InVivoMAb anti-mouse CTLA-4 (CD152)

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8(+) and CD4(+) T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy.

Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-gamma, which is a critical cytokine for host immune responses. However, the role of IFN-gamma signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here, we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-gamma pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-gamma receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-gamma signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-gamma related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.

Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 15-19 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV(+) TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and alpha4-1BB promoted the highest CD8(+) versus regulatory FoxP3(+) T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies.

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Melanoma treatment is being revolutionized by the development of effective immunotherapeutic approaches. These strategies include blockade of immune-inhibitory receptors on activated T cells; for example, using monoclonal antibodies against CTLA-4, PD-1, and PD-L1 (refs 3-5). However, only a subset of patients responds to these treatments, and data suggest that therapeutic benefit is preferentially achieved in patients with a pre-existing T-cell response against their tumour, as evidenced by a baseline CD8(+) T-cell infiltration within the tumour microenvironment. Understanding the molecular mechanisms that underlie the presence or absence of a spontaneous anti-tumour T-cell response in subsets of cases, therefore, should enable the development of therapeutic solutions for patients lacking a T-cell infiltrate. Here we identify a melanoma-cell-intrinsic oncogenic pathway that contributes to a lack of T-cell infiltration in melanoma. Molecular analysis of human metastatic melanoma samples revealed a correlation between activation of the WNT/beta-catenin signalling pathway and absence of a T-cell gene expression signature. Using autochthonous mouse melanoma models we identified the mechanism by which tumour-intrinsic active beta-catenin signalling results in T-cell exclusion and resistance to anti-PD-L1/anti-CTLA-4 monoclonal antibody therapy. Specific oncogenic signals, therefore, can mediate cancer immune evasion and resistance to immunotherapies, pointing to new candidate targets for immune potentiation.

Although adoptive T-cell therapy holds promise for the treatment of many cancers, its clinical utility has been limited by problems in delivering targeted lymphocytes to tumor sites, and the cells’ inefficient expansion in the immunosuppressive tumor microenvironment. Here we describe a bioactive polymer implant capable of delivering, expanding and dispersing tumor-reactive T cells. The approach can be used to treat inoperable or incompletely removed tumors by situating implants near them or at resection sites. Using a mouse breast cancer resection model, we show that the implants effectively support tumor-targeting T cells throughout resection beds and associated lymph nodes, and reduce tumor relapse compared to conventional delivery modalities. In a multifocal ovarian cancer model, we demonstrate that polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have little curative effect. Scaffold-based T-cell delivery may provide a viable treatment option for inoperable tumors and reduce the rate of metastatic relapse after surgery.

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete alphaSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.

Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans-elicited Th17 cells contained a higher frequency of IL-17(+)IFN-gamma(+) producers and a lower frequency of IL-10(+) and IL-10(+)IL-17(+) cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4(+) T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

Recurrent solid malignancies are often refractory to standard therapies. Although adoptive T cell transfer may benefit select individuals, the majority of patients succumb to their disease. To address this important clinical dilemma, we developed a mouse melanoma model in which initial regression of advanced disease was followed by tumor recurrence. During recurrence, Foxp3(+) tumor-specific CD4(+) T cells became PD-1(+) and represented >60% of the tumor-specific CD4(+) T cells in the host. Concomitantly, tumor-specific CD4(+) T effector cells showed traits of chronic exhaustion, as evidenced by their high expression of the PD-1, TIM-3, 2B4, TIGIT, and LAG-3 inhibitory molecules. Although blockade of the PD-1/PD-L1 pathway with anti-PD-L1 Abs or depletion of tumor-specific regulatory T cells (Tregs) alone failed to reverse tumor recurrence, the combination of PD-L1 blockade with tumor-specific Treg depletion effectively mediated disease regression. Furthermore, blockade with a combination of anti-PD-L1 and anti-LAG-3 Abs overcame the requirement to deplete tumor-specific Tregs. In contrast, successful treatment of primary melanoma with adoptive cell therapy required only Treg depletion or Ab therapy, underscoring the differences in the characteristics of treatment between primary and relapsing cancer. These data highlight the need for preclinical development of combined immunotherapy approaches specifically targeting recurrent disease.

Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8(+) T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNgamma secretion in mice and humans. NK cell-derived IFNgamma subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8(+) T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.

Thermal ablation to destroy tumor tissue may help activate tumor-specific T cells by elevating the presentation of tumor antigens to the immune system. However, the antitumor activity of these T cells may be restrained by their expression of the inhibitory T-cell coreceptor CTLA-4, the target of the recently U.S. Food and Drug Administration-approved antibody drug ipilumimab. By relieving this restraint, CTLA-4-blocking antibodies such as ipilumimab can promote tumor rejection, but the full scope of their most suitable applications has yet to be fully determined. In this study, we offer a preclinical proof-of-concept in the TRAMP C2 mouse model of prostate cancer that CTLA-4 blockade cooperates with cryoablation of a primary tumor to prevent the outgrowth of secondary tumors seeded by challenge at a distant site. Although growth of secondary tumors was unaffected by cryoablation alone, the combination treatment was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4(+) T cells and CD8(+) T cells, and there was a significant increase in the ratio of intratumoral T effector cells to CD4(+)FoxP3(+) T regulatory cells, compared with monotherapy. These findings documented for the first time an effect of this immunotherapeutic intervention on the intratumoral accumulation and systemic expansion of CD8(+) T cells specific for the TRAMP C2-specific antigen SPAS-1. Although cryoablation is currently used to treat a targeted tumor nodule, our results suggest that combination therapy with CTLA-4 blockade will augment antitumor immunity and rejection of tumor metastases in this setting.

CTLA-4, an Ig superfamily molecule with homology to CD28, is one of the most potent negative regulators of T-cell responses. In vivo blockade of CTLA-4 exacerbates autoimmunity, enhances tumor-specific T-cell responses, and may inhibit the induction of T-cell anergy. Clinical trials of CTLA-4-blocking antibodies to augment T-cell responses to malignant melanoma are at an advanced stage; however, little is known about the effects of CTLA-4 blockade on memory CD8(+) T-cell responses and the formation and maintenance of long-term CD8(+) T-cell memory. In our studies, we show that during in vivo memory CD8(+) T-cell responses to Listeria monocytogenes infection, CTLA-4 blockade enhances bacterial clearance and increases memory CD8(+) T-cell expansion. This is followed by an accumulation of memory cells that are capable of producing the effector cytokines IFN-gamma and TNF-alpha. We also demonstrate that in a vaccination setting, blocking CTLA-4 during CD8(+) T-cell priming leads to increased expansion and maintenance of antigen-specific memory CD8(+) T cells without adversely affecting the overall T-cell repertoire. This leads to an increase in memory cell effector function and improved protective immunity against further bacterial challenges. These results indicate that transient blockade of CTLA-4 enhances memory CD8(+) T-cell responses and support the possible use of CTLA-4-blocking antibodies during vaccination to augment memory formation and maintenance.

Imatinib mesylate targets mutated KIT oncoproteins in gastrointestinal stromal tumor (GIST) and produces a clinical response in 80% of patients. The mechanism is believed to depend predominantly on the inhibition of KIT-driven signals for tumor-cell survival and proliferation. Using a mouse model of spontaneous GIST, we found that the immune system contributes substantially to the antitumor effects of imatinib. Imatinib therapy activated CD8(+) T cells and induced regulatory T cell (T(reg) cell) apoptosis within the tumor by reducing tumor-cell expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido). Concurrent immunotherapy augmented the efficacy of imatinib in mouse GIST. In freshly obtained human GIST specimens, the T cell profile correlated with imatinib sensitivity and IDO expression. Thus, T cells are crucial to the antitumor effects of imatinib in GIST, and concomitant immunotherapy may further improve outcomes in human cancers treated with targeted agents.

Adoptive transfer of large numbers of tumor-reactive CD8(+) cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8(+) CTLs, with much less emphasis on the role and contribution of CD4(+) T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4(+) T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4(+) T cells developed cytotoxic activity, and tumor rejection was dependent on class II-restricted recognition of tumors by tumor-reactive CD4(+) T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4(+) T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4(+) T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4(+) cells in vivo over current protocols favoring in vitro expansion and differentiation.