Identifying T-cell Targets for TAP-Impaired Tumors
Shared Neoantigens as T-Cell Targets for TAP-Impaired Tumors (Revised as of 10:15 a.m. CDT, 6/15/2022)
Sjoerd H. van der Burg (Leiden University Medical Center) presented efforts focused on exploiting a divergent mode of antigen presentation caused by TAP repression in cancer cells for cancer immunotherapy. TAP is responsible for loading peptides on MHC-class I in the ER. Downregulation of TAP in cancer cells occurs frequently and is associated with shorter survival and resistance to immunotherapy. Cancer-selective impairment of TAP function leads to alternate loading of MHC-I of N-terminal ‘signal’ and C-terminal ‘tail’ peptides that leads to a unique repertoire of MHC class I presented self-antigens, representing a potential target for immune surveillance and/or immunotherapy. These unique epitopes are referred to as T cell epitopes associated with impaired peptide processing (TEIPP). Growth of TAP deficient tumors in mice was effectively controlled by TEIPP-specific T cell therapy and vaccination without toxicity. Van der Burg and colleagues sought to identify self-antigens uniquely presented by human cancer cells lacking TAP by querying predicted self N-terminal and C-terminal peptides generated by known cellular proteases, filtering for peptides predicted to bind to common HLA haplotypes, followed by selecting candidate peptides by comparing HLA bound peptides identified on human tumor vs healthy tissue control samples. One candidate identified by this approach, an HLA-A2 binding LRPAP1 peptide, was expressed ubiquitously in many human cancers and used to demonstrate proof-of-principle of targeting TEIPP epitopes for cancer immunotherapy. LRPAP1-specific T cells selectively killed TAP-deficient cancer cells but not TAP-proficient cells. Demonstrating the presence of TEIPP-specific T cells in humans, LRPAP1-specific T cells were detected in human PBMCs of healthy individuals and in patients with lung cancer via LRPAP1 specific tetramer. An optimized synthetic long peptide (SLP) vaccine strategy to target LRPAP1 was developed, to induce cross presentation by dendritic cells, enabling the stimulation of human T cells that were able to efficiently kill LRPAP1 peptide presenting cancer cells. This optimized vaccine approach is currently being testing in a clinical trial for advanced NSCLC patients after immune checkpoint blockade failure.
Tracking tumor evolution and immunoediting in pancreatic cancer
High Quality Neoantigens are Immunoedited in Long-Term Pancreatic Cancer Survivors
Vinod P. Balachandran (Memorial Sloan Kettering Cancer Center) presented a longitudinal study in pancreatic cancer patients to identify the nature of high quality neoantigens to inform cancer vaccine design. Seventy human pancreatic cancers were examined over a 10-year period for mutational load, neoantigen density and quality, and mutational heterogeneity. The team previously reported that the rare long-term survivors of pancreatic cancer harbor higher intratumor T cell densities. In this study, it was determined that tumors of long-term survivors of pancreatic cancer evolved to be less heterogeneous with fewer neoantigens, and overall lower tumor mutational burden, implying immunoediting. To quantify whether these changes were mediated by immune selection, neoantigen quality was defined by their 1) similarity to known ‘non-self’ immunogenic antigens and 2) predicting difference in affinity between the mutation wildtype versions of the peptide (a prediction that was informed by empirical TCR:peptide binding affinity studies). Tumors from long-term survivors harbored significantly lower high quality neoantigens, providing evidence that the immune system selectively edits high quality neoantigens. Based upon these studies, an improved neoantigen quality model that identifies immunogenic features in neoantigens was developed, and a clinical trial of personalized neoantigen mRNA vaccines in combination with PD-L1 inhibitors in pancreatic cancer recently completed phase I clinical testing wherein vaccine-specific T cell responses were observed in patients; follow-up for survival in this trial is ongoing.
Antitumor functions of CD4 T cells
Role of CD4 T Cell Help, and Impact of CD27 Costimulation
Jannie Borst (Leiden University) highlighted the critical importance of CD4 T cells in cancer immune surveillance, and in unleashing the full potential of antitumor CD8 T cells. DNA vaccination with HPV E7 MHC-I peptides with and without companion MHC-II epitopes was used by the team to decipher antitumor mechanisms of CD4 T cells, and establish functional consequences CD4 T cell help in CD8 T cells. CD4 T cell help improved the cytotoxic function of antitumor T cells, reduced exhaustion of effector CD8 T cells, and promoted T cell trafficking to the tumor. CD70 expression on DCs is induced by CD4 T cell help, in part by CD40L:CD40 interactions between CD4 T cells and DCs, respectively. Blocking CD70 binding to CD27 in the context of CD4 T cell help reversed ‘helped’ CD8 T cell phenotypes and conversely, providing CD27 activating antibody partially recapitulated ‘helped’ CD8 T cell phenotypes in mice that received vaccines lacking MHC class-II epitopes. Combining CD27 agonism with PD1 blockade in mice vaccinated against tumor antigen without CD4 T cell help achieved the level of antitumor efficacy observed in mice with helper epitope vaccination. A potential explanation for the synergy between PD1 blockade and CD27 agonism was that of IL-2 induction induced by CD27; IL2 production has previously been shown to accentuate PD1 blockade efficacy. Based upon this work, current efforts are focused on translating a CD27 activating antibody candidate for cancer immunotherapy.
Spatial analysis of the proteome of the TME
Spatial Proteome Analysis (CODEX) of Effective Cancer Immunotherapy
Ruan Viera Medan (Washington University School of Medicine) used spatial analyses (CODEX; also called ‘Phenocycler’) of a progressing T3 sarcoma tumor model that is fully rejected by PD1 + CTLA4 blockade (ICB) to study how the tumor microenvironment evolves with and without successful immunotherapy. CODEX is a high dimensional immunohistochemistry platform that functions via iterative staining and imaging cycles of 3 DNA barcoded antibodies + DAPI that can perform spatial analysis of the proteome on fixed (FFPE) or frozen tissue. Prior to complete tumor rejection with ICB tumors were harvested and compared to control treated (progressing) tumors. The tumor microenvironment was analyzed for 24 targets, including a marker for tumor and all common immune cell types. In progressing tumors T cells were largely restricted to the tumor border, with very few infiltrating the tumors. After ICB, higher densities of T cells infiltrated the tumor and expressed higher levels of Ki67 (indicating proliferation). Several changes in myeloid populations were observed with increased neutrophil influx in areas of apparent tumor death; increased iNOS expression in myeloid cells within the tumor; and increased CD103+ cDC1-like cells. Intriguingly, areas of neutrophil influx associated with caspase 3 positive regions that lacked robust T cell densities, relative to portions of the tumor that contained T cells expressing granzyme B. These spatial differences may imply active areas of T cell immune surveillance distinct from areas where tumor cells have already been killed, where neutrophils are recruited to engulf dying tumor cells after successful immune surveillance. These findings inform the understanding of spatial and temporal mechanisms of immune surveillance. Ongoing efforts are focused on applying higher dimensional CODEX analysis of this model across various time points to define the nature of successful tumor immune rejection.
HLA-class II molecules and CD4 Tregs promote immune evasion in melanoma
The Landscape of Helper and Regulatory CD4+ T cells in Human Melanoma
Giacomo Oliveira (Dana-Farber Cancer Institute) reported on efforts to characterize the population of CD4+ tumor-infiltrating lymphocytes (TILs) in melanoma and the how these cells may be coopted by HLA class II expressing cancer cells during immune evasion. A large panel of melanoma cell lines were tested for HLA class II expression; where induction of MHC-class II expression was observed after IFNg treatment on a majority of cell lines. In human tumors, HLA-class II expression was associated with expansion of CD4 T regulatory (Tregs) cells. Single-cell characterization of CD4+ TILs from human melanomas, T cell receptor (TCR) reconstruction, and antigen specificity screening identified three distinct phenotypes among CD4+ clonotypes: memory, exhausted, and regulatory T cells. Tregs were present in high numbers of HLA-class II-positive melanomas, and these Tregs were elicited through presentation of neoantigens by antigen-presenting cells. A large number of tumor-reactive Treg TCRs were stimulated by HLA-class II-positive melanoma and exhibited specificity for melanoma neoantigens. A subset of exhausted cytotoxic CD4 T cells were also observed that intriguingly had specificity to MHC class I presented neoantigens. HLA-class II positive tumors were associated with higher densities of CD4 Tregs, harbored higher levels of neoantigen load, but were more responsive to immune checkpoint blockade than HLA-class II low tumors. These data indicate presentation of neoantigens by HLA-class II molecules and engagement of tumor-reactive CD4+ Treg cells are novel mechanisms of immune evasion in HLA-class II-positive melanoma.
Elucidating roles for dendritic cell subsets in the anti-tumor response
Not All T Cell Responses are Generated Equal: The Role of Dendritic Cells in Anti-Tumor Immunity
Stefani Spranger (Massachusetts Institute of Technology) presented ongoing work using murine models to dissect the roles of dendritic cell subsets, and their phenotypes, in mediated T cell immune surveillance. Comparing model CD8 T cell epitope (SIY) expressing murine tumor models that were either progressed or were rejected by the host (regressed), the requirements of specific DC subsets (e.g. cDC1 vs cDC2) in mediating antitumor T cell activity were tested. Relative to progressing tumors regressing tumors harbored higher levels of cDC1 infiltration, regardless of the presence (wildtype) or absence (RAG2-/-) of T cells, implying DC recruitment as a potentially causal mechanism of tumor regression. Surprisingly, despite their sentinel roles in mediating tumor antigen cross presentation, cDC1s were not required for tumor regression. Instead, cDC2s with an interferon stimulated gene (ISG) signature were able to cross present tumor antigen and successfully prime antitumor T cells (‘cross dressing’). Selective deletion of the IFN receptor (IFNAR) on cDC2s prevented tumor rejection, implying a critical role of IFN signaling in cDC2s for spontaneous tumor rejection. The team hypothesized this was due to tumor intrinsic differences in progressor vs regressor cell lines, and discovered higher levels of baseline IFNb production in the regressor tumor line. Tumor derived IFNb was required for this process: knocking out IRF3-/- ablated spontaneous tumor regression, and coating the progressing tumor with IFNb prior to implant led to tumor regression. These data demonstrate a novel route for IFN experienced cDC2s to mediate priming of, and cross presentation to, antitumor CD8 T cells. Separately the group recently published a study deciphering mechanisms of ICB resistance in NSCLC using low tumor mutational burden KRAS driven genetically engineered model. Interestingly, lung tumors in this model do not respond to ICB, but the same tumor model is rejected when implanted in the flank of mice, implying a difference in tissue site in controlling ICB response. Differences in T cell proliferation between the two sites were not observed; however, marked differences in the tumor draining lymph node (mediastinal lymph node for lung vs inguinal for flank). These findings indicate distinct immunobiology of T cells in different lymphatic and tissue locations.
Identifying neoantigens in the immunopeptidome
Deciphering the Immunopeptidome in vivo Reveals Novel Tumor Antigens
Alex M. Jaeger (Moffitt Cancer Center) presented a novel approach to defining the immunopeptidome, the collective of MHC-class I-associated peptides, in vivo using mouse models of autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD). A Cre-inducible affinity tag in mouse MHC-I gene (H2-K1) and targeted to KrasLSL-G12D/+; p53fl/f (KP) mouse model (KP/KbStrep) were used to isolate and characterize MHC-I + peptide complexes from PDAC and LUAD after in vivo tumor evolution. Changes in the immunopeptidome over tumor evolution were observed, and novel immunopeptides, which had not been found in previous studies in vitro, were identified. Some cancer-specific peptides were associated with minimal levels of preceding mRNA, indicating a discordance between relative transcript abundance and peptide presentation. In addition, using expression of the affinity tagged MHC-I in normal cells in vivo also enabled characterization of the healthy tissue immunopeptidome. Highlighting the danger of using transcriptome abundance to predict neoantigen presentation for vaccine design, some cancer testis antigen transcripts were significantly enriched in tumor vs normal, yet were presented efficiently by both cancer and normal cells. This new mouse model will enable the discovery of novel, tumor specific MHC-I-associated peptides in vivo. The tagged MHC-I allele is compatible with other Cre-driver lines, providing opportunities to further study antigen presentation in the context of immunology, infectious disease, and autoimmunity.
Neoantigen-targeting cellular therapies for fibrolamellar carcinoma
Developing T Cell Immunotherapies Targeting Conserved Fusion Neoantigens in Fibrolamellar Carcinoma
Allison M. Kirk (St. Jude Children's Research Hospital) reported on a study of CD8 T cell responses to DNAJB1-PRKACA fusion neoantigens. Fibrolamellar carcinoma (FLC), a rare liver cancer, is driven by gene fusion of DNAJB1 to PRKACA. The fusion protein sequence identical in > 90% of patients, making it potential target for immunotherapy. Net MHC (a neoantigen prediction pipeline) was used to predict 10 putative neoepitopes. Tumor infiltrating lymphocytes (TILs) from FLC tumor biopsies were expanded ex vivo using peptides containing these eiptopes, and the fusion neoepitope HLA-A HLA-A*68:02-EIFDRYGEEV was found to dominantly elicit a functional CD8 T cell response. Two functional patient T cell receptors (TCR) for this neoepitope was identified. PBMCs from health donors and FLC patients underwent an antigen-specific expansion protocol to expand additional fusion-specific TCR clonotypes. TCRs were cloned and expressed in Jurkat or primary T cells. Exposure to the fusion neoantigen elicited IFN gamma and TNF alpha production by cells expressing the fusion-specific TCR. Fusion–specific TCRs mediated the killing of fusion-presenting target cells in vitro and showed little cross-reactivity against wild-type antigens. Experiments to characterize the activity of T cells expressing the fusion-specific TCR against established tumors in mice are ongoing.
Characterization of tumor-infiltrating lymphocytes in non-small cell lung cancer (NSCLC)
Discovery and Characterization of Tumor-Reactive TIL
Kellie N. Smith (Johns Hopkins University School of Medicine) presented work comparing the phenotypes, functionalities, and TCR characteristics of bystander antiviral T cells—known to infiltrate tumors despite lacking reactivity to tumor antigens—as compared to antitumor T cells in NSCLC. Most NSCLC tumors do not respond to immune checkpoint blockade; PD-L1 expression and high TMB are imperfect biomarkers. Thus, identification of determinants of response and/or mechanisms of resistance are needed. Tumor specimens were collected pre- and post- treatment with ICB in a neoadjuvant ICB clinical trial design and subjected to single cell TCR and RNA sequencing. Fourteen distinct CD8 T cell transcriptional clusters were identified. TCR clonotype sequences were cloned and expressed in Jurkat cells, using EBV and flu specific peptides to identify positive control/comparative TCRs. The avidities of tumor specific TCRs were determined and used to track transcriptional programs associated with antitumor vs bystander antiviral T cells. Tumor antigen specific TILs exhibited suboptimal cytolytic phenotypes, tissue-resident memory T cell phenotypes, and low levels of IL-7 receptor. Antitumor TILs had impaired responses to IL-7 relative to bystander antiviral T cells in vitro. Moreover, patients responsive to PD1 blockade in the study had higher expression of the IL-7 receptor, genes associated with memory and effector function, and lower expression of T cell dysfunctional phenotypes.
Anti-tumor activity associated with childhood vaccine-specific CD4 T cells
Intratumor Childhood Vaccine-specific CD4+ T Cell Recall Coordinates Antitumor Type I and II Immunity
Michael C. Brown (Duke University) presented work revealing antitumor potential of intratumorally delivered, childhood vaccine associated antigens to engage antitumor functions of CD4 T cells. In studying the mechanism of recombinant poliovirus (PVSRIPO), it was observed that pre-existing immunity to polio from polio vaccination in mice potentiated antitumor efficacy in mice despite dramatically reducing intratumor viral replication. Polio virotherapy in the context of pre-existing anti-polio immunity was associated with increased conventional CD4 T cell and eosinophil influx. Using mice immunized against both polio and tetanus, antitumor effects of intratumor polio capsid and tetanus toxoid antigens occurred only in mice immunized against polio or tetanus, respectively. This effect was shown to be due to CD4 T cells, independent of B cells, and partially dependent upon CD8 T cells. To test the hypothesis that CD4 T cells specific to presented recall antigens could provide help to antitumor CD8 T cells, OT-I (OVA-specific) CD8 T cells were adoptively transferred to mice bearing B16-OVA tumors, and antitumor (OT-I) T cell phenotypes were queried. Both polio and tetanus recall antigens led to enhanced IFNg, TNF and granzyme B expression in antitumor CD8 T cells; and adoptive transfer of T cells from mice treated with either polio or tetanus recall antigen protected naive tumor bearing recipients from tumor growth relative to that of mice receiving T cells from mice treated with a control antigen. To address the functional significance of the observed infiltrating eosinophils, a canonical type II immune effector, after polio recall, eosinophil depletion was used to demonstrate that they also contribute to the antitumor efficacy of intratumor polio recall. These data imply that childhood vaccine-specific CD4+ T cells, hold cancer immunotherapy potential. In the context of PVSRIPO therapy, antitumor and inflammatory effects of polio vaccine-specific CD4+ T cell recall supersedes inhibitory effects of attenuated intratumor viral replication, and represents a novel mechanism to simultaneously engage both type I and II antitumor immunity.
Genetic factors that affect the tumor microenvironment and immunotherapy response
The Tumor Microenvironment and Immunotherapy Efficacy
Thomas Gajewski (University of Chicago) presented ongoing work focused on rectifying insufficient T cell inflammation within melanoma tumors that mediate resistance to immunotherapy. Considerable heterogeneity in the tumor microenvironments of melanoma patients have long been noted, yet molecular and cellular explanations of such heterogeneity that determine immunotherapy response remain undefined. Factors demonstrated by Gejewski and colleagues to influence such heterogeneity include tumor somatic mutations associated with beta-catenin signaling, PTEN deletion, and DNA repair machinery; differences in commensal microbiota constitution; and germline genetic differences. Regarding the latter, germline PKC-delta signaling mutations that led to repression of PKC-delta activity were shown to sensitize tumors to immune checkpoint blockade (ICB). PKC-delta is selectively expressed in myeloid cells, and was shown to promote M1-like phenotypes in murine tumor associated myeloid cells upon deletion. These studies may imply utility of PKC delta inhibitors for cancer immunotherapy. More broadly, this collective research demonstrates both genetic and environmental influences on tumor microenvironment phenotypes that dictate immune surveillance: further investigations into such factors are anticipated to reveal personalized routes to sensitize malignant tumors to cancer immunotherapy.
Anti-cancer vaccine effectiveness depends on dosing of MHC-II neoantigens
Identification of a Novel FoxP3-negative CD4+ T Cell Population that Inhibits Cancer Immunotherapy
Hussein Sultan (Washington University School of Medicine) reported on studies on the development and efficacy of personalized neoantigen vaccines using MHC-II neoantigens. Studies were performed with the T3 sarcoma cell line with the MHC-II neoantigen mltgb1. It had been previously shown that vaccines containing only MHC-I or MHC-II neoepitopes did not induce T3 rejection in vivo. In contrast, vaccines containing T3 MHC-I neoepitopes plus low doses of mltgb1 (LDVax) induced tumor rejection in mice. Vaccination with the same vaccine but higher doses of mlgtb1 (HDVax) was ineffective. Adoptive transfer of T cells from HDVax mice conferred unresponsiveness in anti-PD-1-treated unvaccinated tumor-bearing mice, suggesting HDVax inhibits anti-tumor activity of anti-PD-1. HDVax induces high expression of PD-1 on CD4 T cells and alters expression of Foxp3 and CXCR5/BCL6. PD-1-high CD4 T cells suppressed anti-tumor activity CD4 and CD8 T cells in vitro and blocked T3 tumor rejection in RAG -/- mice after adoptive transfer of T cells from LDVax treated mice. PD-1-high CD4 T cells are defective in IL2 production. Exogenous expression of wild type IL2 does not reverse immunosuppression caused by these PD-1-high CD4 T cells, but expression of the cis-targeted IL2 mutein that directs the non-alpha form of IL2 to CD8 T cells (CD8-IL2) restores tumor rejection. These data indicate that neoantigen vaccine effectiveness is dependent on dosing of MHC-II neoantigens and that directing IL2 to CD8+ cells can restore vaccine effectiveness.
Targeting macrophages in triple negative breast cancer
Elucidating Macrophage Diversity to Uncover Novel Anti-Cancer Therapeutic Strategies
Jennifer Guerriero (Brigham and Women's Hospital) highlighted the vast diversity and plasticity of myeloid cell phenotypes in cancer, and presented proof-of-principle evidence on how they can be rationally targeted to improve therapy in breast cancer patients. Tumor associated myeloid cells associate with poor prognosis in breast cancer, mediate resistance to chemotherapy, and are predominately derived from bone marrow—though a subset of tissue resident macrophages are derived from yok sac precursors with tissue-specific transcriptional programs. Such differences in ontogeny and plasticity render myeloid cells difficult to both define and target. Triple negative breast cancer (TNBC) is highly infiltrated with both macrophages and T cells, yet remains relatively refractory to conventional immunotherapy. Recent work indicates that PARP inhibitor therapy (PARPi) leads to STING pathway activation, mediates antitumor effects in a CD8 T cell dependent manner, and synergizes with immune checkpoint blockade. Accordingly, numerous trials have commenced to test the efficacy of PARP inhibitors combined with immunotherapy. In vivo, PARPi led to an influx of myeloid cells and CD8 T cells; with macrophages expressing higher levels of CSF1R and PDL1. PARPi was found to directly modulate CSF1R and PDL1 expression on human macrophages, indicating a potential immunosuppressive role of tumor associated macrophages after PARPi. Indeed, inhibiting CSF1R on macrophages to deplete/reprogram them away from tumor promoting phenotypes led synergized in restricting tumor growth. Lipid metabolism was found to be induced by PARPi alone, indicating that fatty acid synthesis mechanisms known to drive immunosuppressive macrophage phenotypes were engaged. Thus, targeting SREBP1 with fatostatin to block lipid metabolism was shown to prevent PDL1 and CSF1R induction on macrophages after PARPi treatment, implicating SREBP1 in mediating resistance to PARPi therapy. Ongoing efforts are focused on identifying native states of tumor associated macrophages that restrict tumor immune surveillance and mediate therapy resistance in breast cancer.
Using picornaviruses for intratumoral virotherapy
Cancer Immunotherapy through Endogenous Type-I IFN Dominant Signaling in Dendritic Cells
Matthias Gromeier (Duke University School of Medicine) presented mechanistic insights and rationale for targeting the tumor microenvironment with a recombinant poliovirus (PVSRIPO). PVSRIPO is currently in clinical testing as an intratumor virotherapy approach for gliomas, melanoma, and breast cancer. Long-standing evidence from wildtype polio investigations in primates indicates a preference for polio replication in lymphatic tissue, with tropism for myeloid cells in natural infectious settings. PVSRIPO was derived from the live attenuated Sabin vaccine strain of polio, and was further modified by replacing the Sabin IRES (internal ribosomal entry site) with that of the Human Rhinovirus type II (HRV2) IRES. This recombinant poliovirus was found to be stably neuro-attenuated, but maintain capacity to kill cancer cells, and lead to activation of tumor associated myeloid cells, including dendritic cells. Relative to other viral species, picornaviruses lead to selective activation of MDA5, a RIG-I like cytosolic pattern recognition receptor, and exhibit selective tropism for tumor associated myeloid cells, as opposed to malignant cells. Indeed, in murine models, infection of the tumor microenvironment, but not malignant cells, was required for antitumor efficacy after polio virotherapy. Moreover, human tumor slice culture assays revealed minimal ‘oncolysis’, but sustained type I/III IFN responses localized to tumor associated myeloid cells. Relative to TLR agonists, polio virotherapy led to less substantial T cell influx; however, anti-tumor T cells had significantly greater polyfunctional phenotypes and mediated more robust antitumor efficacy upon adoptive transfer to naïve tumor bearing mice. These findings indicate higher functionality of antitumor T cells after intratumor MDA5 engagement. In murine glioma models, PVSRIPO replication was observed both in spleen and cervical lymph nodes (tumor draining) after therapy, coinciding with T cell inflammation and microglial proliferation. Altogether this work indicates unique mechanisms of action for leveraging picornaviruses as in situ vaccine approaches.
Effects of synthetic long peptide (SLP) personalized vaccines on the tumor microenvironment
SLPs for personal neoantigen-targeting vaccines
Catherine J. Wu (Dana-Farber Cancer Institute) and her team have a long-standing interest in developing personalized vaccine approaches that can effectively drive T cells into tumors and increase the fraction of patients responding to immune checkpoint blockade. Towards this end, her team and collaborators are exploring the pivotal questions of defining the baseline composition of tumor infiltrating leukocytes (TILs) in diverse cancer types, and elucidating how vaccination changes the densities and character of tumor antigen specific TILS within the tumor microenvironment. Identification of both neoantigens and TCR specificities are now feasible and have been extensively used by the group to address these questions. In general, clonally expanded and exhausted T cells within the tumor microenvironment are specific to tumor associated antigens (as opposed to bystander T cells). Wu and colleagues have developed a synthetic long peptide (SLP) personalized vaccine platform using computational prediction of neoantigen epitopes that are synthesized for vaccination with poly IC-LC after subcutaneous administration, delivering up to 20 peptides per patient. Interestingly, CD4 T cell responses to these vaccines are more dominantly engaged, however CD8 T cell responses to neoepitopes are also induced. Cases studies within ongoing clinical trials provided evidence that recurrent tumors after SLP vaccination are sensitized to PD1 blockade. Analysis of TCR clonotypes and anti-vaccine T cell responses reveals new TCR clonotype emergence after vaccines specific to tumor neoantigens, persisting several years after vaccination. Novel vaccination approaches currently being implemented include montanide as an added vaccine adjuvant (in addition to Poly IC-LC) with local Ipilimumab administered near the vaccination site. Studies beyond melanoma implementing the SLP personalized vaccine platform are in progress and ongoing studies are determining both peripheral and TIL changes induced by vaccination at single cell resolution.
T cell hierarchies in mouse models of lung cancer
Antigen dominance hierarchies shape anti-tumor T cell phenotypes and immunotherapy response
Megan L. Burger (Massachusetts Institute of Technology) provided mechanistic insights into immunodominance, wherein epitopes with high affinity and/or stability are dominantly recognized by T cells preferentially above that of other concurrently presented epitopes. Using a genetically engineered mouse model of lung cancer expressing both SIINFEKL and SIY, two model antigens, indicated that antigen dominance hierarchies exist in this model system, and CD8 T cell expansion predominantly occurs against neoantigens that most stably bind to MHC. This dominance hierarchy limits T cell diversity and suppresses differentiation of T cells that respond to subdominant neoantigens. Subdominant T cell responses are associated with a TCF1+ progenitor cell phenotype known to associate with response to immune checkpoint blockade (ICB). However, a dysfunctional subset of CCR6+ TCF1+ cells expressing IL17 (consistent with a Tc17 phenotype) interfered with response to immune checkpoint blockade in the context of a subdominant T cell response. Vaccination against dominant and subdominant neoantigens abolishes the dominant T cell hierarchy, improves subdominant T cell response and eliminates CCR6+ TCF1+ cells. Since vaccination was able to eliminate subdominant antigen-specific T cell dysfunction, it is hypothesized that the CCR6+ TCF1+ subset represents inappropriately or sub-optimally primed T cell populations. Thus, vaccination may serve to overcome limitations of immunodominance during natural immune surveillance.
Personalized neoantigen vaccine combined with immune checkpoint blockade for kidney cancer
A personalized neoantigen vaccination incorporating locally delivered ipilimumab induces tumor-specific immunity in renal cell carcinoma
David A. Braun (Dana-Farber Cancer Institute) reported on a phase I clinical trial (NCT02950766) of personalized neoantigen vaccine combined with ipilimumab for surgically resected, stage III/IV renal cell carcinoma (RCC). Nine patients (7 grade III, 2 grade IV) received a synthetic long peptide vaccine targeting up to 20 personal tumor neoantigens mixed with adjuvant poly ICLC -/+ co-administration with ipilimumab (two arms) at the vaccination site. Single-cell RNA sequencing and T cell receptor (TCR) sequencing analysis of immune populations at the vaccine site reveal an increase in T cell clonotypes, and in all patients, predicted neoantigens generate an antigen-specific response in peripheral T cells ex vivo. Peripheral T cells from most patients recognized autologous tumor cells ex vivo, and clinical follow-up is ongoing. These results indicate that personalized neoantigen vaccines, when co-administered locally with immune checkpoint inhibitors, is safe and can generate anti-tumor immune responses in RCC.
Predictive biomarkers for neoadjuvant therapy in head and neck cancers
Neoadjuvant PD1 therapy
Ravi Uppalura (Brigham and Women's Hospital) updated on clinical progress and identification of biomarkers of response in a cohort of patients with HPV unrelated HNSCC cancer treated with pre- and post- surgical PD1 blockade. Neoadjuvant immune checkpoint blockade was safe, and was associated with encouraging clinical responses. Pre-treatment features associated with response included PD-L1 expression, CD8 T cell density, and IFN-gamma gene expression signatures. A subset of responder vs non-responder tumor tissues were analyzed by single cell RNA-sequencing. Twelve clusters of CD8 TIL phenotypes were observed, with two predominant phenotypes reflecting terminal exhaustion phenotypes. Exhausted CD8 TIL phenotypes were more frequent in non-responders and reductions in terminally exhausted T cells were primarily observed in responders relative to other T cell phenotypes. In responders, TCR expansion was primarily restricted within non-exhausted TIL populations. These data indicate potential for neoadjuvant immune checkpoint blockade to benefit patients with HNSCC, and indicate that baseline features influence immunotherapy responsiveness.
Peripheral blood for neoantigen identification
Exploiting circulating lymphocytes and cell-free DNA as a source to develop minimally invasive personalized T-cell therapies
Andrea Garcia-Garijo (Vall d'Hebron Institute of Oncology) presented results on a study to use peripheral blood as a source for neoantigen-specific T lymphocytes and neoantigen identification through cell-free DNA (cfDNA). CD8+ and CD4+ peripheral blood lymphocytes were sorted from 6 patients with various epithelial cancers. Cells were sorted based on PD-1 expression and in combination with other cell-surface receptors (CD27, CD39, CD38, HLA-DR and 4-1BB). 35 CD4+ and 35 CD8+ populations were identified and expanded ex vivo, and the populations were further screened for neoantigen specificity. Neoantigen-specific T cells were detected in 5 of 6 patients using this approach. CD8+ PD-1 high CD39+ T cells were the most frequently represented neoantigen-specific lymphocytes compared to any other combination of markers in the CD8+ population. With regards to neoantigen identification, non-synonymous mutations (NSMs), the overlap between NSMs identified in tumor DNA vs. cfDNA varied between patients. 2 of 6 patients displayed no overlap in NSMs between tumor DNA and cfDNA, but cfDNA did identify 11/16 neoantigens in patients with at least some overlap of NSMs in tumor DNA and cfDNA. These data identify peripheral blood as a potential source for the identification of neoantigens and CD8+ and CD4+ neoantigen-specific T lymphocytes and T cell receptors in patients with epithelial cancers.
Delivery of IL-12 to the tumor microenvironment
Modified vaccinia virus Ankara expressing IL-12 promotes potent anti-tumor activity through effective reprogramming of the tumor microenvironment
Elisa Scarselli (Nouscom Srl) presented on local administration of IL-12 to boost anti-tumor immunity in the tumor microenvironment (TME). IL-12, an inflammatory cytokine, was administered locally in order to bypass adverse events associated with systemic administration. Modified vaccinia ankara (MVA) vector, which induces a pro-inflammatory response, encoding IL-12 was delivered to tumors with novel “Vector Aided Microenvironment Programming” (VAMP). Administration of VAMP-IL-12 resulted in complete remission of established CPI-resistant tumors in mouse models as monotherapy and in combination with anti-PD-1 therapy. Tumors treated with VAMP-IL-2 exhibited reduced immunosuppressive M2 macrophages, increased M1 macrophages, and recruitment of dendritic cells suggesting VAMP-IL-12 as a novel and promising approach to reprogramming an immunosuppressive TME.
Optimizing TCR-CD3 interactions
Modulating extracellular TCR-CD3 interactions to identify new immunotherapy targets
Yogambigai Velmurugu (NYU Grossman School of Medicine) reported on a novel strategy to optimize T cell receptor (TCR)-CD3 interaction to enhance T cell effector function and anti-tumor activity and to increase specificity against tumor-specific antigens. Retroviral TCR display libraries in T cell hybridoma were created, and mutations were induced at specific extracellular TCR sites known to interact with CD3. C beta helix 3, C beta helix 3 and F strand. Single-cell sorting with soluble CD3 tetramers identified TCRs with increased binding specificity to CD3. Select TCR mutations with enhanced CD3 binding are associated with increased IL-2 production and rapid phosphorylation kinetics of Lck, indicating increased T cell functionality and signal transduction. Select TCR mutants are currently being tested in vivo in mouse tumor models. Findings from this study show promise in enhancing efficacy and specificity of T cell therapy.