Judith M. Ramage

Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis

Many colorectal tumors lose or downregulate cell surface expression of MHC class I molecules conferring resistance to T‐cell‐mediated attack. It has been suggested that this phenomenon is due to in vivo immune‐tumor interactions. However, evidence of the impact of MHC class I loss on outcomes from colorectal cancer is scarce. In our study of more than 450 colorectal cancers in tissue microarray format, we have shown that both high levels of MHC class I expression and absent MHC class I expression are associated with similar disease‐specific survival times, possibly due to natural killer cell‐mediated clearance of MHC class I‐negative tumor cells. However, tumors with low level expression of MHC class I were found to confer a significantly poorer prognosis, retaining independent significance on multivariate analysis. The existence of these poor prognosis tumors, which may avoid both NK‐ and T‐cell‐mediated immune surveillance, has important implications for the design of immunotherapeutic strategies in colorectal cancer.

The chemokine, CXCL12, is an independent predictor of poor survival in ovarian cancer.

Background:The chemokine CXCL12 and its cognate receptor, CXCR4, have been implicated in numerous tumour types where expression promotes tumour growth, angiogenesis, metastasis and suppresses tumour immunity.Methods:Using a tissue microarray of 289 primary ovarian cancers coupled to a comprehensive database of clinicopathological variables, the expression of CXCL12 and CXCR4 was assessed by immunohistochemistry and its impact in terms of survival and clinicopathological variables was determined.Results:Patients whose tumours expressed high levels of CXCL12 had significantly poorer survival (P=0.026) than patients whose tumours failed to produce this chemokine. Lack of CXCL12 expression within tumours was associated with a 51-month survival advantage for patients when compared with patients whose tumours expressed high levels of CXCL12. FIGO stage, adjuvant chemotherapy and the absence of macroscopic disease after surgery were all shown to predict prognosis independently of each other in this cohort of patients. CXCL12 was independently predictive of prognosis on multivariate analysis (P=0.016). There was no correlation between CXCL12 and any clinicopathological variable.Conclusion:The chemokine CXCL12 is an independent predictor of poor survival in ovarian cancer. High expression of CXCL12 was seen in only 20% of the tumours, suggesting a role for anti-CXCL12/CXCR4 therapy in the management of these patients.

Costimulation via CD55 on Human CD4+ T Cells Mediated by CD97

Decay-accelerating factor (CD55) is a complement regulatory protein, which is expressed by most cells to protect them from complement-mediated attack. CD55 also binds CD97, an EGF-TM7 receptor constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells upon activation. Early results suggested that CD55 could further enhance T cell proliferation induced by phorbol ester treatment. The present study demonstrates that coengagement of CD55, using either cross-linking mAbs or its natural ligand CD97, and CD3 results in enhanced proliferation of human peripheral blood CD4+ T cells, expression of the activation markers CD69 and CD25, and secretion of IL-10 and GM-CSF. Recently, an increase in T cell responsiveness in CD55−/− mice was shown to be mediated by a lack of complement regulation. In this study, we show that direct stimulation of CD55 on CD4+ T cells with CD97 can modulate T cell activation but does not interfere with CD55-mediated complement regulation. Our results support a multifaceted role for CD55 in human T cell activation, constituting a further link between innate and adaptive immunity.

Complement decay accelerating factor (DAF)/CD55 in cancer

The complement system is a powerful innate mechanism involved in protection of the host against pathogens. It also has a role in the clearance of apoptotic cells and has been implicated in a range of pathologies including autoimmunity and graft rejection. The control of complement is mediated through the complement regulatory proteins (CRPs). These are present on most cells and protect normal cells from complement-mediated attack during innate activation. However, in a range of pathologies and cancer, these molecules are up or down regulated, sometimes secreted and even lost. We will review the expression of CRPs in cancer, focussing on CD55 and highlight other roles of the CRPs and their involvement in leukocyte function. We will also provide some data providing a potential mechanism by which soluble CD55 can inhibit T-cell function and discuss some of the implications of this data.

Enhancement of T cell recruitment and infiltration into tumours

Studies have documented that cancer patients with tumours which are highly infiltrated with cytotoxic T lymphocytes show enhanced survival rates. The ultimate goal of cancer immunotherapy is to elicit high‐avidity tumour‐specific T cells to migrate and kill malignant tumours. Novel antibody therapies such as ipilumimab (a cytotoxic T lymphocyte antigen‐4 blocking antibody) show enhanced T cell infiltration into the tumour tissue and increased survival. More conventional therapies such as chemotherapy or anti‐angiogenic therapy and recent therapies with oncolytic viruses have been shown to alter the tumour microenvironment and thereby lead to enhanced T cell infiltration. Understanding the mechanisms involved in the migration of high‐avidity tumour‐specific T cells into tumours will support and provide solutions for the optimization of therapeutic options in cancer immunotherapy.

DNA vaccination with T-cell epitopes encoded within Ab molecules induces high-avidity anti-tumor CD8+ T cells.

Stimulation of high‐avidity CTL responses is essential for effective anti‐tumor and anti‐viral vaccines. In this study we have demonstrated that a DNA vaccine incorporating CTL epitopes within an Ab molecule results in high‐avidity T‐cell responses to both foreign and self epitopes. The avidity and frequency was superior to peptide, peptide‐pulsed DC vaccines or a DNA vaccine incorporating the epitope within the native Ag. The DNA Ab vaccine was superior to an identical protein vaccine that can only cross‐present, indicating a role for direct presentation by the DNA vaccine. However, the avidity of CTL responses was significantly reduced in Fc receptor γ knockout mice or if the Fc region was removed suggesting that cross presentation of Ag via Fc receptor was also important in the induction of high‐avidity CTL. These results suggest that generation of high‐avidity CTL responses by the DNA vaccine is related to its ability to both directly present and cross‐present the epitope. High‐avidity responses were capable of efficient anti‐tumor activity in vitro and in vivo. This study demonstrates a vaccine strategy to generate high‐avidity CTL responses that can be used in anti‐tumor and anti‐viral vaccine settings.

Development of cancer vaccines to activate cytotoxic T lymphocytes.

Cancer vaccines have been shown to stimulate cytotoxic T lymphocyte (CTL) responses in a variety of cancer patients. However, the response is often of low frequency and moderate avidity, and does not result in objective clinical responses. This is related to the target antigens, which are usually over-expressed self-antigens that elicit tolerogenic and regulatory immune responses, resulting in deletion or inactivation of high-avidity T cells. Although moderate-avidity T cells can be efficient killers, tumours are often poor targets as they express a variety of molecules to protect them from cell-mediated immunity. Adoptive transfer of large numbers of high-avidity Tcells has been shown to induce regression of bulky disease, proving that immune responses can effectively eradicate tumours. New approaches that target activated de-ndritic cells invivo, resulting in cross-presentation of CTL epitopes and release of cytokines that suppress regulatory T cells, have resulted in the pr-oduction of T cells with sufficient avidity to kill tumour target cells. These approaches in combination with regimes, such as cytokine therapy, chemotherapy or radiotherapy, that modulate effector costimulatory expression on tumour targets may result in more effective second-generation cancer vaccines.

Identification of an HLA‐A*0201 cytotoxic T lymphocyte epitope specific to the endothelial antigen Tie‐2

Tie‐2 stabilises pericyte–endothelial interactions during angiogenesis and is highly expressed on endothelium during several diseases, including arthritis, age‐related macular degeneration and cancer. A vaccine that targets endothelium overexpressing Tie‐2 may result in vessel damage and stimulate an inflammatory cascade resulting in disease regression. We have identified a region unique to Tie‐2 (amino acids 1–196) that is homologous in humans and mice. Using computer algorithms, several HLA‐A*0201 epitopes that are identical in mice and humans were predicted within this region; however, binding assays showed that the majority of these epitopes were of low affinity. Modification of the anchor residues of 4 epitopes enhanced HLA binding. These epitopes were incorporated by site‐directed mutagenesis into a Tie‐2 DNA construct. Immunisation of HLA*0201 transgenic mice with one of the modified Tie‐2 constructs stimulated CTLs that recognised both wild‐type and modified peptide‐pulsed target cells. In contrast, no CTLs were generated in mice immunised with wild‐type Tie‐2 construct, demonstrating that the modified epitope was necessary in the generation of CTLs. Moreover, CTLs from mice immunised with the modified construct killed HLA‐A*0201 endothelial cells overexpressing Tie‐2. Our study demonstrates that it is possible to break tolerance to the endothelial antigen Tie‐2, suggesting that it may be feasible to design a vaccine to activate CTLs to kill endothelial cells overexpressing Tie‐2.

CD55 Costimulation Induces Differentiation of a Discrete T Regulatory Type 1 Cell Population with a Stable Phenotype

Unlike other helper T cells, the costimulatory ligands responsible for T regulatory type 1 (Tr1) cell differentiation remain undefined. Understanding the molecular interactions driving peripheral Tr1 differentiation is important because Tr1s potently regulate immune responses by IL-10 production. In this study, we show that costimulation of human naive CD4+ cells through CD97/CD55 interaction drives Tr1 activation, expansion, and function. T cell activation and expansion was equipotent with CD55 or CD28 costimulation; however, CD55 costimulation resulted in two IL-10–secreting populations. Most IL-10 was secreted by the minor Tr1 population (IL-10highIFN-γ−IL-4-, <5% cells) that expresses Tr1 markers CD49b, LAG-3, and CD226. This Tr1 phenotype was not restimulated by CD28. However, on CD55 restimulation, Tr1s proliferated and maintained their differentiated IL-10high phenotype. The Tr1s significantly suppressed effector T cell function in an IL-10–dependent manner. The remaining (>95%) cells adopted a Th1-like IFN-γ+ phenotype. However, in contrast to CD28-derived Th1s, CD55-derived Th1s demonstrated increased plasticity with the ability to coexpress IL-10 when restimulated through CD55 or CD28. These data identify CD55 as a novel costimulator of human Tr1s and support a role for alternative costimulatory pathways in determining the fate of the growing number of T helper populations. This study demonstrates that CD55 acts as a potent costimulator and activator of human naive CD4+ cells, resulting in the differentiation of a discrete Tr1 population that inhibits T cell function in an IL-10–dependent manner and maintains the Tr1 phenotype upon restimulation.

SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade.

ABSTRACT Checkpoint blockade has demonstrated promising antitumor responses in approximately 10–40% of patients. However, the majority of patients do not make a productive immune response to their tumors and do not respond to checkpoint blockade. These patients may benefit from an effective vaccine that stimulates high-avidity T cell responses in combination with checkpoint blockade. We have previously shown that incorporating TRP-2 and gp100 epitopes into the CDR regions of a human IgG1 DNA (ImmunoBody®: IB) results in significant tumor regression both in animal models and patients. This vaccination strategy is superior to others as it targets antigen to antigen-presenting cells and stimulates high-avidity T cell responses. To broaden the application of this vaccination strategy, 16 NY-ESO-1 epitopes, covering over 80% of HLA phenotypes, were incorporated into the IB (SCIB2). They produced higher frequency and avidity T cell responses than peptide vaccination. These T cells were of sufficient avidity to kill NY-ESO-1-expressing tumor cells, and in vivo controlled the growth of established B16-NY-ESO-1 tumors, resulting in long-term survival (35%). When SCIB2 was given in combination with Treg depletion, CTLA-4 blockade or PD-1 blockade, long-term survival from established tumors was significantly enhanced to 56, 67 and 100%, respectively. Translating these responses into the clinic by using a combination of SCIB2 vaccination and checkpoint blockade can only further improve clinical responses.