Rachael L. Metheringham

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBodyTM, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBodyTM expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBodyTM are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBodyTM vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.

High Avidity Cytotoxic T Lymphocytes Can Be Selected into the Memory Pool but They Are Exquisitely Sensitive to Functional Impairment

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 µg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.

Novel prostate acid phosphatase-based peptide vaccination strategy induces antigen-specific T-cell responses and limits tumour growth in mice

Treatment options for patients with advanced prostate cancer remain limited and rarely curative. Prostatic acid phosphatase (PAP) is a prostate‐specific protein overexpressed in 95% of prostate tumours. An FDA‐approved vaccine for the treatment of advanced prostate disease, PROVENGE® (sipuleucel‐T), has been shown to prolong survival, however the precise sequence of the PAP protein responsible for the outcome is unknown. As the PAP antigen is one of the very few prostate‐specific antigens for which there is a rodent equivalent with high homology, preclinical studies using PAP have the potential to be directly relevant to clinical setting. Here, we show three PAP epitopes naturally processed and presented in the context of HHDII/DR1 (114–128, 299–313, and 230–244). The PAP‐114‐128 epitope elicits CD4+ and CD8+ T‐cell‐specific responses in C57BL/6 mice. Furthermore, when immunised in a DNA vector format (ImmunoBody®), PAP‐114‐128 prevents and reduces the growth of transgenic adenocarcinoma of mouse prostate‐C1 prostate cancer cell‐derived tumours in both prophylactic and therapeutic settings. This anti‐tumour effect is associated with infiltration of CD8+ tumour‐infiltrating lymphocytes and the generation of high avidity T cells secreting elevated levels of IFN‐γ. PAP‐114‐128 therefore appears to be a highly relevant peptide on which to base vaccines for the treatment of prostate cancer.

Autophagy, citrullination and cancer

ABSTRACT A cell needs to maintain a balance between biosynthesis and degradation of cellular components to maintain homeostasis. There are 2 pathways, the proteasome, which degrades short-lived proteins, and the autophagy/lysosomal pathway, which degrades long-lived proteins and organelles. Both of these pathways are also involved in antigen presentation or the effective delivery of peptides to MHC molecules for presentation to T cells. Autophagy (macroautophagy) is a key player in providing substantial sources of citrullinated peptides for loading onto MHC-II molecules to stimulate CD4+ T cell responses. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. We therefore investigated if citrullinated peptides could stimulate CD4+ T cell responses that would recognize these modifications produced during autophagy within tumor cells. Focusing on the intermediate filament protein VIM (vimentin), we generated citrullinated VIM peptides for immunization experiments in mice. Immunization with these peptides induced CD4+ T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 d after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. These results show how CD4+ cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides produced during autophagy may offer especially attractive vaccine targets for cancer therapy.

Vaccines as early therapeutic interventions for cancer therapy: neutralising the immunosuppressive tumour environment and increasing T cell avidity may lead to improved responses

Importance of the field: Considerable progress has been made in identifying the antigens recognised by the immune system. This has led to the success of monoclonal antibody therapy and the recent approval of prophylactic vaccines that give excellent protection against cervical cancer. Provenge will shortly be the first therapeutic vaccine to be approved. Areas covered in this review: Our aim is to discuss the recent success with prophylactic cancer vaccines for prevention of cancer and the progress with therapeutic vaccines design to eradicate established tumours. Therapeutic vaccines need to stimulate high-avidity T cell responses that can recognise and kill tumours. How this can be achieved in cancer patients is discussed. The immunosuppressive tumour environment also needs to be modified to allow extravasation and efficacy of the vaccine induced T cells. What the reader will gain: An insight into the limitations of present cancer vaccine approaches and how they can be manipulated to give more effective anti-tumour responses. Take home message: A combination of more effective vaccines that stimulate high-avidity T cells, in combination with drugs or monoclonal antibodies that neutralize immunosuppressive factors within the tumour environment are needed to improve the efficacy of immunotherapy of cancer.

SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors

Purpose We have previously shown that supraoptimal signaling of high avidity T cells leads to high expression of PD-1 and inhibition of proliferation. This study was designed to see if this effect could be mitigated by combining a vaccine that stimulates high avidity T cells with PD-1 blockade. Experimental Design We investigated the anti-tumor effect of a huIgG1 antibody DNA vaccine (SCIB1) and PD-1 blockade. Results Vaccination of HLA-DR4 transgenic mice with SCIB1 induced high frequency and avidity T cell responses that resulted in survival (40%) of mice with established B16F1-DR4 tumors. SCIB1 vaccination was associated with increased infiltration of CD4 and CD8 T cells within the tumor but was also associated with upregulation of PD-L1 within the tumor environment. PD-1 blockade also resulted in increased CD8 T cell infiltration and an anti-tumor response with 50% of mice showing long term survival. In line with our hypothesis that PD-1/PD-L1 signaling results in inhibition of proliferation of high avidity T cells at the tumor site, the combination of PD-1 blockade with vaccination, enhanced the number and proliferation of the CD8 tumor infiltrate. This resulted in a potent anti-tumor response with 80% survival of the mice. Conclusions There is a benefit in combining PD-1 blockade with vaccines that induce high avidity T cell responses and in particular with SCIB1.

Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial

ABSTRACT A DNA vaccine, SCIB1, incorporating two CD8 and two CD4 epitopes from TRP-2/gp100 was evaluated in patients with metastatic melanoma. Each patient received SCIB1 via intramuscular injection with electroporation. The trial was designed to find the safest dose of SCIB1 which induced immune/clinical responses in patients with or without tumour. Fifteen patients with tumor received SCIB1 doses of 0.4-8 mg whilst 20 fully-resected patients received 2–8 mg doses. Twelve patients elected to continue immunization every 3 months for up to 39 months. SCIB1 induced dose-dependent T cell responses in 88% of patients with no serious adverse effects or dose limiting toxicities. The intensity of the T cell responses was significantly higher in patients receiving 4 mg doses without tumor when compared to those with tumor (p < 0.01). In contrast, patients with tumor showed a significantly higher response to the 8 mg dose than the 4 mg dose (p < 0.03) but there was no significant difference in the patients without tumor. One of 15 patients with measurable disease showed an objective tumor response and 7/15 showed stable disease. 5/20 fully-resected patients have experienced disease recurrence but all remained alive at the cut-off date with a median observation time of 37 months. A positive clinical outcome was associated with MHC-I and MHC-II expression on tumors prior to therapy (p = 0.027). We conclude that SCIB1 is well tolerated and stimulates potent T cell responses in melanoma patients. It deserves further evaluation as a single agent adjuvant therapy or in combination with checkpoint inhibitors in advanced disease.

Abstract CT331: Phase I/II trial of a novel antibody DNA immunotherapy, targeting CD64, in the treatment of Melanoma

SCIB1 is a novel DNA immunotherapy that has epitopes from gp100 and TRP-2 melanoma antigens, engineered into a human IgG1 antibody. The therapy works by direct transfection and cross presentation via CD64 of dendritic cells. Vaccination results in high avidity T cells and tumour elimination in preclinical models. (1,2) A clinical trial was conducted to determine its safety and its ability to induce cellular immune responses. Patients and Methods: The vaccine was administered via Intramuscular injection with electroporation at 3 weekly intervals for 3 vaccinations then at 3 and 6 months. In part 1 of the study, nine patients with Stage III/IV melanoma were given escalating doses of SCIB1. Due to lack of toxicity the 2mg cohort were allowed to receive 4mg doses in their booster immunisations and the 4mg cohorts were allowed to continue with 3-6 monthly immunisations for 5 years. The 4mg dose was selected for an expansion cohort (part 2). To date 8 patients with fully resected stage III and 6 with fully resected stage IV melanoma have been treated and 7/14 patients are receiving ongoing vaccination. Results: No systemic dose-limiting toxicities were observed. The most common adverse event was injection site pain. 4/6 patients in the 2mg/4mg cohorts who received >3 doses of SCIB1, are still alive with a median survival time of 24 months. One patient had multiple tumour lesions (several in her lungs). All decreased in size or disappeared following treatment except for one lesion which was resected. Immunohistochemistry demonstrated strong expression of PD.L1 on the tumour cells. All patients in part 2 remain alive and only three have progressed. The median survival time in Part 2 is 15 months from study entry and 19 months from diagnosis of metastatic disease. In part 1, one patient in the 0.4mg cohort, all three patients in the 2mg/4mg dose cohort and two patients in the 4mg dose cohort mounted a measurable immune response to the vaccine-encoded antigens. In part 2, all 14 patients responded immunologically. 12/14 patients in the proliferation assay, 13/14 patients responded after T cell expansion in-vitro followed by ELISPOT assay and 11/14 patients responded in both assays. Responses were seen against both the CD8 epitopes and against the CD4+ epitopes. Six patients responded to all four epitopes, five patients responded to three epitopes and three patients responded to two epitopes. Significant responses (p>0.0001) were seen after three, four or five immunisations, indicating that at least three doses are required for a strong immune response to develop. Conclusion: We demonstrate that SCIB1 is safe in melanoma patients. 19/20 patients showed immune responses to repeat dosing at 2 or 4 mg. Detection of an objective clinical response and overall survival times are encouraging. 1. Pudney et al (2010). Eur J Immunol 40: 899. 2. Brentville et al (2012). Plos one 7:e4111 Citation Format: Lindy G. Durrant, Christian H. Ottensmeier, Paul Lorigan, Clive Mulatero, Ruth Plummer, Michelle Cunell, Rachael Metheringham, Victoria Brentville, Lee Machado, Poulam Patel. Phase I/II trial of a novel antibody DNA immunotherapy, targeting CD64, in the treatment of Melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT331. doi:10.1158/1538-7445.AM2014-CT331

Abstract A035: Citrullinated α-enolase as a novel target for cancer immunotherapy

Stressful conditions in the tumor microenvironment induce autophagy in cancer cells to promote their survival, however, autophagy also causes post-translational modification of proteins which are recognized by the immune system. In particular, modified self-antigens can trigger CD4+ T cell responses that can be exploited to boost antitumor immune defenses. We have previously investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. These studies showed that vimentin, which is frequently expressed in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, is citrullinated and is a good target for anti-tumor immunity (Ref. 1). Immunization with citrullinated vimentin peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long term survival in 60-90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8+ T cells. Due to its ubiquitous expression and abundance in most cells, the glycolytic enzyme α-enolase is a protein that is often citrullinated and degraded during autophagy and may represent a further novel antitumor target. In this study we demonstrate that immunization of C57Bl, HLA-DR4 and HLA-DP4 transgenic mice with citrullinated enolase peptides induces strong Th1/cytotoxic CD4 responses that efficiently target tumor cells. The Th1 cell repertoire to citrullinated enolase is also detectable in healthy donors and cancer patients. Immunization of mice with citrullinated enolase peptides led to tumor therapy in HLA-DP4 mice with established B16-DP4 tumors (70% survival p = 0.0058) and in HLA-DR4 transgenic mice with established B16F1-DR4 melanoma (50% survival; p = 0.0048) or Pan02-DR4 pancreatic tumors (survival 50%; p = 0.0076). The response was partially mediated by CD4 cytotoxic T cells as tumor therapy was observed against the HLA-DR4-expressing lung tumor LLC2 (40% survival; p = 0.0142) but no survival advantage was witnessed against LLC2 tumors which do not express class II MHC. As MHC-II is not expressed by the majority of tumors unless induced by IFNγ we designed an HLA-DR4 construct under expression of an IFNγ inducible promoter. Immunization of HLA-DR4 mice with citrullinated enolase peptides led to tumor therapy against the established B16F1-IFNγ inducible DR4 melanoma (90% survival p>0.0001). These results suggest that, similar to citrullinated vimentin, citrullinated α-enolase is a promising novel target for human cancer immunotherapy. References1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60. Citation Format: Katherine Cook, Ian Daniels, Victoria Brentville, Rachael Metheringham, Wei Xue, Peter Symonds, Tracy Pitt, Mohammed Gijon, Lindy G. Durrant. Citrullinated α-enolase as a novel target for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A035.

Abstract A015: Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer

Citrullination of proteins is associated with a number of autoimmune diseases. Protein Arginine Deiminases (PADs) are a family of Ca2+ dependent enzymes that under cellular stress post-translationally convert arginine to citrulline within protein substrates to generate self-modified neo-antigens. It has been shown that presentation of citrullinated peptides on MHC class II stimulates CD4 T cells to mediate potent anti-tumor immunity (1). In this study we focus on the role of the PADI2 and PADI4 family members in colorectal cancer. Using a tissue microarray of colorectal cancers and compiling a comprehensive database of clinicopathological variables, the expression of PADI2 and PADI4 was assessed by immunohistochemistry in a study cohort. This data was used to correlate PADI2 and PADI4 expression with patient survival. In the study cohort 462 colorectal tumors were stained for PADI2 and PADI4. Of these 171 (37%) and 231 (50%) could not be evaluated for PADI2 and PADI4 staining respectively due to the absence of enough tissue core or no evaluable tumor cells (i.e. all stroma) in the core. Of the 291 evaluable colorectal tumors stained with a PADI2 specific antibody, only 18/291 (6.2%) tumors failed to stain. A further 153/291 (52.5%) stained weakly, 102/291 (35.1%) moderate and 18/291 (6.2%) stained strongly. Of the 231 evaluable colorectal tumors stained with a PADI4 specific antibody, no tumors failed to stain. All cases stained strongly for PADI4 expression within the nucleus. In the cytoplasm 63/231 (27.3%) stained weakly, 143/231 (61.9%) moderate and 25/231 (10.8%) stained strongly. PADI2 expression did not correlate with any clinicopathological variables whereas nuclear but not cytoplasmic PADI4 showed a strong association with histological type (p = 0.008). Kaplan-Meier analysis showed there was a correlation of PADI2 and cytoplasmic PADI4 expression with improved survival. Expression of PADI2 gave an increase in survival time from 44.8 months (95% CI 24.3 to 65.4) to 76.2 months (95% CI 69.9 to 82.4, log rank test, p = 0.012). Expression of cytoplasmic PADI4 increased survival time from 57.9 months (95% CI 43.6 to 72.3) to 77.3 months (95% CI 69.6 to 85.1, log rank test, p = 0.012). No significant correlation was observed between PADI2 and the cytoskeletal protein Vimentin or the glycolytic enzyme α-enolase both reported to be citrullinated by PAD enzymes. PADI2 expression was significantly associated with expression of the Nuclear antigen Ki67 (p = 0.046) a cellular marker for proliferation. Nuclear PADI4 significantly correlated with the cytoplasmic glycolytic enzyme α-enolase only (p = 0.001) and cytoplasmic PADI4 was highly significantly associated with α-enolase located in both the cytoplasm (p References 1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60 Citation Format: R. Metheringham, M. Gijon, I. Daniels, K. Cook, P. Symonds, T. Pitt, W. Xue, V. Brentville, L. Durrant. Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A015.