overexpressed genes, but not DRibbles from normal kidney cells. Since no autologous tumors were isolated from these patients, we are looking for ways to evaluate the tumor-killing abilities of these T cells. Current studies are examining this strategy in cancers where autologous tumor cells are available. Support