Cell Responses In Cancer Patients Research Articles

Immune responses to SARS-CoV-2 in vaccinated patients receiving checkpoint blockade immunotherapy for cancer.

Vaccination against SARS-CoV-2 has been successful in protecting patients with cancer from severe infections, but how immune responses against COVID-19 vaccination interact with those elicited during cancer immunotherapy has not been fully described. Immune checkpoint blockade (ICB) disrupts inhibitory pathways in immune cells to improve function and induce tumor immunity but can often cause serious immune related adverse events (IRAEs). Because COVID-19 vaccination and ICB both boost immune responses, it is imperative to understand if combining these regimens causes synergistic enhancement of the immune system. Specifically, whether ICB impacts anti-vaccine immunity in previously vaccinated patients is important since a large percentage of newly diagnosed cancer patients eligible for immunotherapy will have already been vaccinated against COVID-19. To address this, we investigated the influence of ICB on SARS-CoV-2-spike protein (SP) antibody titers and T cell responses in cancer patients previously vaccinated against COVID-19. Human blood samples were collected from 29 vaccinated patients and 12 unvaccinated control patients at baseline (prior to ICB) and following two rounds of ICB infusion. Anti-SARS-CoV-2-SP IgG titers and T cell responses were quantified. Compared to responses at baseline, there was no significant difference in these immune responses after immunotherapy in vaccinated individuals (P=0.4583, P=0.4571, respectively). We interpret these results as evidence that ICB immunotherapy does not significantly enhance SARS-CoV-2-specific antibody titers or T cell responses. Although our study lacks corresponding IRAE rates, the results provide humoral and cellular immunological data that support recent reports documenting the clinical safety and efficacy of COVID-19 vaccination in patients receiving ICB. Additional longitudinal prospective studies, such as the VOICE study (ClinicalTrials.gov identifier NCT04715438) and CAPTURE study (ClinicalTrials.gov identifier NCT03226886), are warranted and will provide broader safety and immunological data defining the effect of systemic cancer therapies on COVID-19 immunity.

Brief Research Report: Anti-SARS-CoV-2 Immunity in Long Lasting Responders to Cancer Immunotherapy Through mRNA-Based COVID-19 Vaccination.

Cancer patients (CPs) have been identified as particularly vulnerable to SARS-CoV-2 infection, and therefore are a priority group for receiving COVID-19 vaccination. From the patients with advanced solid tumors, about 20% respond very efficiently to immunotherapy with anti-PD1/PD-L1 antibodies and achieve long lasting cancer responses. It is unclear whether an efficient cancer-specific immune response may also correlate with an efficient response upon COVID-19 vaccination. Here, we explored the antiviral immune response to the mRNA-based COVID-19 vaccine BNT162b2 in a group of 11 long-lasting cancer immunotherapy responders. We analysed the development of SARS-CoV-2-specific IgG serum antibodies, virus neutralizing capacities and T cell responses. Control groups included patients treated with adjuvant cancer immunotherapy (IMT, cohort B), CPs not treated with immunotherapy (no-IMT, cohort C) and healthy controls (cohort A). The median ELISA IgG titers significantly increased after the prime-boost COVID vaccine regimen in all cohorts (Cohort A: pre-vaccine = 900 (100-2700), 3 weeks (w) post-boost = 24300 (2700-72900); Cohort B: pre-vaccine = 300 (100-2700), 3 w post-boost = 8100 (300-72900); Cohort C: pre-vaccine = 500 (100-2700), 3 w post-boost = 24300 (300-72900)). However, at the 3 w post-prime time-point, only the healthy control group showed a statistically significant increase in antibody levels (Cohort A = 8100 (900-8100); Cohort B = 900 (300-8100); Cohort C = 900 (300-8100)) (P < 0.05). Strikingly, while all healthy controls generated high-level antibody responses after the complete prime-boost regimen (Cohort A = 15/15 (100%), not all CPs behaved alike [Cohort B= 12/14 (84'6%); Cohort C= 5/6 (83%)]. Their responses, including those of the long-lasting immunotherapy responders, were more variable (Cohort A: 3 w post-boost (median nAb titers = 95.32 (84.09-96.93), median Spike-specific IFN-γ response = 64 (24-150); Cohort B: 3 w post-boost (median nAb titers = 85.62 (8.22-97.19), median Spike-specific IFN-γ response (28 (1-372); Cohort C: 3 w post-boost (median nAb titers = 95.87 (11.8-97.3), median Spike-specific IFN-γ response = 67 (20-84)). Two long-lasting cancer responders did not respond properly to the prime-boost vaccination and did not generate S-specific IgGs, neutralizing antibodies or virus-specific T cells, although their cancer immune control persisted for years. Thus, although mRNA-based vaccines can induce both antibody and T cell responses in CPs, the immune response to COVID vaccination is independent of the capacity to develop an efficient anti-cancer immune response to anti PD-1/PD-L1 antibodies.

Abstract 4159: Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses

Abstract The immunogenicity and efficacy of RNA-based vaccine platforms has been abundantly shown through their application in prophylactic SARS-CoV2 vaccines. Contrasting to mRNA based vectors, self amplifying mRNA platforms may offer dose-sparing and superior induction of T cell responses, and may also trigger distinct innate immune pathways, which may exert adjuvanting or inhibiting effects on vaccine-induced immunity. Optimal dosing for a novel self-amplifying mRNA (SAM) in a heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and SAM boosts was evaluated in two first-in-human phase 1/2 clinical trials assessing personalized neoantigen vaccines in patients with metastatic cancer (NCT03639714, NCT03953235). SAM vaccine dose escalation was performed to assess safety, tolerability, and immunogenicity, including administration of up to 8 SAM doses at 30, 100, or 300µg following a fixed dose of ChAd (1012 vp) over the course of a year. SAM was safe and well tolerated at all 3 dose levels, with no evidence of increasing reactogenicity with sequential doses. However, while immune monitoring via IFNγ ELISpot revealed that the 30µg SAM dose boosted T cell responses induced by the ChAd prime, the 100µg and 300µg SAM doses resulted in maintenance of T cell levels, without a clear T cell boost, suggesting a non-linear and likely bell-shaped dose-response curve to SAM in humans. Follow-up studies in non-human primates (NHPs) using a model antigen revealed dose-dependent increases in serum IFNa levels following administration of increasing SAM doses. Similarly, while multiple inflammatory cytokines were transiently increased following both ChAd and SAM administration in patients, serum IFNa levels were only increased 24h post SAM administration and correlated positively with SAM dose. Increased IFNa levels post SAM dosing suggested activation of mRNA-sensing innate immune pathways that may reduce the amplification of, and/or antigen expression by, the SAM vector and thus blunt T cell boosting at higher SAM doses. In addition, analysis of T cell responses in patients and NHPs showed increased boosting of T cell responses with longer intervals. These data lead to a reduction of the SAM dose to 30µg and adjusting SAM dosing intervals to 8 weeks in the Phase 2 portion of these clinical studies. Multiple patients have been dosed with the adjusted vaccine regimen, and preliminary data suggest robust boosting of ChAd-induced neoantigen-specific T cell responses with the selected SAM dosing regimen and the 30µg dose. We anticipate that this translational approach of adjusting clinical vaccine regimens based on strong translational immune data will increase the potency of our heterologous neoantigen vaccine, and subsequently provide more durable clinical benefit to patients with cancer. Citation Format: Christine D. Palmer, Amy R. Rappaport, Meghan G. Hart, Lauren D. Kraemer, Sonia Kounlavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Annie Shen, Gregory R. Boucher, Melissa A. Kachura, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Alexander I. Spira, Chrisann Kyi, Daniel V. Catenacci, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Lower doses of self-amplifying mRNA drive superior neoantigen-specific CD8 T cell responses in cancer patients versus high doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4159.

Abstract 1376: Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions

Abstract T cells have the potential to carry out potent anti-tumor responses. Most studies examining T cell-cancer interactions have been directed toward late-stage tumors, but much less is known about how T cells respond to premalignant lesions. Using a clinically relevant liver cancer mouse model (Albumin-Stop-Large T Antigen; AST) in which the SV40 Large T Antigen (TAG) serves as oncogene and tumor neoantigen, we previously demonstrated that TAG-specific CD8 T cells (TCR-TAG) rapidly become dysfunctional (unable to produce cytokines and cytolytic molecules) after entering premalignant liver lesions, although they persist long-term. We hypothesized that peripheral tolerance mechanisms, which prevent autoimmunity against self-antigens, drive T cell dysfunction in premalignant lesions. To test this, we compared CD8 T cell (TCR-GAG) responses to a liver self-antigen (GAG) to TCR-TAG responses to the TAG neoantigen in premalignant hepatocytes. We found that liver-reactive TCR-GAG were unable to produce cytokines, similar to neoantigen-specific TCR-TAG; however, in contrast, they failed to persist. To test whether oncogenic transformation of hepatocytes promoted T cell persistence, we crossed Alb-GAG and AST mice to generate a model in which GAG and TAG was expressed in premalignant hepatocytes (ASTxAlb-GAG). We found that both TCR-GAG and TCR-TAG transferred into ASTxAlb-GAG mice bearing premalignant lesions driven by the TAG oncogene rapidly lost the ability to produce cytokines; however, only TCR-TAG persisted while TCR-GAG quickly disappeared. To understand why the TCR-GAG failed to persist, we assessed proliferation and cell cycling after T cells encountered their cognate antigen. While both TCR-GAG and TCR-TAG were rapidly activated and began proliferating, TCR-GAG exited cell cycle and began decreasing in number after four cell divisions, while TCR-TAG continued to divide and increase in number. Antigen amount and localization may regulate T cell proliferation and persistence. We found much higher levels of TAG-encoding versus GAG-encoding mRNA in the liver, suggesting that higher antigen expression may be required for T cell persistence. T cell responses can be directed against both shared tumor self-antigens and neoantigens. By dissecting the determinants of CD8 T cell function and persistence in response to self and non-self-antigens, these studies could inform strategies to boost anti-tumor CD8 T cell responses in cancer patients. Citation Format: Jessica J. Roetman, Megan M. Erwin, Minna K. Apostolova, Mary Philip. Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1376.

Role of the ITAM-Bearing Receptors Expressed by Natural Killer Cells in Cancer.

Natural Killer (NK) cells are innate lymphoid cells (ILCs) capable of recognizing and directly killing tumor cells. They also secrete cytokines and chemokines, which participate in the shaping of the adaptive response. NK cells identify tumor cells and are activated through a net positive signal from inhibitory and activating receptors. Several activating NK cell receptors are coupled to adaptor molecules containing an immunoreceptor tyrosine-based activation motif (ITAM). These receptors include CD16 and the natural cytotoxic receptors NKp46, NKp44, NKp30 in humans. The powerful antitumor NK cell response triggered by these activating receptors has made them attractive targets for exploitation in immunotherapy. In this review, we will discuss the different activating receptors associated with ITAM-bearing cell surface receptors expressed on NK cells, their modulations in the tumor context and the various therapeutic tools developed to boost NK cell responses in cancer patients.

CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration.

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

Abstract S03-03: Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection

Abstract Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17); all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA−PD1+TIM3+), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls; interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population. Citation Format: Michael Chiorazzi, Erin Silva, Kristina Brower, Patrick Wong, Carolina Lucas, Jon Klein, Feimei Liu, Maura Nakahata, Jun Zhao, Nur-Taz Rahman, Camila Odio, Santos Bermejo, Shelli F. Farhadian, Charles Dela Cruz, Arnau Casanovas-Massana, John Fournier, Catherine Muenker, Anne L. Wyllie, Chantal B.F. Vogels, Chaney C. Kalinich, Mary E. Petrone, Isabel M. Ott, Anne E. Watkins, Adam J. Moore, Tara Alpert, Yuval Kluger, Aaron Ring, Nathan D. Grubaugh, Akiko Iwasaki, Albert I. Ko, Roy S. Herbst. Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S03-03.

Peripheral Innate Lymphoid Cells Are Increased in First Line Metastatic Colorectal Carcinoma Patients: A Negative Correlation With Th1 Immune Responses

Several distinct innate lymphoid cell (ILC) populations have been recently identified and shown to play a critical role in the immediate immune defense. In the context of tumors, there is evidence to support a dual role for ILCs with pro- or antitumor effects, depending on the ILC subset and the type of cancer. This ambivalent role has been particularly well-described in colorectal cancer models (CRC), but the presence and the evolution of ILCs in the peripheral blood of metastatic CRC (mCRC) patients have not yet been explored. Here, we investigated the distribution of ILC subsets in 96 mCRC patients who were prospectively included in the “Epitopes-CRC02” trial. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at metastatic diagnosis and after 3-months of treatment. The treatments consisted of Oxaliplatin-based chemotherapies for 76% of the patients or Folfiri (5FU, Irinotecan) chemotherapies for 14% of patients. Compared to healthy donors, the frequency of total ILCs was dramatically increased at metastatic diagnosis. CD56+ ILC1-like cells were expanded, whereas ILC2, NCR− ILCP and NCR+ ILCP subsets were decreased. Combined analysis with the systemic anti-telomerase hTERT Th1 CD4 response revealed that patients with low anti-TERT Th1 CD4 responses had the highest frequencies of total ILCs at diagnosis. Of those, 91% had synchronous metastases, and their median progression-free survival was 7.43 months (vs. 9.17 months for the other patients). In these patients, ILC1 and ILC2 were significantly decreased, whereas CD56+ ILC1-like cells were significantly increased compared to patients with low frequency of total ILCs and high anti-TERT responses. After treatment, the NCR+ ILCP were further decreased irrespective of the chemotherapy regimen, whereas the balance between ILC1 and CD56+ ILC1-like cells was modulated mainly by the Folfiri regimen in favor of ILC1. Altogether our results describe the effects of different chemotherapies on ILCs in mCRC patients. We also establish for the first time a link between frequency of ILCs and anti-tumor CD4 T cell responses in cancer patients. Thus, our study supports an interest in monitoring ILCs during cancer therapy to possibly identify predictive biomarkers in mCRC.

Abstract IA22: Reprogramming the tumor microenvironment and T cells for ovarian cancer immunotherapy

Abstract Immunologic destruction of tumors is a multistep, coordinated process that can be modulated or targeted at several critical points to elicit tumor rejection. These steps in the cancer immunity cycle include: (i) generation of sufficient numbers of effector T cells with high avidity recognition of tumor antigens in vivo; (ii) trafficking and infiltration into the tumor; (iii) overcoming inhibitory networks in the tumor microenvironment; (iv) direct recognition of tumor antigens and generation of an effector antitumor response; and (v) persistence of the antitumor T cells. Although several immune-based interventions have gained regulatory approval in many solid tumors and hematologic malignancies, there are currently no approved immune therapies for ovarian cancer. A major barrier to successful deployment of cancer immunotherapy for ovarian cancer patients is the immunosuppressive tumor microenvironment. Even if large numbers of tumor-specific T cells are generated in patients by active immunization or adoptive transfer, these T cells may not persist to readily destroy tumor targets in vivo. Previous studies by our group have defined some of the dominant immune resistance mechanisms in ovarian cancer to include extrinsic suppression of CD8+ effector cells by regulatory T cells (Tregs) (1, 2); metabolic deregulation via tryptophan catabolism by the immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO) (3, 4); engagement of inhibitory receptors such as PD-1 by the ligand PD-L1/B7-H1 (5-7); development of antigen loss variants (8); myeloid-derived suppressor cells (9); and inhibitory cytokines such as TGF-β. Collectively, this redundant immunosuppressive network facilitates tumor progression by actively restraining antitumor immunity and must be overcome in order to implement efficacious immunotherapeutic strategies. This presentation will be focused on our efforts at: (i) overcoming IDO-mediated innate and adaptive immune resistance as a strategy to reprogram the ovarian tumor microenvironment and (ii) novel “next-generation” strategies for reprograming T cells for adoptive immunotherapy of ovarian cancer.

PD-1 Blockade Promotes Emerging Checkpoint Inhibitors in Enhancing T Cell Responses to Allogeneic Dendritic Cells

Immune checkpoint inhibitors, which target coinhibitory T cell molecules to promote anticancer immune responses, are on the rise to become a new pillar of cancer therapy. However, current immune checkpoint-based therapies are successful only in a subset of patients and acquired resistances pose additional challenges. Finding new targets and combining checkpoint inhibitors might help to overcome these limitations. In this study, human T cells stimulated with allogeneic dendritic cells (DCs) were used to compare immune checkpoint inhibitors targeting TIM-3, BTLA, LAG-3, CTLA-4, and TIGIT alone or in combination with a PD-1 antibody. We found that PD-1 blockade bears a unique potency to enhance T cell proliferation and cytokine production. Other checkpoint inhibitors failed to significantly augment T cell responses when used alone. However, antibodies to TIM-3, BTLA, LAG-3, and CTLA-4 enhanced T cell proliferation in presence of a PD-1 antibody. Upregulation of coinhibitory T cell receptors upon PD-1 blockade was identified as a potential mechanism for synergistic effects between checkpoint inhibitors. Donor-specific variation in response to immune checkpoint inhibitors was attributed to the T cells rather than DCs. Additionally, we analyzed the regulation of checkpoint molecules and their ligands on T cells and allogeneic DCs in coculture, which suggested a PD-1 blockade-dependent crosstalk between T cells and APC. Our results indicate that several immune checkpoint inhibitors have the capacity to enhance T cell responses when combined with PD-1 blockade. Additional in vitro studies on human T cells will be useful to identify antibody combinations with the potential to augment T cell responses in cancer patients.

Abstract A66: Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation

Abstract Tumor-recruited myeloid cells represent a significant portion of inflammatory cells within the tumor microenvironment and influence nearly all steps of tumor progression. Although tumor-associated neutrophils (TANs) are present in large numbers, the role of TANs in cancer progression remains unclear and has only been recently investigated in murine models. The goal of this study was to provide a phenotypic and functional characterization of TANs in freshly harvested surgically resected lung cancer samples. Tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. Importantly, fresh lung tumors from patients were processed within 20 minutes of removal from the patient. We developed an approach to prepare human lung tumors for immunological studies by combining gentle mechanical manipulation with an optimized cocktail of enzymes used at low doses. This approach retrieved all major tumor-associated cell populations with high cell yield and maintained the expression of cell surface markers and the functions of isolated cells. In multicolor tracings, TANs were defined as CD15hi/CD66b+/CD11b+/MPO+/Arg1+/IL-5Ra-/ cells. We found that TANs comprised 5—25% of cells isolated from all digested human lung tumors (n=42). Compared to blood neutrophils, TANs displayed an activated phenotype (CD62Llo/CD54hi) and a novel repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. They also produced substantial quantities of the pro-inflammatory factors MCP-1, IL-8, MIP-1a, and IL-6, as well as the anti-inflammatory IL-1R antagonist. TANs were not hypofunctional cells, since they were able to phagocytize bacteria and produce reactive oxygen species. Understanding the role of TANs in regulating T cell responses in cancer patients is particularly important because cytotoxic T lymphocytes are the chief effector cells mediating antigen-driven anti-tumor immunity. We found that TANs were able to stimulate antigen non-specific T cell proliferation and IFN-γ release. Crosstalk between TANs and activated T cells led to substantial up-regulation of CD54, CD86, OX40L, and 4-1BBL co-stimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive feedback loop. This stimulatory activity of TANs appeared to be related to tumor size, as TANs from the majority of the smaller tumors (&amp;lt; 3cm) were more stimulatory than those from larger tumors. We also identified a novel subset of TANs with a hybrid “composite” phenotype of neutrophils and antigen-presenting cells (MPO+CD66b+CD14+HLA-DR+CCR7+CD86+). We found that this hybrid subset had the ability to: 1) dramatically stimulate antigen non-specific T cell proliferation, 2) induce the proliferation of allogeneic T cells in mixed lymphocyte reaction and 3) trigger antigen-specific memory T cell response to a mixture of T cell epitopes from human CMV, Epstein-Barr, flu viruses, as well as tetanus toxoid from all the common HLA types. Thus, our data suggest that in patients with early-stage lung cancer, TANs do not significantly contribute to inhibition of T cell responses. Rather, the majority of neutrophils recruited into the tumor microenvironment undergo phenotypic and functional changes that result in the formation of cells that resemble the murine anti-tumor “N1 TANs” and could potentially augment and support T cells responses. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Tom Stephen, Anjana Ranganathan, Charuhas Deshpande, Tatiana Akimova, Anil Vachani, Leslie Litzky, Wayne Hancock, Jose Conejo-Garcia, Michael Feldman, Sunil Singhal, Steven Albelda. Tumor-associated neutrophils in early stage human lung cancer are not immunosuppressive, but exhibit an inflammatory phenotype and provide accessory signals for T cell activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A66.

Cancer immunotherapy. Neo approaches to cancer vaccines.

A neoantigen-based vaccine elicits T cell responses in cancer patients [Also see Report by Carreno et al. ]

Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.

Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy

In this Timeline, we describe the characteristics of tumour antigens that are recognized by spontaneous T cell responses in cancer patients and the paths that led to their identification. We explain on what genetic basis most, but not all, of these antigens are tumour specific: that is, present on tumour cells but not on normal cells. We also discuss how strategies that target these tumour-specific antigens can lead either to tumour-specific or to crossreactive T cell responses, which is an issue that has important safety implications in immunotherapy. These safety issues are even more of a concern for strategies targeting antigens that are not known to induce spontaneous T cell responses in patients.

Abstract 2834: Cancer testis antigens-specific CD4+ T-cell immunity augmented by CTL-epitopes vaccination in cancer patients.

Abstract We identified novel cancer testis antigens (CTAs) including CDCA1, LY6K (URLC10) and KIF20A that are overexpressed in lung cancer, head and neck cancer (HNC), and other various malignancies, but not expressed in many adult normal tissues except for testis and fetal organs by using genome-wide cDNA microarray analyses. We identified CTAs-derived peptides that can activate tumor-reactive and HLA-A2 or -A24-restricted CTLs in both in vitro preclinical and in vivo clinical studies of peptide vaccine. It is well known that IL-2 and IFN-γ produced by Th1 cells have pivotal roles in efficient induction of memory CTLs and efficient infiltration of CTLs into the tumor. We identified these CTAs-derived promiscuous 20∼26-mer long peptides (LPs) that can induce both Th1 cells restricted by several frequent HLA class II and tumor-reactive CTLs restricted by HLA-A2 or -A24. The LPs were naturally processed from CTAs and presented by dendritic cells. In addition, these LPs induced propagation of CTA-specific CTLs by cross presentation in both human in vitro and HLA-class I transgenic mice in vivo. Furthermore, these LPs and Th1-cell clone enhanced induction of CTA-specific CTLs in vitro. Significant frequencies of CTA-specific Th1 cells were detected after short-term in vitro stimulation with LPs in HNC patients vaccinated with CTA-derived CTL-epitope peptides and cancer patients before vaccination but not in healthy donors; responses were augmented by repeated vaccinations. These results show for the first time the presence of novel CTAs-specific Th1 cell responses in cancer patients and suggest the potential utility of these LPs for propagation of CTAs-specific Th1 cells and CTLs. Citation Format: Yusuke Tomita, Akira Yuno, Yoshihiro Yoshitake, Hirotake Tsukamoto, Atsushi Irie, Satoru Senju, Koji Yoshida, Takuya Tsunoda, Yusuke Nakamura, Masanori Shinohara, Yasuharu Nishimura. Cancer testis antigens-specific CD4+ T-cell immunity augmented by CTL-epitopes vaccination in cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2834. doi:10.1158/1538-7445.AM2013-2834

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma.

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients.

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.

Evaluation of T7 and lambda phage display systems for survey of autoantibody profiles in cancer patients

In the current study we attempted to evaluate the suitability of T7 Select 10-3b and λKM8 phage display systems for the identification of antigens eliciting B cell responses in cancer patients and the production of phage-displayed antigen microarrays that could be exploited for the monitoring of autoantibody profiles. Members of 15 tumour-associated antigen (TAA) families were cloned into both phage display vectors and the TAA mini-libraries were immunoscreened with 22 melanoma patients' sera resulting in the detection of reactivity against members of 5 antigen families in both systems, yet with variable sensitivity. T7 phage display system showed greater sensitivity for the detection of antibodies against members of CTAG, MAGEA and GAGE families, both systems showed equal performance in detecting the reactivity against MAGEC and SSX2 while only λKM8 allowed the detection of anti-CTAGE5 antibodies. The biological properties of both phages turned out to be equally suitable for the production of antigen microarrays however in line with the plaque assay the sensitivity for the detection of various autoantibodies differed between the vectors. However, presumably due to the higher variability of the background signals in the microarray assay, it turned out to have comparable, in some cases even slightly lower sensitivity than the plaque assay.Next, we explored the repertoire of antigens that could be identified by screening T7 phage-displayed testis cDNA library with sera from melanoma patients. From the 243 antigens identified, only 24 represented known genes translated in their natural reading frame and included known TAAs like Annexin XI-A and a novel potential CT antigen SPAG8. Another 12 were uncharacterised genes but the remaining clones contained DNA fragments in non-natural reading frames that most likely represent mimotopes, nevertheless, they may turn out to be valid biomarkers.

P8 T cell responses in cancer patients after vaccination with a cryptic hTERT peptide (Vx-001)

P8 T cell responses in cancer patients after vaccination with a cryptic hTERT peptide (Vx-001)

Soluble Ig-Like Transcript 3 Inhibits Tumor Allograft Rejection in Humanized SCID Mice and T Cell Responses in Cancer Patients

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.