Tumor-specific T-cell Responses Research Articles

Viral Therapy for Glioblastoma

Viral Therapy for Glioblastoma

A Virus-Infected, Reprogrammed Somatic Cell-Derived Tumor Cell (VIReST) Vaccination Regime Can Prevent Initiation and Progression of Pancreatic Cancer.

Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.

CD200-CD200R Pathway in the Regulation of Tumor Immune Microenvironment and Immunotherapy.

Tumor-associated inflammation and immune responses are key components in the tumor microenvironment (TME) which regulate tumor growth, progression, and metastasis. Tumor-associated myeloid cells (TAMCs) are a group of cells that play multiple key roles including induction of tumor-associated inflammation/angiogenesis and regulation of tumor-specific T-cell responses. Thus, identification and characterization of key pathways that can regulate TAMCs are of critical importance for developing cancer immunotherapy. Recent studies suggest that CD200-CD200 receptor (CD200R) interaction may be important in regulating the TME via affecting TAMCs. In this chapter, we will give a brief overview of the CD200-CD200R axis, including the biology behind CD200-CD200R interaction and the role(s) it plays in tumor microenvironment and tumor growth, and activation/effector functions of T cells. We will also discuss CD200-CD200R's role as potential checkpoint molecules for cancer immunotherapy. Further investigation of the CD200-CD200R pathway will not only advance our understanding of tumor pathogenesis and immunity but also provide the rationale for CD200-CD200R-targeted immunotherapy of human cancer.

Abstract KN01: Dissecting antitumor immune response in ovarian cancer

Abstract In epithelial ovarian cancer (EOC), intratumoral or intraepithelial tumor infiltrating lymphocytes (TILs), i.e. T cells specifically infiltrating tumor islets, occur in approximately half of the patients and correlate with longer survival. As with many other solid tumors, despite promising results in mouse models, response of EOC to PD-1 blockade has been quite limited in the clinic, but the mechanisms underlying therapeutic failures in immunoreactive human tumors harboring TILs are not well understood. Our work has sought to answer the following key questions: a) whether ovarian TILs are tumor-specific; b) if so, what are the relevant antigens recognized by TILs; c) whether the PD-1/PD-L1 pathway is relevant; d) if so, what are the mechanisms underlying response to PD-1 blockade; and finally e) what mechanisms govern TIL recruitment and engraftment. The data presented will show that there is indeed a spontaneous and tumor-specific T-cell response in a proportion of EOC, accurately heralded by intraepithelial CD8+ TILs. Tumor-specific antigens will be discussed. Our work also shows that the PD-1/PD-L1 is a central immune checkpoint pathway in these tumors. However, only a fraction of such tumors with ieTILs respond to PD-1. We dissected the mechanisms underlying response to PD-1 blockade and therapeutic approaches to enhance response to PD-1 in human and mouse preclinical models. Therapeutic approaches to achieve effective immunotherapy in ovarian cancer will be discussed, including combination checkpoint blockade, adoptive T cell therapy and new rational combinations. Finally, successful T-cell engraftment in ovarian cancer requires tumor cell-derived CCL5 and is amplified by myeloid cell-secreted IFNg-inducible CXCL9, dependent on activation of tumor-infiltrating lymphocytes (TILs). Spontaneous, epigenetically-mediated loss or stable knockdown of ccl5 leads to CD8+ T-cell loss and immune desertification in the mouse, while forced ccl5 expression prevents loss of cxcl9 and TILs, and leads to attenuated tumor growth. Accordingly, CCL5-CXCL9 co-expression reveals immunoreactive tumors with prolonged survival and response to PD-1 blockade. The impact of homologous recombination and BRCA loss on the establishment of immunoreactive tumors will be also discussed. Citation Format: George Coukos. Dissecting antitumor immune response in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr KN01. doi:10.1158/1535-7163.TARG-19-KN01

Tumor-Specific Regulatory T Cells from the Bone Marrow Orchestrate Antitumor Immunity in Breast Cancer.

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen-reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen-specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector-encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.

Listeria monocytogenes cancer vaccines: bridging innate and adaptive immunity.

Immunotherapy has emerged as a promising cancer treatment, however success in only select clinical indications underscores the need for novel approaches. Recently Listeria monocytogenes-based vaccines have been developed to drive tumor specific T-cell responses. Here, we discuss recent preclinical studies using L. monocytogenes vaccines, innate immune pathways that influence T-cell priming, and new vaccine strategies in clinical trials. Recent studies indicate that in addition to inducing antigen specific T-cell responses, L. monocytogenes vaccines remodel the TME. In addition, several innate immune pathways influence adaptive immune responses to L. monocytogenes and modulating these pathways holds promise to enhance anti-tumor T-cell responses. The interplay between innate and adaptive immune responses to L. monocytogenes is poorly understood. Understanding these interactions will facilitate the design of better anti-cancer vaccines and improved use of combination therapies.

Maria-I: A Deep-Learning Approach for Accurate Prediction of MHC Class I Tumor Neoantigen Presentation

Background: Therapeutic cancer vaccines targeting neoantigens have shown promise in early phase clinical trials for inducing tumor-specific T-cell responses in diverse tumor types. Identification of the small number of optimal cancer vaccine targets is essential to limit cost and improve efficacy of patient-specific vaccine design. Therefore, there is a need to narrow neoantigen selection to those peptides with the highest potential to induce anti-tumor immune responses, and to maximize the associated clinical benefits. The presentation of these antigens by Major Histocompatibility Complex (MHC) class I is key for prioritizing candidates. Previous algorithms to predict antigen presentation have had limited success and have primarily been trained on in-vitro binding affinity assays, or are commercial platforms not widely available for use. Methods: We previously developed MARIA (MHC Analysis with Recurrent Integrated Architecture) as a method for predicting MHC-II neoantigens [Chen B et al. 2017 ASH, and Chen et al. 2019, Nature Biotechnology, in press]; here we adapt and develop the same MARIA design for MHC-I and benchmark its performance. Specifically, we used deep-learning for predicting the likelihood of neoantigen presentation trained on presented lymphoma peptides as identified by mass-spectrometry (LC-MS/MS). MARIA-I was initially trained on >47,000 ligands we previously identified by mass-spectrometry through antigen presentation profiling of patients with mantle cell lymphomas [Khodadoust et al. 2017, Nature]. Recurrent neural network (RNN) layers within MARIA-I integrate 4 key features: peptide sequence-MHC key residues relationship, peptide-MHC binding, peptide cleavage pattern, and gene expression. Results: We internally validated MARIA-I using 10-fold cross validation, where we observed an average AUC of 0.99 for the integrated model (Fig 1a); the peptide-MHC key residues relationships conferred the largest contribution to MARIA-I performance. We also further tested MARIA-I using an external dataset of ~19,000 diverse peptides from MHC-I antigen presentation profiles of four cancer cell lines (ACC1143, HCT116, HCC1937, and SUP-B15). MARIA-I maintains above 0.92 AUC across four diverse cancer types (AUC = 0.92, 0.92, 0.96, and 0.95, respectively). Since presentation of immunogenic peptides is essential for activation of T cell effector functions, we separately tested MARIA-I's ability to identify CD8 tumor infiltrating lymphocyte responses using corresponding data from 62 patients and 7587 neoantigens [Parkhurst et al. 2019, Cancer Discovery]. Despite MARIA-I not being trained on T cell reactivity data, neoantigens known to elicit positive T cell responses had significantly higher MARIA-I scores (Fig 1b, p=6.92e-5, Mann-Whitney U test). Conclusion: MARIA-I enables accurate prediction of neoantigen presentation for MHC class I at scale. Given its generalizability, we expect MARIA-I to yield insights for development of therapeutic cancer vaccines as well as applications in transplantation and autoimmune pathology. Figure 1. MARIA-I performance on antigen presentation and identifying immunogenic peptides. a) MARIA-I and sub-model predictors evaluated on 10% held-out validation set. Merging peptide sequence, gene expression, binding, and cleavage scores yielded improvements in performance as compared to each predictor's individual ability. b) MARIA-I assigns significantly higher scores to peptides known to elicit positive CD8 T cell response (p=6.92e-5, Mann-Whitney U test). Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Davis:Vir Biotechnology: Consultancy, Equity Ownership, Honoraria; PACT Bio: Consultancy, Equity Ownership, Honoraria; Adicet Inc: Consultancy, Equity Ownership, Honoraria; Chuga Pharmabody: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Atreca: Consultancy, Equity Ownership, Honoraria; Juno: Consultancy, Equity Ownership, Honoraria. Levy:Five Prime: Membership on an entity's Board of Directors or advisory committees; Corvus: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Nohla: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees. Altman:Personalis: Consultancy; Pfizer: Consultancy; Karius: Consultancy.

RARE-26. WHOLE GENOME SEQUENCING OF AN OSSEOUS METASTASIS DURING CHECKPOINT-CONTROLLED INTRACRANIAL GLIOBLASTOMA REVEALS NEW INSIGHTS INTO POTENTIAL MECHANISMS OF IMMUNE ESCAPE

Abstract Glioblastoma (GBM) has a devastating prognosis and recent advances in the treatment of a variety of cancer entities, e.g. through checkpoint inhibition, could so far not be translated into improved outcome in newly-diagnosed GBM. Characterizing rare cases of peripheral metastases which succeeded in overcoming immune control, can help to understand the mechanisms of immune escape. Here we describe the first reported case of a detailed genetic and immunological characterization of a peripheral bone metastasis from a GBM which was controlled intracranially by anti-PD1 checkpoint inhibition We performed whole genome sequencing (WGS) of the primary- and recurrent tumor, as well as the bone metastasis. Genomic data was analyzed for copy number variations and mutational profiles. In addition, immune monitoring with flow cytometric phenotyping and next-generation sequencing of the peripheral T-cell repertoire was used. A 70-year old patient developed multiple osseous metastases in the spine, while his IDHwt GBM recurrence was immunologically controlled with checkpoint inhibition. Biopsy confirmed peripheral GBM metastases. Immunophenotyping reflected the effective activation of the peripheral T-cell response, with, however, simultaneous upregulation of regulatory T-cells during disease progression. WGS sequencing demonstrated a distinct molecular profile of the GBM metastasis, with amplifications in chromosome 3 and 9, as well as genomic loss on chromosomes 4, 10 and 11. The peripheral metastasis was distinguished by mutations in mismatch repair genes, such as MSH4 and MLH1, associated with a hypermutated phenotype. Among the mutated genes we found potential candidates involved in immune escape of circulating tumor cells. This case represents a unique opportunity to analyze potential mechanisms of GBM-mediated immune escape during immune activation with anti-PD1 checkpoint therapy. It highlights the fact, that although an effective, disinhibited immune response can control the cranial GBM disease, hypermutated tumor clones can evade the tumor-specific T-cell response and disseminate to distant organs.

Pathogen Molecular Pattern Receptor Agonists: Treating Cancer by Mimicking Infection.

Immunotherapies such as checkpoint blockade have achieved durable benefits for patients with advanced stage cancer and have changed treatment paradigms. However, these therapies rely on a patient's own a priori primed tumor-specific T cells, limiting their efficacy to a subset of patients. Because checkpoint blockade is most effective in patients with inflamed or "hot" tumors, a priority in the field is learning how to "turn cold tumors hot." Inflammation is generally initiated by innate immune cells, which receive signals through pattern recognition receptors (PRR)-a diverse family of receptors that sense conserved molecular patterns on pathogens, alarming the immune system of an invading microbe. Their immunostimulatory properties can reprogram the immune suppressive tumor microenvironment and activate antigen-presenting cells to present tumors antigens, driving de novo tumor-specific T-cell responses. These features, among others, make PRR-targeting therapies an attractive strategy in immuno-oncology. Here, we discuss mechanisms of PRR activation, highlighting ongoing clinical trials and recent preclinical advances focused on therapeutically targeting PRRs to treat cancer.

1423P - Inferring the correlation between incidence rates of melanoma and the average tumour-specific epitope binding ability of HLA class I molecules in different populations

BackgroundHuman Leucocyte Antigen (HLA) molecules are encoded by the most polymorphic genes in the human genome. The genetic variation of these genes are considerable across different geographic subpopulations. We hypothesised that this genetic variation might contribute to the risk of melanoma both at population and subject level. MethodsWe developed a cancer risk predictor based on the complete HLA class I genotype of individuals. The HLA-score, used in the predictor describes the ability of the HLA class I alleles of an individual to bind epitopes derived from 48 selected tumor antigens as an indicator of the breadth of the tumor-specific T-cell responses. We collected HLA data for subjects from 20 different geographic regions (ethnic populations) (n=3278) as well as the corresponding melanoma incidence rates. The average HLA-scores were compared to the incidence rates. We also classified a mixed US population consisting of melanoma and healthy subjects based on their HLA-score. ResultsOn population level, we found significant correlation between the incidence rates of melanoma and average HLA-scores in different geographic regions (R2 = 0.5005; p<0.001; n=20; df=18). The highest average HLA-scores (range 75-140) were obtained for the Far East Asian and Pacific regions, where the incidence rates are low (0.4-3.4 per 100,000 per year). The lowest average HLA-scores (range 50-90) were obtained in the European and US regions, where the rates are high (12.6-13.8 per 100,000 per year). On subject level, the risk ratio between the riskiest (HLA-score <34) and the most protected groups (HLA-score ≥96) was 5.69 comparing the top and bottom 20% of the HLA-score distribution (p<0.05). These HLA-score ranges are consistent with the threshold values separating populations with low and high incidence rates of melanoma. ConclusionsBy developing a novel HLA-score determined by autologous HLA allele binding epitopes of tumor antigens, we showed that individuals with HLA allele sets supporting broader tumor-specific T-cell responses have lower risk of developing melanoma. These results imply that the HLA genotype and HLA-score could be used to determine the immunogenetic risk of melanoma. Legal entity responsible for the studyTreos Bio Zrt. FundingTreos Bio Zrt. DisclosureL. Molnar: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. J. Toth: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. O. Lorincz: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. Z. Csiszovszki: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. P. Pales: Full / Part-time employment: Treos Bio Ltd. K. Pántya: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. M. Megyesi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. E. Somogyi: Shareholder / Stockholder / Stock options, Full / Part-time employment: Treos Bio Ltd. E.R. Tőke: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Treos Bio Ltd. All other authors have declared no conflicts of interest.

PD-L1 blockade engages tumor-infiltrating lymphocytes to co-express targetable activating and inhibitory receptors

BackgroundThe clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination immunotherapy offers an attractive avenue to develop more effective cancer therapies by improving the efficacy and duration of the tumor-specific T-cell response. Here, we aimed at deciphering the mechanisms governing the response to PD-1/PD-L1 checkpoint blockade to support the rational design of combination immunotherapy.MethodsMice bearing subcutaneous MC-38 tumors were treated with blocking PD-L1 antibodies. To establish high-dimensional immune signatures of immunotherapy-specific responses, the tumor microenvironment was analyzed by CyTOF mass cytometry using 38 cellular markers. Findings were further examined and validated by flow cytometry and by functional in vivo experiments. Immune profiling was extended to the tumor microenvironment of colorectal cancer patients.ResultsPD-L1 blockade induced selectively the expansion of tumor-infiltrating CD4+ and CD8+ T-cell subsets, co-expressing both activating (ICOS) and inhibitory (LAG-3, PD-1) molecules. By therapeutically co-targeting these molecules on the TAI cell subsets in vivo by agonistic and antagonist antibodies, we were able to enhance PD-L1 blockade therapy as evidenced by an increased number of TAI cells within the tumor micro-environment and improved tumor protection. Moreover, TAI cells were also found in the tumor-microenvironment of colorectal cancer patients.ConclusionsThis study shows the presence of T cell subsets in the tumor micro-environment expressing both activating and inhibitory receptors. These TAI cells can be targeted by combined immunotherapy leading to improved survival.

Therapeutic vaccine strategies to induce tumor-specific T-cell responses.

Haploidentical stem cell transplantation is increasingly used worldwide as an alternative donor transplantation method. Although novel preparative regimens and T-cell deletion techniques have improved engraftment rates and viral safety, relapses of the underlying leukemia/lymphoma are still frequent, thus representing a significant and unsolved problem. Recent technological advances now enable us to individually decipher the MHC-associated immunopeptidome of cancer cells in reasonable time. These tumor-specific peptides can then be used to skew the early immune reconstitution toward anti-leukemia T-cell responses. In this meeting contribution, we summarize recent innovations in the field and present preliminary data on using this technique for cancer epitope discovery in a paradigmatic pediatric brain tumor with very low mutational burden.

A Sampling of Highlights from the Literature

Prostaglandin E2 (from WikiMedia Commons)Neutrophils can be immunosuppressive when located in the microenvironment of tumors. GM-CSF in the tumor microenvironment stimulates STAT5-upregulation of fatty-acid transporter protein FATP2 primarily on tumor neutrophils. This increases arachidonic acid uptake and the generation of PGE2, with subsequent suppression of tumor-specific T-cell responses. By combining inhibition of FATP2 with blockade of CTLA-4, tumor growth can be effectively inhibited in two mouse tumor models.Veglia F, …, Gabrilovich DI. Nature 2019 Apr 17;569:73–8.Neo open reading frame peptides (from Koster et al., Fig. 2)Cancer cell genomes can contain frameshift mutations, from which long neopeptides can be translated. Examination of over 10,000 TGCA tumor samples finds 143,444 frame-shift mutations, and 70,000 unique peptides ≥ 10 amino acids. Tumor-drivers are commonly frame-shifted genes. The neo-open-reading-frame peptides (NOPs) converge on common peptide sequences, in that by including only 6 driver genes, 1244 new peptides could be found in 30% of the patients. NOPs offer a new source of commonly shared potential neoantigens that could inform production of an “off-the-shelf” library of vaccine reagents.Koster J and Plasterk RHA. Sci Rep 2019 Apr 29;9:6577.Antibody binding sites on PD-1 (from Fenwick et al., Fig. 2F)Clinically useful mAbs to PD-1 block the PD-L1 binding site and thereby block the inhibition signal. Fenwick and colleagues find two new mAbs that bind to PD-1, but structural analysis indicates that binding is on locations away from the PD-1 binding site. The nonblocking mAbs act through the CD28 coreceptor, restoring signaling through the AKT–NF-κB pathway, and helping further rescue exhausted T cells. Combining both types of antibodies in tumor models enhances tumor clearance over monotherapy.Fenwick C, …, Pantaleo G. J Exp Med 2019 May 23. DOI: 10.1084/jem.20182359.Effects of DDR and PD-L1 inhibition (from Sen et al., Fig. 7E)Immune checkpoint blockade is successful only in some cases of small cell lung cancer (SCLC). Sen et al. find that FDA-approved small molecule inhibitors of CHK1 and PARP, which participate in the DNA damage response, can enhance PD-L1 blockade of the CD8+ T cell antitumor response. This is mediated in part through activation of the STING pathway that leads to increased production of T cell chemoattractants like CXCL10 and CCL5. The results suggest dual therapy could improve immunotherapy in SCLC.Sen T, …, Byers LA. Cancer Discov 2019 May 1;9:646–61.Melanoma-associated lymph node (from Broggi et al., Fig. 6A)Two papers show the promise of using tumor-draining lymph node exudate as the substrate for tumor biomarkers and evaluation of antitumor immune responses. Garcia-Silva et al. make use of extracellular vesicles (EVs) to detect markers of melanoma progression and the status of BRAF mutations. Broggi et al. compare plasma to draining lymph node exudate and find the exudate and EVs to be enriched in metastatic tumor–derived factors with protein signatures that can differentiate early from advanced metastasis and provide insights into tumor interactions with the immune system.García-Silva S, …, Peinado H. J Exp Med 2019 MAY 6;216: 1061–70.Broggi MAS, …, Swartz MA. J Exp Med 2019 May 6;216:1091–107.Moderately differentiated gastric tumor (by Nephron via Wikimedia Commons)The ramifications of PD-1 blockade on regulatory T cells (Tregs) are not clear. Almost 10% of patients with gastric cancer treated with anti–PD-1 respond with rapid progression of their tumors. In these patients, treatment increases the proliferative rate and immune suppressive activity of their Tregs. Tregs from PD-1–deficient mice lack PD-1 and both proliferate strongly and have enhanced immunosuppressive activity of antitumor responses. This association of hyperprogressive disease with PD-1 treatment suggests that Tregs be routinely monitored during checkpoint blockade therapy to identify this patient subset.Kamada T, …, Nishikawa H. Proc Natl Acad Sci U S A 2019 May 14;116:9999–10008.

Abstract CT217: An open-label, Phase I/IIa study of VB10.NEO (DIRECT-01) in combination with checkpoint blockade in patients with locally advanced or metastatic solid tumors including melanoma, NSCLC, renal cell carcinoma, urothelial cancer or SSCHN

Abstract BACKGROUND: Neoantigens are ideal for developing personalized cancer vaccines as they are tumor specific and not subject to central tolerance. Targeting neoantigens to generate potent tumor-specific T-cell responses has shown both promising preclinical efficacy as well as clinical responses. VB10.NEO is a DNA plasmid vaccine with intrinsic adjuvant effect designed for efficient delivery of personalized neoepitopes and can hold up unique neoepitopes specific for each patient’s tumor. Here we describe VB N-01 (DIRECT-01), an open-label phase I/IIa study, designed to evaluate the safety, immunogenicity and efficacy of administrating personalized VB10.NEO in combination with checkpoint blockade in up to 40 patients with locally advanced or metastatic solid tumors including melanoma, NSCLC, renal cell carcinoma, urothelial cancer or head and neck cancer. The study is open at 3 clinical study sites in Germany (NCT03548467). METHODS: The primary objectives are to assess the safety/tolerability of VB10.NEO immunotherapy and determine the feasibility of the VB10.NEO vaccine manufacturing. Secondary objectives are to make a preliminary assessment of the efficacy, to assess the immunogenicity of VB10.NEO immunotherapy, and the correlation between immunological response and clinical efficacy. Eligible patients who have not achieved a confirmed complete response by week 12 on checkpoint blockade as respective standard of care (SOC) will be enrolled in the study. A tumor biopsy and blood sample will be collected and used to identify the patient-specific tumor mutations. A personalized VB10.NEO holding up to 20 unique neoantigens will be designed based on Vaccibody’s proprietary neoepitope selection algorithm NeoSelect™. Patients will receive multiple vaccinations of VB10.NEO at pre-specified time points; an induction phase consisting of 3 injections during the first 6 weeks followed by a maintenance phase with vaccinations every 4th week up to one year from first immunization. VB10.NEO will be administered IM using the PharmaJet® Stratis Needle-free Injection System. A 24 month follow up period will follow the treatment phase. Patients will be assessed for safety, including AEs, vital signs, safety laboratory assessments and ECOG status. Tumor response will be assessed according to the iRECIST criteria. The study aims to characterize the cellular immune response against the individual neoepitopes by IFNγ ELISpot or other immune assays. A descriptive analysis of serial biopsies will also be assessed, including analysis of the mutagenic landscape by exome and RNA sequencing, and IHC to study tumor microenvironment. The trial is currently recruiting patients and initial accrual goal was meet. Citation Format: Jurgen Krauss, Angela Krackhardt, Elke Jager, Anja Williams, Hedda Wold, Lisa Gerner, Monika Sekelja, Karoline Schjetne, Agnete B. Fredriksen, Mads Axelsen. An open-label, Phase I/IIa study of VB10.NEO (DIRECT-01) in combination with checkpoint blockade in patients with locally advanced or metastatic solid tumors including melanoma, NSCLC, renal cell carcinoma, urothelial cancer or SSCHN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT217.

Functional characterization of tumor antigen-specific T-cells isolated from the tumor microenvironment of sleeping beauty induced murine glioma models.

Diffuse Gliomas represent 80% of brain tumors with an average survival of the most aggressive form glioblastoma (GBM) 15-22 months from the time of diagnosis. The current standard of care includes tumor resection, chemotherapy and radiation, nevertheless, the incidence of recurrence remains high and there is a critical need for developing new therapeutic strategies. T-cell mediated immunotherapy that triggers an anti-tumor T cell-mediated memory response is a promising approach since it will not only attack the primary tumor but also prevent recurrence. Multiple immunotherapeutic strategies against glioma are currently being tested in clinical trials. We have developed an immune-mediated gene therapy (Thymidine kinase plus Fms-like tyrosine kinase 3 ligand: TK/Flt3L) which induces a robust anti-tumor T cell response leading to tumor regression, long-term survival and immunological memory in GBM models. Efficacy of the anti-glioma T cell therapy is determined by anti-tumor specific effector T cells. Therefore, assessing effector T cell activation status and function are critical readouts for determining the effectiveness of the therapy. Here, we detail methodologies to evaluate tumor specific T-cell responses using a genetically engineered Sleeping Beauty transposase-mediated glioma model. We first describe the glioma model and the generation of neurospheres (NS) that express the surrogate antigen cOVA. Then, we describe functional assays to determine anti-tumor T-cell response.

Determination of the immunogenetic risk of developing cancer.

e13132 Background: Association between certain HLA types and cancer is well known. We hypothesized that the number of epitopes of tumor antigens presented by autologous HLAs characterizes a patient’s capacity to kill tumor cells. These CD8+ T cell epitopes are presented by 6 out of &gt;13,000 known HLA class I alleles and induce extremely variable tumor-specific T-cell responses. Methods: We predicted HLA-binding epitopes from 48 frequently expressed tumor antigens in subjects characterized with 6 HLA class I alleles. To develop the “HLA Score” cancer risk predictor we used epitopes binding to multiple HLAs of a subject. The predictor was trained on 5,789 non-American subjects. To identify populations with high and low immunogenetic risk to develop cancer we compared the HLA Score of American cancer subjects to a general population of American subjects (1,400 subjects). The performance of the predictor was characterized with AUC and Risk Ratio. Due to Bonferroni correction, AUC values with p-value p&lt;0.007 was accepted as significant. Results: “HLA Score” predictor significantly separated cancer patients from the general population in six out of the seven investigated cancer types. The Risk Ratios between the most protected and most at risk subpopulations ranged between 2.38 and 5.67 (Table). Cancer risk prediction with HLA Score. Conclusions: Subjects with certain HLA class I alleles have high risk of developing cancer. The novel “HLA Score” predictor we introduced here could complement current testing used for determination of the genetic risk of cancer.[Table: see text]

Programmed Cell Death 1 (PD-1) Inhibitors in Renal Transplant Patients with Advanced Cancer: A Double-Edged Sword?

Given advancements in cancer immunity, cancer treatment has gained breakthrough developments. Immune checkpoint inhibitors, such as programmed cell death 1 (PD-1) inhibitors, are the most promising drugs in the field and have been approved to treat various types of cancer, such as metastatic melanoma, head and neck squamous cell carcinoma, and urothelial carcinoma. However, whether PD-1 inhibitors should be administered to renal transplant patients with advanced cancer remains unclear because the T-cells produced after administration of these inhibitors act against not only tumor antigens but also donor alloantigens. Thus, the use of PD-1 inhibitors in kidney-transplanted patients with advanced cancer is limited on account of the high risk of graft failure due to acute rejection. Hence, finding optimal treatment regimens to enhance the tumor-specific T-cell response and decrease T-cell-mediated alloreactivity after administration of a PD-1 inhibitor is necessary. Thus far, no recommendations for the use of PD-1 inhibitors to treat cancer in renal transplant patients are yet available, and very few cases reporting kidney-transplanted patients treated with PD-1 inhibitors are available in the literature. Therefore, in this work, we review the published cases and suggest feasible approaches for renal transplant patients with advanced malignancy treated by a PD-1 inhibitor. Of the 22 cases we obtained, four patients maintained intact grafts without tumor progression after treatment with a PD-1 inhibitor. Among these patients, one maintained steroid dose before initiation of anti-PD1, two received immunosuppressive regimens with low-dose steroid and calcineurin inhibitor (CNI)-elimination with sirolimus before initiation of anti-PD-1 therapy, and one received combined anti-PD-1, anti-vascular endothelial growth factor (VEGF), and chemotherapy with unchanged immunosuppressive regimens. mammalian target of rapamycin (mTOR) inhibitors and anti-VEGF may act as regulators of tumor-specific and allogenic T-cells. However, more studies are necessary to explore the optimal therapy and ensure the safety and efficacy of PD-1 inhibitors in kidney-transplanted patients.

Immune pressure against ovarian cancer depends on antigen-specific TRM T cells

Abstract Conventional memory T cells classically include central memory T (TCM) cells, residing in lymphoid organs, and effector memory T (TEM) cells, circulating through various tissues. Recently, a novel population of memory T cells has gained interest, namely tissue resident memory T (TRM) cells, which persist in tissues and do not recirculate. It is described that these cells reside in human tumors playing a role in tumor-specific T-cell responses. We found by flow cytometry that between 50%–80% of the CD8+ T cells in human ovarian carcinomas are CD103+CD69+TRM cells. RNA-seq of TRM and their re-circulating counterparts from 7 different human carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector (GZMB, IFNG) and exhaustion (PD-1, TIM3) markers, along with transcription factors and signaling molecules likely involved in the acquisition of the TRM phenotype. Unexpectedly, we found very little overlap between the TCR repertoire of both populations in multiple tumors, and TRM T cells consistently showed significantly higher clonality. Tumor antigen-specific TRM T cells intratumorally transferred into syngeneic mice were more effective at delaying tumor growth compared with their tumor antigen-specific recirculating counterparts. Finally, both the acquisition and the maintenance of a TRM phenotype within the CD8 T cell compartment at tumor beds are dependent on exposure to tumor cognate antigen. Together, our data indicate that TRM CD8+ T cells, but not their CD103− counterparts, represent tumor antigen-specific effector lymphocytes actively exerting anti-tumor immune pressure in the ovarian cancer microenvironment.

IL2/Anti-IL2 Complex Combined with CTLA-4, But Not PD-1, Blockade Rescues Antitumor NK Cell Function by Regulatory T-cell Modulation.

High-dose IL2 immunotherapy can induce long-lasting cancer regression but is toxic and insufficiently efficacious. Improvements are obtained with IL2/anti-IL2 complexes (IL2Cx), which redirect IL2 action to CD8+ T and natural killer (NK) cells. Here, we evaluated the efficacy of combining IL2Cx with blockade of inhibitory immune pathways. In an autochthonous lung adenocarcinoma model, we show that the IL2Cx/anti-PD-1 combination increases CD8+ T-cell infiltration of the lung and controls tumor growth. In the B16-OVA model, which is resistant to checkpoint inhibition, combination of IL2Cx with PD-1 or CTLA-4 pathway blockade reverses that resistance. Both combinations work by reinvigorating exhausted intratumoral CD8+ T cells and by increasing the breadth of tumor-specific T-cell responses. However, only the IL2Cx/anti-CTLA-4 combination is able to rescue NK cell antitumor function by modulating intratumoral regulatory T cells. Overall, association of IL2Cx with PD-1 or CTLA-4 pathway blockade acts by different cellular mechanisms, paving the way for the rational design of combinatorial antitumor therapies.

Abstract P2-09-04: Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer

Abstract Background: Recent studies have demonstrated that the number of tumor infiltrating lymphocytes (TILs) positively correlates with outcome and response to chemotherapy in patients with HER2+ and Triple-Negative Breast Cancer (TNBC). Furthermore, first studies of immune-checkpoint inhibitors showed promising results in those patients. However, the targets of those TILs remain unknown. Neoantigens, which arise in the process of tumorigenesis, appear as potential targets. They can elicit high avidity, tumor-specific T-cell responses. Thus, it is the aim of our study to ascertainif these TILs are directed against tumor-specific mutations. Methods: TILs from breast cancer biopsies taken at the time point of diagnosis were expanded by unspecific stimulation. Additionally, we used the Gentle Macs Dissociator in combination with flow cytometry to investigate the number of TILs in the tumor tissue. Furthermore, we performed whole-genome sequencing of tumor tissue and as reference autologous blood cells to determine tumor-specific mutations. Mutations leading to a non-synonymous amino acid change were analyzed for RNA expression of the encoding gene as well as to determine potential neoantigens. Neoantigens were evaluated for their potential binding to the patient's specific HLA molecules. Peptides for potential neoantigens were synthesized, loaded onto autologous antigen presenting cells (APCs) and cocultured with TILs. All IFNγ producing T-cells were clonally expanded and retested for peptide specificity to identify neoantigen specific T-cell clones. Results: Our flow cytometric analysis of the tumor biopsy for more than 300 patients showed higher frequencies of TILs in TNBC as compared to other types of breast cancer or patients without malignancy. Screening for neoantigen specific T-cells in one patient led to identification of three peptide-specific CD4+ T-cell clones isolated from HER2+ breast cancer tissue taken at the time point of diagnosis. All T-cell clones specifically recognized the same tumor-specific mutation and not the wildtype counterpart. Furthermore, we demonstrated that these T-cell clones also recognized the endogenously expressed mutated antigen. This verified the ability of processing and presentation of the respective protein. Interestingly, we could also isolate a T-cell clone recognizing the same neoantigen in the resected tumor tissue after neoadjuvant therapy. Based on CDR3 sequencing we could prove that the four T-cell clones represented individual clones. This confirms the polyclonal nature of the immune response. Moreover, we showed that the same neoepitope was presented in two different HLA restriction molecules of the patient with three of the clones recognizing it in HLA-DPB1*0401 and one in HLA-DPB1*0201. These results further underline the immunogenicity of this neoantigen. Conclusion: In conclusion, our data demonstrate tumor-specificity of TILs in a patient with HER2+ breast cancer. Furthermore, we show the feasibility to identify individual cancer specific T-cell targets in breast cancer patients. These results may contribute to the development of targeted patient-specific immunotherapies in the future. Citation Format: Reimann H, Nguyen A, Hübner H, Erber R, Bausenwein J, Van der Meijden ED, Lux MP, Jud S, Griffioen M, Rauh C, Sanborn JZ, Benz SC, Rabizadeh S, Beckmann MW, Mackensen A, Rübner M, Fasching PA, Kremer AN. Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-04.