T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

OX40 agonist stimulation increases and sustains humoral and cell-mediated responses to SARS-CoV-2 protein and saRNA vaccines.

To prevent SARS-CoV-2 infections and generate long-lasting immunity, vaccines need to generate strong viral-specific B and T cell responses. Previous results from our lab and others have shown that immunizations in the presence of an OX40 agonist antibody lead to higher antibody titers and increased numbers of long-lived antigen-specific CD4 and CD8 T cells. Using a similar strategy, we explored the effect of OX40 co-stimulation in a prime and boost vaccination scheme using an adjuvanted SARS-CoV-2 spike protein vaccine in C57BL/6 mice. Our results show that OX40 engagement during vaccination significantly increases long-lived antibody responses to the spike protein. In addition, after immunization spike protein-specific proliferation was greatly increased for both CD4 and CD8 T cells, with enhanced, spike-specific secretion of IFN-γ and IL-2. Booster (3rd injection) immunizations combined with an OX40 agonist (7 months post-prime) further increased vaccine-specific antibody and T cell responses. Initial experiments assessing a self-amplifying mRNA (saRNA) vaccine encoding the spike protein antigen show a robust antigen-specific CD8 T cell response. The saRNA spike-specific CD8 T cells express high levels of GrzmB, IFN-γ and TNF-α which was not observed with protein immunization and this response was further increased by the OX40 agonist. Similar to protein immunizations the OX40 agonist also increased vaccine-specific CD4 T cell responses. In summary, this study compares and contrasts the effects and benefits of both protein and saRNA vaccination and the extent to which an OX40 agonist enhances and sustains the immune response against the SARS-CoV-2 spike protein.

PD-1 and ICOS coexpression identifies tumor-reactive CD4+ T cells in human solid tumors.

CD4+ Th cells play a key role in orchestrating immune responses, but the identity of the CD4+ Th cells involved in the antitumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4+ Th cells distinct from FOXP3+ Tregs that coexpressed programmed cell death 1 (PD-1) and ICOS. These tumor-infiltrating lymphocyte CD4+ Th cells (CD4+ Th TILs) had a tissue-resident memory phenotype, were present in MHC class II-rich areas, and proliferated in the tumor, suggesting local antigen recognition. The T cell receptor repertoire of the PD-1+ICOS+ CD4+ Th TILs was oligoclonal, with T cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4+ Th TILs were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4+ Th TILs directly ex vivo that will help define their role in the antitumor immune response and potentially improve future adoptive T cell therapy approaches.

Neoadjuvant anti-OX40 (MEDI6469) therapy in patients with head and neck squamous cell carcinoma activates and expands antigen-specific tumor-infiltrating T cells.

Despite the success of checkpoint blockade in some cancer patients, there is an unmet need to improve outcomes. Targeting alternative pathways, such as costimulatory molecules (e.g. OX40, GITR, and 4-1BB), can enhance T cell immunity in tumor-bearing hosts. Here we describe the results from a phase Ib clinical trial (NCT02274155) in which 17 patients with locally advanced head and neck squamous cell carcinoma (HNSCC) received a murine anti-human OX40 agonist antibody (MEDI6469) prior to definitive surgical resection. The primary endpoint was to determine safety and feasibility of the anti-OX40 neoadjuvant treatment. The secondary objective was to assess the effect of anti-OX40 on lymphocyte subsets in the tumor and blood. Neoadjuvant anti-OX40 was well tolerated and did not delay surgery, thus meeting the primary endpoint. Peripheral blood phenotyping data show increases in CD4+ and CD8+ T cell proliferation two weeks after anti-OX40 administration. Comparison of tumor biopsies before and after treatment reveals an increase of activated, conventional CD4+ tumor-infiltrating lymphocytes (TIL) in most patients and higher clonality by TCRß sequencing. Analyses of CD8+ TIL show increases in tumor-antigen reactive, proliferating CD103+ CD39+ cells in 25% of patients with evaluable tumor tissue (N = 4/16), all of whom remain disease-free. These data provide evidence that anti-OX40 prior to surgery is safe and can increase activation and proliferation of CD4+ and CD8+ T cells in blood and tumor. Our work suggests that increases in the tumor-reactive CD103+ CD39+ CD8+ TIL could serve as a potential biomarker of anti-OX40 clinical activity.

OX40 Agonist Tumor Immunotherapy Does Not Impact Regulatory T Cell Suppressive Function.

OX40 is a costimulatory molecule from the TNFR family. In mice, it is expressed on Foxp3+ regulatory T cells (Tregs) constitutively and on conventional CD4 (Tconv) and CD8 T cells after Ag encounter. OX40 agonists are in clinical development to enhance antitumor immune responses, and one proposed mechanism of action is loss of Treg suppressive function. Studies have postulated that agonist OX40 therapy can impair Treg suppressive function. Using tools developed since the initial studies were published, we evaluated a direct effect of OX40 agonism on Treg function. We conclude that OX40 agonist Abs do not intrinsically impair Treg function but rather enhance Tconv cell IL-2 production, increasing Treg and Tconv cell proliferation. OX40-stimulated Tregs retain suppressive function, but also gain IFN-γ, TNF-α, and granzyme B expression. These data help resolve mechanistic questions regarding OX40 agonist immunotherapy and thus are relevant to developing combination therapies that target distinct T cell functions.

Multi-antigen Vaccination With Simultaneous Engagement of the OX40 Receptor Delays Malignant Mesothelioma Growth and Increases Survival in Animal Models.

Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues. This vaccine, that we term p-Tvax, is comprised of a series of immunogenic peptides presented by both MHC-I and -II to generate robust immune responses. The peptides were designed using in silico algorithms that discriminate between highly immunogenic T cell epitopes and other harmful epitopes, such as suppressive regulatory T cell epitopes and autoimmune epitopes. Vaccination of mice with p-Tvax led to antigen-specific immune responses that involved both CD8+ and CD4+ T cells, which exhibited cytolytic activity against MM cells in vitro. In mice carrying MM tumors, p-Tvax increased tumor infiltration of CD4+ T cells. Moreover, combining p-Tvax with an OX40 agonist led to decreased tumor growth and increased survival. Mice treated with this combination immunotherapy displayed higher numbers of tumor-infiltrating CD8+ and CD4+ T cells and reduced T regulatory cells in tumors. Collectively, these data suggest that the combination of p-Tvax with an OX40 agonist could be an effective strategy for MM treatment.

Neoantigen-specific immunity in low mutation burden colorectal cancers of the consensus molecular subtype 4.

BACKGROUND: The efficacy of checkpoint blockade immunotherapies in colorectal cancer is currently restricted to a minority of patients diagnosed with mismatch repair-deficient tumors having high mutation burden. However, this observation does not exclude the existence of neoantigen-specific T cells in colorectal cancers with low mutation burden and the exploitation of their anti-cancer potential for immunotherapy. Therefore, we investigated whether autologous neoantigen-specific T cell responses could also be observed in patients diagnosed with mismatch repair-proficient colorectal cancers. METHODS: Whole-exome and transcriptome sequencing were performed on cancer and normal tissues from seven colorectal cancer patients diagnosed with mismatch repair-proficient tumors to detect putative neoantigens. Corresponding neo-epitopes were synthesized and tested for recognition by in vitro expanded T cells that were isolated from tumor tissues (tumor-infiltrating lymphocytes) and from peripheral mononuclear blood cells stimulated with tumor material. RESULTS: Neoantigen-specific T cell reactivity was detected to several neo-epitopes in the tumor-infiltrating lymphocytes of three patients while their respective cancers expressed 15, 21, and 30 non-synonymous variants. Cell sorting of tumor-infiltrating lymphocytes based on the co-expression of CD39 and CD103 pinpointed the presence of neoantigen-specific T cells in the CD39+CD103+ T cell subset. Strikingly, the tumors containing neoantigen-reactive TIL were classified as consensus molecular subtype 4 (CMS4), which is associated with TGF-ß pathway activation and worse clinical outcome. CONCLUSIONS: We have detected neoantigen-targeted reactivity by autologous T cells in mismatch repair-proficient colorectal cancers of the CMS4 subtype. These findings warrant the development of specific immunotherapeutic strategies that selectively boost the activity of neoantigen-specific T cells and target the TGF-ß pathway to reinforce T cell reactivity in this patient group.

Late-Stage Tumor Regression after PD-L1 Blockade Plus a Concurrent OX40 Agonist.

The protective capability of tumor antigen-specific T cells is regulated by costimulatory and inhibitory signals. Current approaches in cancer immunotherapy seek to restore the function of unresponsive T cells by blocking inhibitory pathways. In contrast, providing exogenous costimulatory signals to T cells also enhances antitumor functionality. By combining these two clinical approaches, we demonstrate the synergy of targeting PD-L1 together with the costimulatory molecule OX40, to enhance antitumor immunity. Concurrently blocking PD-L1 and providing a costimulatory agonist to OX40 increased the presence and functionality of tumor antigen-specific CD8+ T cells with simultaneous enhancement of T-helper type 1 (Th1)-skewed CD4+ T cells. This shift was functionally supported by increased glucose metabolism of antigen-specific CD8+ T cells and the acquisition of granzyme B by regulatory T cells. Together, this mechanism promoted tumor regression of late-stage tumors beyond that achieved by either blockade as monotherapy. These findings indicate that targeting both T-cell intrinsic (OX40) and extrinsic (PD-L1) regulatory molecules increases the bioenergetic potential of T cells, thereby expanding functional and tumor antigen-specific T cells.

Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors.

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies.

Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression.

It is well-known that vaccines comprising of irradiated whole tumor cells or tumor-derived heat shock proteins can generate tumor-specific immune responses. In contrast, we showed recently that vaccines composed of autophagosomes (DRibbles) derived from syngeneic sarcomas could induce cross-reactive T-cell responses and cross-protection against the tumor. This unusual property of DRibbles was related to the selective recruitment of defective ribosomal products (DRiPs) and other short-lived proteins (SLiPs) into autophagosomes via sequestosome (SQSTM1, p62) mediated association of ubiquitinated SLiPs to the autophagy gene product LC3. Here, we extend our observations to mammary carcinomas from mice of different genetic background. We demonstrated that combined of intranodal administration of autologous or allogeneic DRibbles together with anti-OX40 antibody led to robust proliferation, expansion, and differentiation of memory and effector T cells. We also showed that SLiPs is an excellent source of antigen for cross-priming of CD8+ T-cells that recognize shared tumor antigens in the context of host MHC class I molecules. Thus, our results provide a strong basis for novel clinical trials that combine allogeneic "off-the-shelf" DRibble vaccines together with antibodies against co-stimulatory molecules.

Cytoreductive surgery for head and neck squamous cell carcinoma in the new age of immunotherapy.

Cytoreductive surgery is an approach to cancer treatment that aims to reduce the number of cancer cells via resection of primary tumor or metastatic deposits, in an effort to minimize a potentially immunosuppressive tumor burden, palliate symptoms, and prevent complications. Furthermore, it provides a platform for investigation of biomarkers with the goal of optimizing immunotherapy to reverse the immunosuppressive tumor microenvironment and enhance adaptive immune responses. Ultimately, our group aims to exploit the concept that successful cancer therapy is dependent upon an effective immune response. Surgery will remain an integral part of head and neck squamous cell carcinoma (HNSCC) treatment in the future, even as checkpoint inhibitors, co-stimulatory molecules, vaccines, adoptive T cell therapy and other novel agents enter clinical routine. Cytoreductive resection may provide an effective platform for immunotherapy and biomarker directed interventions to improve outcomes for patients with HNSCC.

Immunotherapy Expands and Maintains the Function of High-Affinity Tumor-Infiltrating CD8 T Cells In Situ.

Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.

OX40 signaling in head and neck squamous cell carcinoma: Overcoming immunosuppression in the tumor microenvironment.

OX40 is a member of the tumor necrosis factor (TNF) receptor family and a potent co-stimulatory pathway that when triggered can enhance T-cell memory, proliferation and anti-tumor activity in patients with metastatic cancer. Ongoing investigations at our institution have demonstrated that OX40 expressing T cells are found in abundance in the tumors of patients with advanced stage head and neck squamous cell carcinoma (HNSCC). This has led to the initiation of human clinical trials investigating OX40-directed therapy for patients with HNSCC in both the metastatic and curative setting. The purpose of this review is to explore what is known about OX40 signaling and discuss how this pathway potentially can be modulated to improve outcome for patients with HNSCC.

STAT3 Signaling Is Required for Optimal Regression of Large Established Tumors in Mice Treated with Anti-OX40 and TGFß Receptor Blockade.

In preclinical tumor models, αOX40 therapy is often successful at treating small tumors, but is less effective once the tumors become large. For a tumor immunotherapy to be successful to cure large tumors, it will most likely require not only an agonist to boost effector T-cell function but also inhibitors of T-cell suppression. In this study, we show that combining αOX40 antibodies with an inhibitor of the TGFß receptor (SM16) synergizes to elicit complete regression of large established MCA205 and CT26 tumors. Evaluation of tumor-infiltrating T cells showed that SM16/αOX40 dual therapy resulted in an increase in proliferating granzyme B(+) CD8 T cells, which produced higher levels of IFNγ, compared with treatment with either agent alone. We also found that the dual treatment increased pSTAT3 expression in both CD4 and CD8 T cells isolated from tumors. Because others have published that STAT3 signaling is detrimental to T-cell function within the tumor microenvironment, we explored whether deletion of STAT3 in OX40-expressing cells would affect this potent combination therapy. Surprisingly, we found that deletion of STAT3 in OX40-expressing cells decreased the efficacy of this combination therapy, showing that the full therapeutic potential of this treatment depends on STAT3 signaling, most likely in the T cells of tumor-bearing mice.

Detailed characterization of tumor infiltrating lymphocytes in two distinct human solid malignancies show phenotypic similarities.

BACKGROUND: We examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC). METHODS: Tumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4(+) T cells were also analyzed by qPCR. RESULTS: Overall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4(+)/CD25(+)/FoxP3(+)) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8(+) T cells (HLA-DR(+)/CD38(+)) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)). CONCLUSION: This study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR(+), CD39(+), CTLA-4(+) and Helios(+)) and activated cytotoxic T cells (CD8(+)/HLA-DR(+)/CD38(+)) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.

Caloric restriction maintains OX40 agonist-mediated tumor immunity and CD4 T cell priming during aging.

Immune responses wane during aging, posing challenges to the potential effectiveness of cancer immunotherapies. We previously demonstrated that in the context of a promising immunotherapeutic, OX40 agonist (αOX40), older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. In this study, we hypothesized that tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a calorically restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed after challenging with either sarcoma or breast tumor cells followed by αOX40 treatment. Our results show that CR, but not RES, maintained OX40-mediated anti-tumor immunity. In addition, CR fully sustained antigen-specific CD4 T cell priming in aged hosts (12 months old), whereas tumor-specific CD8 T cell priming was not fully maintained compared to young reference animals (2 months old). Thus, CR appears to maintain immunological fitness of the CD4 T cell priming environment during aging, which is critical for optimal OX40-mediated responses.

Early-onset age-related changes in dendritic cell subsets can impair antigen-specific T helper 1 (Th1) CD4 T cell priming.

Decline in CD4 T cell immune responses is associated with aging. Although a number of immunological defects have been identified in elderly mice (>18 months old), a key early-onset immune defect at middle age could be a driver or contributor to defective CD4 T cell responses. Our studies demonstrate that age-related alterations in DC subsets within the priming environment of middle-aged mice (12 months old) correlate with and can directly contribute to decreases in antigen-specific CD4 T cell Th1 differentiation, which measured by T-bet and IFN-γ expression, was decreased significantly in T cells following VSV infection or s.c. immunization with a protein antigen in the context of immune stimulation via OX40. The deficient Th1 phenotype, observed following protein antigen challenge, was found to be the result of an age-related decrease in an inflammatory DC subset (CD11b+ Gr-1/Ly6C+) in the dLN that corresponded with T cell dysfunction. In the virus model, we observed significant changes in two DC subsets: mDCs and pDCs. Thus, different, early age-related changes in the DC profile in the priming environment can significantly contribute to impaired Th1 differentiation, depending on the type of immunological challenge.

OX40 is a potent immune-stimulating target in late-stage cancer patients.

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy.

T cell-mediated rejection of tumors requires signals from the T cell receptor and co-stimulatory molecules to license effector functions of tumor-antigen specific T cells. There is also an array of immune suppressive mechanisms within the tumor microenvironment that can suppress anti-tumor immunity. The use of monoclonal antibodies to overcome this suppression and/or enhance tumor-antigen specific T cell responses has shown promise in clinical trials. In particular, targeting co-stimulatory members of the tumor necrosis factor receptor (TNFR) family with agonist Abs enhances T cell function, which has led to encouraging therapeutic results in cancer-bearing hosts. These encouraging data establish TNFRs as important targets for enhancing tumor-specific immune responses in mice and man. This review will focus on agonists that target the TNFRs OX40, 4-1BB, and CD40.