Murrium Ahmad

Escape from immunotherapy: possible mechanisms that influence tumor regression/progression

Tumor escape is one major obstacle that has to be addressed prior to designing and delivering successful immunotherapy. There is compelling evidence to support the notion that immunogenic tumors, in murine models and cancer patients, can be rejected by the immune system under optimum conditions for activating adaptive and nonadaptive antitumor immune responses. Despite this capability, a large number of tumors continue to grow and evade recognition and/or destruction by the immune system. The limited success in current immunotherapeutic strategies may be due to a variety of reasons: failure of effector cells to compete with the growing tumor burden, production of humoral factors by tumors that locally block cytotoxicity, antigen/MHC loss, T-cell dysfunction, production of suppressor T cells—to name but a few causes for therapeutic ineffectiveness for the particular malignancy being treated. To optimize immunotherapy strategies, correction of immune-activating signals, eradication of inhibitory factors, and the evasion from newly developed immunoresistant tumor phenotypes need to be simultaneously considered.

The identification of human tumour antigens: current status and future developments

The biggest challenge facing us today in cancer control and prevention is the identification of novel biomarkers for detection and improved therapeutic interventions to reduce mortality and morbidity rates. Biomarkers are important indicators to inform us of the physiological state of the cell at a specific time. It is now clear that malignant transformation occurs by changes in cellular DNA and protein expression with subsequent clonal proliferation of the altered cells. The affected genes and their expressed protein products or biomarkers are those involved in the normal growth and maintenance of the cancerous cells. These biomarkers could prove pivotal for the identification of early cancer and people at risk of developing cancer. Altered proteins or changes in gene expression in malignant cells may lead to the expression of tumour antigens recognised by host immune system. In this review we discuss current research into the molecular technologies making possible the global genomic-wide analysis of changes in DNA (genotyping), RNA expression (transcriptomics) and protein expression (proteomics) that have accelerated the rate of new biomarker/tumour antigen discovery. To gain a comprehensive understanding of the physiology and pathophysiology of cancer an approach that harmoniously integrates the various ‘omic’ platforms are key to unraveling the complexity ‘needle-in-a-haystack’ quality of biomarker/tumour antigen discovery.

Immunotherapeutic potential of DISC-HSV and OX40L in cancer

Several vectors, viral and bacterial, have been developed over the past few years for means of generating an effective antitumor immune response. We have developed and studied a “model for immunotherapy” using a viral vector disabled infectious single cycle-herpes simplex virus (DISC-HSV), which efficiently transduces various tumor cell lines and offers a useful vehicle for the further development of cell-based vaccines. The immunotherapeutic potential of DISC-HSV encoding granulocyte macrophage colony stimulating factor (GM-CSF) was demonstrated in a number of murine carcinoma models, leading to complete regression of well-established tumors in up to 70% of the mice. Moreover, the therapeutic potential of DISC-HSV-GM-CSF was significantly enhanced when used in combination therapy with either OX40L or dendritic cells (DC), even in a poorly immunogenic tumor model. The ability of this vector to accept large gene inserts, its good safety profile, its ability to undergo only a single round of infection, the inherent viral immunostimulatory properties and its ability to infect various tumor cell lines efficiently, make DISC-HSV an ideal candidate vector for immunotherapy. The DISC- CT-26 tumor model was used to investigate the mechanisms associated with immunotherapy induced tumor rejection. Although CTL induction, was positively correlated with regression, MHC class I down regulation and accumulation of immature Gr1+ myeloid cells were shown to be the main immuno-suppressor mechanisms operating against regression and associated with progressive tumor growth. The CTL response was associated with the immuno-dominant AH-1 peptide of the retroviral glycoprotein gp70. This model of immunotherapy has provided an opportunity to dissect further the immunological events associated with tumor-rejection and escape. Since other antigens may be important in initiating tumor rejection, we have investigated the expression of MTA-1, an antigen that appears to be expressed widely in human and murine tumors. The immunogenicity of MTA-1 was studied and its potential as a tumor rejection antigen is under investigation.

Trafficking of tumor peptide-specific cytotoxic T lymphocytes into the tumor microcirculation

The major histocompatibility complex class I‐restricted CD8+ cytotoxic T‐lymphocyte (CTL) effector arm of the adaptive immune response can specifically recognize and destroy tumor cells expressing peptide antigens. Although adoptive T‐cell therapy has been successfully used for the treatment of viral and malignant diseases, little is known of the trafficking and fate of adoptively transferred antigen‐specific T cells. In the present study, splenocytes derived from mice that rejected their tumors (CT26 or CT26‐clone 25 tumors) in response to direct intratumor injection of disabled infectious single‐cycle herpes simplex virus (DISC‐HSV) encoding murine GM‐CSF were restimulated with peptide in vitro. CTLs specific for the AH‐1 and β‐gal peptides expressed by CT26 and CT26‐clone 25 tumor cells, respectively, were generated and used for adoptive cellular therapy and trafficking studies. Intravenous administration of AH‐1‐specific CTLs 3 days following i.v. injection of CT26 cells resulted in significant tumor growth inhibition, whereas administration of control CTLs generated against a bacterial β‐gal peptide did not inhibit the growth of tumors. Trafficking of AH‐1‐specific lymphocytes and their interaction with the CT26 tumor microcirculation was analyzed using real‐time in vivo microscopy (IVM). AH‐1‐specific but not β‐gal‐specific CTLs adhered and localized in the CT26 tumor microvasculature, but neither population adhered to the endothelium of the normal microcirculation. This study provides direct visual evidence suggesting that AH‐1‐specific CTLs that mediate a therapeutic response traffic to and localize within the tumor microenvironment.

Tumour-associated antigens: considerations for their use in tumour immunotherapy.

Since their discovery, tumour-associated antigens (TAA) have provided highly inviting targets for cancer therapy, especially immunotherapy. Evidence now points to their involvement in the malignant phenotype of transformed cells and heightens their importance for being targeted by different treatments. TAA vary in their nature and pattern of expression and this influences the way therapy is directed towards them. While large numbers of these antigens have been isolated from solid tumours, fewer are linked with haematological malignancies. Those TAA found in this latter group of cancers, referred to as leukaemia-associated antigens (LAA), also appear to have significant potential for promoting the malignant phenotype and have been described in detail in terms of expression and therapy. Interestingly, the action of some of LAA in blood cancers, which are stem cell derived, could act as model for solid tumours, which are increasingly thought to be also derived from a cancer stem cell origin. In this review, TAA and their use in immunotherapy will be discussed. The nature and expression of these antigens will be described together with the events that provide tumours, including haematological cancers, with the ability to avoid immune deletion.

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.

The helicase HAGE prevents interferon-α-induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK–STAT (janus kinase–signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK–STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.

Serum biomarkers which correlate with failure to respond to immunotherapy and tumor progression in a murine colorectal cancer model

Purpose: To advance our understanding of mechanisms involved in tumor progression/regression, a CT26 colorectal mouse model treated intra‐tumorally with DISC‐herpes simplex virus as immunotherapy was used in the discovery and validation phases to investigate and ultimately identify biomarkers correlating with the failure to respond to immunotherapy.

Immunity to Tumour Antigens

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

Regulation of CTL responses to MHC-restricted class i peptide of the gp70 tumour antigen by splenic parenchymal CD4+ T cells in mice failing immunotherapy with DISC-mGM-CSF

Direct intratumour injection of the disabled infectious single‐cycle–herpes simplex virus–encoding murine granulocyte/macrophage colony‐stimulating factor (DISC‐HSV–mGM‐CSF) into established colon carcinoma CT26 tumours induced complete tumour rejection in up to 70% of treated animals (regressors), while the remaining mice developed progressive tumours (progressors). This murine Balb/c model was used to dissect the cellular mechanisms involved in tumour regression or progression following immunotherapy. CTLs were generated by coculturing lymphocytes and parenchymal cells from the same spleens of individual regressor or progressor animals in the presence of the relevant AH‐1 peptide derived from the gp70 tumour‐associated antigens expressed by CT26 tumours. Tumour regression was correlated with potent CTL responses, spleen weight and cytokine (IFN‐γ) production. Conversely, progressor splenocytes exhibited weak to no CTL activity and poor IFN‐γ production, concomitant with the presence of a suppressor cell population in the progressor splenic parenchymal cell fraction. Further fractionation of this parenchymal subpopulation demonstrated that cells inhibitory to the activation of AH‐1‐specific CTLs, restimulated in vitro with peptide, were present in the nonadherent parenchymal fraction. In vitro depletion of progressor parenchymal CD3+/CD4+ T cells restored the CTL response of the cocultured splenocytes (regressor lymphocytes and progressor parenchymal cells) and decreased the production of IL‐10, suggesting that CD3+CD4+ T lymphocytes present in the parenchymal fraction regulated the CTL response to AH‐1. We examined the cellular responses associated with tumour rejection and progression, identifying regulatory pathways associated with failure to respond to immunotherapy.