T-cell Immunogenicity Research Articles

Evaluation of T-cell proliferation and immunogenicity assays with genetically engineered chondrocytes, tissuegene-c

Background & Aim Background The recognition of knee osteoarthritis as potentially mediated by the body's own immune system and inflammatory response has led to the development of non-operative biologic management modalities. TissueGene-C (TG-C), genetically engineered allogenic human chondrocytes (GEC) transduced with a retroviral vector encoding TGF-β1 (designated hChonJb#7) and non-transduced allogeneic human chondrocytes (designated hChonJ), are a more recent development that target immune-mediated pathways. Results from Phase 1, 2, and 3 trials have shown the safety and efficacy of this injection. The purpose of this study was to rule out possibility of an allogeneic response or induction of T-cells capable of responding to chondrocyte antigens or human leukocyte antigens (HLAs) in patients managed with TG-C. We evaluated: 1) T-cell proliferation assay (CD8 +, IFN-γ +); 2) T-cell proliferation assay (CD4 +, IFN-γ +); 3) Panel Reactive Antibody Test on anti-HLA antibodies; 4) Anti-TGF-β1 antibody assay; 5) High-Sensitivity C-Reactive Protein (HSCRP) assay; and 6) Cytokine Assay. Methods, Results & Conclusion Methods Between 1 May 2011 and 31 October 2012, we enrolled 102 patients into a multicenter, double-blinded, placebo-controlled, randomized study of adults with Kellgren-Lawrence grade III osteoarthritis of the knee (NCT01221441) based at 5 institutions. We injected TG-C into the intra-articular knee space. Immunogenicity was evaluated by assays targeting immune pathways. Peripheral blood mononuclear cells (PBMC) from study and control arms were isolated and challenged with TG-C for T-cell proliferation assays. Blood was analyzed for antibody formation, marker protein, and cytokines: 1) GM-CSF; 2) IL-10; 3) IL-1α; 4) IL-2; 5) IL-4; 6) IL-5; 7) IL-6; 8) Interferon (IFN)-γ and 9) TNF-α. Results Laboratory analyses revealed: no induced T-cell response; no clinically significant changes in IFN-γ producing T-cells or HSCRP levels; and no antibody formation to donor HLA and TGF-β1 proteins. No clinically significant changes were observed in the 9 cytokines assessed (GM-CSF, IL-10, IL-1a, IL-2, IL-4, IL-5, IL-6, IFN-γ and TNF-α). Conclusion These results indicate that both hChonJ or hChonJb#7 do not induce T-cell response to the previously TG-C exposed PBMCs. No evidence of immune response in Phase 2 study was noted. Patients exposed to TG-C did not respond to TG-C with in vitro exposure. It can be concluded that TG-C is not immunogenic to humans, and is therefore likely safe for repeat injections. The recognition of knee osteoarthritis as potentially mediated by the body's own immune system and inflammatory response has led to the development of non-operative biologic management modalities. TissueGene-C (TG-C), genetically engineered allogenic human chondrocytes (GEC) transduced with a retroviral vector encoding TGF-β1 (designated hChonJb#7) and non-transduced allogeneic human chondrocytes (designated hChonJ), are a more recent development that target immune-mediated pathways. Results from Phase 1, 2, and 3 trials have shown the safety and efficacy of this injection. The purpose of this study was to rule out possibility of an allogeneic response or induction of T-cells capable of responding to chondrocyte antigens or human leukocyte antigens (HLAs) in patients managed with TG-C. We evaluated: 1) T-cell proliferation assay (CD8 +, IFN-γ +); 2) T-cell proliferation assay (CD4 +, IFN-γ +); 3) Panel Reactive Antibody Test on anti-HLA antibodies; 4) Anti-TGF-β1 antibody assay; 5) High-Sensitivity C-Reactive Protein (HSCRP) assay; and 6) Cytokine Assay. Between 1 May 2011 and 31 October 2012, we enrolled 102 patients into a multicenter, double-blinded, placebo-controlled, randomized study of adults with Kellgren-Lawrence grade III osteoarthritis of the knee (NCT01221441) based at 5 institutions. We injected TG-C into the intra-articular knee space. Immunogenicity was evaluated by assays targeting immune pathways. Peripheral blood mononuclear cells (PBMC) from study and control arms were isolated and challenged with TG-C for T-cell proliferation assays. Blood was analyzed for antibody formation, marker protein, and cytokines: 1) GM-CSF; 2) IL-10; 3) IL-1α; 4) IL-2; 5) IL-4; 6) IL-5; 7) IL-6; 8) Interferon (IFN)-γ and 9) TNF-α.

Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes.

Reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The immune mechanisms of protection against recurrent genital herpes remain to be fully elucidated. In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed HSV-2 envelope and tegument proteins. These viral protein antigens (Ags) were rationally selected for their ability to recall strong CD4+ and CD8+ T-cell responses from naturally "protected" asymptomatic individuals, who, despite being infected, never develop any recurrent herpetic disease. Out of the eight HSV-2 proteins, the envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene), and ribonucleotide reductase subunit 2 protein (RR2; encoded by the UL40 gene) produced significant protection against recurrent genital herpes. The RR2 protein, delivered either intramuscularly or intravaginally with CpG and alum adjuvants, (i) boosted the highest neutralizing antibodies, which appear to cross-react with both gB and gD, and (ii) enhanced the numbers of functional gamma interferon (IFN-γ)-producing CRTAM+ CFSE+ CD4+ and CRTAM+ CFSE+ CD8+ TRM cells, which express low levels of PD-1 and TIM-3 exhaustion markers and were localized to healed sites of the vaginal mucocutaneous (VM) tissues. The strong B- and T-cell immunogenicity of the RR2 protein was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In vivo depletion of either CD4+ or CD8+ T cells significantly abrogated the protection. Taken together, these preclinical results provide new insights into the immune mechanisms of protection against recurrent genital herpes and promote the tegument RR2 protein as a viable candidate Ag to be incorporated in future genital herpes therapeutic mucosal vaccines.IMPORTANCE Recurrent genital herpes is one of the most common sexually transmitted diseases, with a global prevalence of HSV-2 infection predicted to be over 536 million worldwide. Despite the availability of many intervention strategies, such as sexual behavior education, barrier methods, and the costly antiviral drug treatments, eliminating or at least reducing recurrent genital herpes remains a challenge. Currently, no FDA-approved therapeutic vaccines are available. In this preclinical study, we investigated the immunogenicity and protective efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed herpes envelope and tegument proteins. We discovered that similar to the dl5-29 vaccine, based on a replication-defective HSV-2 mutant virus, which has been recently tested in clinical trials, the RR2 protein-based subunit vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with an increase in numbers of functional tissue-resident IFN-γ+ CRTAM+ CFSE+ CD4+ and IFN-γ+ CRTAM+ CFSE+ CD8+ TRM cells that infiltrate healed sites of the vaginal tissues. Our study sheds new light on the role of TRM cells in protection against recurrent genital herpes and promotes the RR2-based subunit therapeutic vaccine to be tested in the clinic.

In silico Designed Ebola Virus T-Cell Multi-Epitope DNA Vaccine Constructions Are Immunogenic in Mice.

Background: The lack of effective vaccines against Ebola virus initiates a search for new approaches to overcoming this problem. The aim of the study was to design artificial polyepitope T-cell immunogens—candidate DNA vaccines against Ebola virus and to evaluate their capacity to induce a specific immune response in a laboratory animal model. Method: Design of two artificial polyepitope T-cell immunogens, one of which (EV.CTL) includes cytotoxic and the other (EV.Th)—T-helper epitopes of Ebola virus proteins was carried out using original TEpredict/PolyCTLDesigner software. Synthesized genes were cloned in pcDNA3.1 plasmid vector. Target gene expression was estimated by synthesis of specific mRNAs and proteins in cells transfected with recombinant plasmids. Immunogenicity of obtained DNA vaccine constructs was evaluated according to their capacity to induce T-cell response in BALB/c mice using IFNγ ELISpot and ICS. Results: We show that recombinant plasmids pEV.CTL and pEV.Th encoding artificial antigens provide synthesis of corresponding mRNAs and proteins in transfected cells, as well as induce specific responses both to CD4+ and CD8+ T-lymphocytes in immunized animals. Conclusions: The obtained recombinant plasmids can be regarded as promising DNA vaccine candidates in future studies of their capacity to induce cytotoxic and protective responses against Ebola virus.

Abstract B080: Improved neoantigen vaccine selection by combining prediction of pMHC presentation and T-cell epitopes

Abstract The OpenVax group has helped initiate two neoantigen vaccine clinical trials (NCT02721043, NCT02721043) at Mount Sinai based on a simple multiplicative ranking criterion which assigns equal weight to expression and predicted Class I MHC binding affinity of mutated peptides (1). This poster seeks to better ground our ranking method for selecting the contents of neoantigen vaccines in several sources of immunological data. We built a better model of MHC-I presentation on the cell surface by relating RNA expression and MHC affinity to pMHC ligands identified with mass spectrometry (2). Secondly, we trained a model of overall T-cell immunogenicity whose primary input is the predicted pMHC presentation score of any peptide-MHC combination, alongside other features such as similarity to the self proteome. This model is trained on T-cell response data deposited in the Immune Epitope Database (3). Lastly, we assembled a small dataset of peptide sequences used in neoantigen vaccine trials (1,4,5), which are labeled by whether they achieved a CD8+ or CD4+ T-cell response. This dataset allows us to explore several hypotheses about the relationship between immunogenic response and sequence similarity to both the self proteome and pathogenic proteomes.

Abstract B089: Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker

Abstract Precision cancer immunotherapy targeting mutations expressed by cancer cells has proven to effectively control the tumor of patients in multiple clinical trials (Sahin et al., Nature 2017; Ott et al., Nature 2017). However, the selection of immunogenic T-cell neo-epitopes remains challenging and many epitopes selected using traditional methodologies fail to induce effector T-cell responses. Poor performance may partially be due to inclusion of mutated epitopes cross-conserved with self-epitopes recognized by regulatory (Treg), anergic, or deleted T-cells. Vaccination with self-epitopes can lead to weak effector responses, active immune suppression, and toxicity due to immune-mediated adverse effects. In addition, most cancer vaccine studies focus on the selection of CD8 T-cell neo-epitopes due to an apparent lack of robust and accurate CD4 T-cell epitope prediction tools. We have developed Ancer, an integrated and streamlined neo-epitope selection pipeline, that accelerates the selection of both CD4 and CD8 T-cell neo-epitopes from next-generation sequencing (NGS) data. Ancer leverages EpiMatrix and JanusMatrix, predictive algorithms that have been extensively validated in prospective vaccine studies for infectious diseases (Moise et al., Hum Vaccines Immunother 2015; Wada et al., Sci Rep 2017). Distinctive features of Ancer are its ability to accurately predict Class II HLA ligands, or CD4 epitopes, with EpiMatrix, and to identify tolerated or Treg epitopes with JanusMatrix. In addition, screening candidate sequences with JanusMatrix enables to the removal of neo-epitopes that may trigger off-target events, which have in some cases abruptly halted the development of promising cancer therapies. Ancer was applied to NGS data derived from the BLCA bladder cancer cohort from The Cancer Genome Atlas (TCGA) database. On average, 55 out of 204 missense mutations in bladder cancer patients’ tumors met Ancer’s quality control standards, in an initial analysis carried out for a representative set of 11 patients. This subset of high-quality missense variants was then screened using Ancer settings defined by the unique HLA of each patient, to derive the best vaccine candidate sequences encompassing these mutations. A median number of 24 (interquartile range: 15-64) candidate sequences were generated for each patient under study. The time required to select sequences for all of the patients in this study was less than two days. This initial analysis of eleven BLCA bladder cancer cohort patients demonstrates the capacity of Ancer to define a sufficient number of candidate sequences for vaccinating bladder cancer patients in a precision immunotherapy setting. We also assessed Ancer’s ability to predict patient outcomes on a larger subset of 58 individuals. While the disease-free status of BLCA patients could not be explained by their tumor mutational burden (AUC = 0.55, p-value = 0.1328), nor by their load of missense mutations (AUC = 0.54, p-value = 0.1740), the number of neoepitopes highly different from self, as defined by Ancer, significantly segregated disease-free patients from patients who recurred or progressed (AUC = 0.68, p-value = 0.0214). These results suggest that defining the number of true neoepitopes using Ancer may represent a novel biomarker for more robust antitumor immune response and higher likelihood of disease-free survival.Our analysis of the BLCA cohort from the TCGA database showcases the value of Ancer in clinical settings. Ancer can be used to identify high-value candidate sequences for inclusion in personalized therapies while removing potentially tolerated or tolerogenic self-epitopes from consideration. Our next step will be to investigate whether Ancer-defined neoepitope load will serve as a biomarker for prognosis and response to therapy in the full BLCA cohort. Citation Format: Guilhem Richard, Randy F. Sweis, Leonard Moise, Matthew Ardito, William A. Martin, Gad Berdugo, Gary D. Steinberg, Anne S. De Groot. Application of precision cancer immunotherapy design tools to bladder cancer: Non-self-like neoepitopes as a prognostic biomarker [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B089.

Generation of Zika Virus-Specific T-Cells for Adoptive Immunotherapy

Background Zika virus (ZIKV) infection during pregnancy can cause severe birth defects, with no effective treatment. Adoptive transfer of virus-specific T-cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for ZIKV infection. However, this approach for ZIKV infection has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T-cells and characterized the cellular immune responses against ZIKV antigens. Methods T-cell products were generated from blood of ZIKV-exposed donors (n=3), ZIKV-naive adult donors (n=6), and umbilical cord blood (n=7) by stimulation with overlapping peptide libraries (15mers overlapping by 11 amino acids) spanning four ZIKV polyproteins (C, M, E, and NS1) using a GMP-compliant protocol. Results We successfully generated T-cells targeting ZIKV antigens with a mean 56.0-fold T cell expansion at 17 days for ZIKV-exposed donors and 21 days for ZIKV-naive donors and cord blood donors. The resultant products specifically recognized C, M, E, and NS1 proteins by IFN-γ ELISpot, with mean 142.5 spot forming cells (SFCs)/1 × 105 cells, 81.7 SFCs/1 × 105 cells, 132.1 SFCs/1 × 105 cells, and 180.7 SFCs/1 × 105 cells, respectively, compared to 5.4 SFCs/1 × 105 cells for irrelevant antigen (Fig 1). ZIKV-specific T-cells were polyclonal (16.2±2.8% CD3+CD4+ and 32.4±2.4% CD3+CD8+ T-cells) and expressed markers for both central and effector memory phenotype (3.5±0.6% CD45RO+CD62L+ and 48.1±1.9% CD45RO+CD62L–), with minimal expression of PD-1, Tim-3, Lag-3, and CTLA-4. Notably, both CD4+ and CD8+ subsets were polyfunctional comprising ZIKV-directed T-cell populations, which secreted multiple cytokines (16.7±0.6% CD4+IFN-γ+TNF-α+ and 7.4±0.4% CD8+IFN-γ+TNF-α+) as measured by intracellular cytokine staining. Moreover, multiplex cytokine assay demonstrated specific production of IFN-γ (928.7±239.8 pg/mL), TNF-α (1495.5±453.3 pg/mL), IL-2 (38.2±8.4 pg/mL), and GM-CSF (67.0±22.2 pg/mL) in response to ZIKV antigens. Importantly, ZIKV-specific T-cells selectively killed autologous monocytes infected with ZIKV confirming functionality of this unique T cell therapeutic. Epitope mapping using peptide arrays in IFN-γ ELISpot identified several novel HLA class I or class II-restricted epitopes within NS1, which is essential for viral replication and immune evasion. Conclusions Our findings demonstrate that it is feasible to generate potent ZIKV-specific T-cells from a variety of cell sources including virus naive donors for future clinical use in an “off the shelf’ setting.

In Silico screening of T-cell and B-cell Epitopes of Rotavirus VP7 and VP4 proteins for Effective Vaccine Design

Rotavirus is one of the deadliest causative agents of childhood diarrhea which causes half a million child death across the globe, mostly in developing countries. However, effective vaccine strategies against rotavirus are yet to be established to prevent these unwanted premature deaths. In this regard, in silico vaccine design for rotavirus could be a promising alternative for developing countries due to its efficiency in shortening valuable time and cost. The present study described an epitope-based peptide vaccine design against rotavirus, using a combination of T-cell and B-cell epitope predictions and molecular docking approach. To perform this, sequences of rotavirus VP7 and VP4 proteins were retrieved from the NCBI database and subjected to different bioinformatics tools to predict most immunogenic T-cell and B-cell epitopes. From the identified epitopes, the sequence VMSKRSRSL of VP7 and TQFTDFVSL of VP4 was identified as the most potential epitopes based on their antigenicity, conservancy and interaction with major histocompatability complex I (MHC-I) alleles. Moreover, the peptide VMSKRSRSL interacted with human leukocyte antigen, HLA-B*08:01 and TQFTDFVSL interacted with HLA-A*02:06 with considerable binding energy and affinity score. Combined population coverage for our identified epitopes was found 70.53% and 45.64% for world population and South Asian population respectively. All these results suggest that, the epitopes identified in this study could be a very good vaccine candidate for the strains of rotavirus circulating in Bangladesh. However, as this study is completely dependent on computational prediction algorithms, further in vivo screening is required to come up in a precise conclusion about these epitopes for effective rotavirus vaccination.
 Bangladesh J Microbiol, Volume 35 Number 1 June 2018, pp 45-55

Epitope Stealing as a Mechanism of Dominant Protection by HLA-DQ6 in Type 1 Diabetes.

The heterozygous DQ2/8 (DQA1*05:01-DQB1*02:01/DQA1*03:01-DQB1*03:02) genotype confers the highest risk in type 1 diabetes (T1D), whereas the DQ6/8 (DQA1*02:01-DQB1*06:02/DQA1*03:01-DQB1*03:02) genotype is protective. The mechanism of dominant protection by DQ6 (DQB1*06:02) is unknown. We tested the hypothesis that DQ6 interferes with peptide binding to DQ8 by competition for islet epitope ("epitope stealing") by analysis of the islet ligandome presented by HLA-DQ6/8 and -DQ8/8 on dendritic cells pulsed with islet autoantigens preproinsulin (PPI), GAD65, and IA-2, followed by competition assays using a newly established "epitope-stealing" HLA/peptide-binding assay. HLA-DQ ligandome analysis revealed a distinct DQ6 peptide-binding motif compared with the susceptible DQ2/8 molecules. PPI and IA-2 peptides were identified from DQ6, of DQ6/8 heterozygous dendritic cells, but no DQ8 islet peptides were retrieved. Insulin B6-23, a highly immunogenic CD4 T-cell epitope in patients with T1D, bound to both DQ6 and DQ8. Yet, binding of InsB6-23 to DQ8 was prevented by DQ6. We obtained first functional evidence of a mechanism of dominant protection from disease, in which HLA molecules associated with protection bind islet epitopes in a different, competing, HLA-binding register, leading to "epitope stealing" and conceivably diverting the immune response from islet epitopes presented by disease-susceptible HLA molecules in the absence of protective HLA.

A Designer Cross-reactive DNA Immunotherapeutic Vaccine that Targets Multiple MAGE-A Family Members Simultaneously for Cancer Therapy.

Cancer/testis antigens have emerged as attractive targets for cancer immunotherapy. Clinical studies have targeted MAGE-A3, a prototype antigen that is a member of the MAGE-A family of antigens, in melanoma and lung carcinoma. However, these studies have not yet had a significant impact due to poor CD8+ T-cell immunogenicity, platform toxicity, or perhaps limited target antigen availability. In this study, we develop an improved MAGE-A immunogen with cross-reactivity to multiple family members. In this study, we analyzed MAGE-A expression in The Cancer Genome Atlas and observed that many patients express multiple MAGE-A isoforms, not limited to MAGE-A3, simultaneously in diverse tumors. On the basis of this, we designed an optimized consensus MAGE-A DNA vaccine capable of cross-reacting with many MAGE-A isoforms, and tested immunogenicity and antitumor activity of this vaccine in a relevant autochthonous melanoma model. Immunization of this MAGE-A vaccine by electroporation in C57Bl/6 mice generated robust IFNγ and TNFα CD8+ T-cell responses as well as cytotoxic CD107a/IFNγ/T-bet triple-positive responses against multiple isoforms. Furthermore, this MAGE-A DNA immunogen generated a cross-reactive immune response in 14 of 15 genetically diverse, outbred mice. We tested the antitumor activity of this MAGE-A DNA vaccine in Tyr::CreER;BRAFCa/+;Ptenlox/lox transgenic mice that develop melanoma upon tamoxifen induction. The MAGE-A DNA therapeutic vaccine significantly slowed tumor growth and doubled median mouse survival. These results support the clinical use of consensus MAGE-A immunogens with the capacity to target multiple MAGE-A family members to prevent tumor immune escape.

Broad CD8+ T cell cross-recognition of distinct influenza A strains in humans

Newly-emerged and vaccine-mismatched influenza A viruses (IAVs) result in a rapid global spread of the virus due to minimal antibody-mediated immunity. In that case, established CD8+ T-cells can reduce disease severity. However, as mutations occur sporadically within immunogenic IAV-derived T-cell peptides, understanding of T-cell receptor (TCRαβ) cross-reactivity towards IAV variants is needed for a vaccine design. Here, we investigate TCRαβ cross-strain recognition across IAV variants within two immunodominant human IAV-specific CD8+ T-cell epitopes, HLA-B*37:01-restricted NP338-346 (B37-NP338) and HLA-A*01:01-restricted NP44-52 (A1-NP44). We find high abundance of cross-reactive TCRαβ clonotypes recognizing distinct IAV variants. Structures of the wild-type and variant peptides revealed preserved conformation of the bound peptides. Structures of a cross-reactive TCR-HLA-B37-NP338 complex suggest that the conserved conformation of the variants underpins TCR cross-reactivity. Overall, cross-reactive CD8+ T-cell responses, underpinned by conserved epitope structure, facilitates recognition of distinct IAV variants, thus CD8+ T-cell-targeted vaccines could provide protection across different IAV strains.

Adoptive T-Cell Therapy with TCL1-Specific TCR for B-Cell Lymphomas

Chimeric antigen receptor (CAR)-modified T-cell therapy targeting CD19 induces high response rates in patients with relapsed or refractory B-cell lymphomas. However, about 60% of patients experience primary or secondary resistance after CD19-targeted CAR T-cell therapy and a major of cause of failure appears to be due to loss of CD19 expression on the tumor. Therefore, novel targets for adoptive T-cell therapeutic approaches are needed to further improve clinical outcome in these patients.T-cell leukemia/lymphoma antigen1 (TCL1) is an oncoprotein that is overexpressed in multiple B-cell malignancies including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). Importantly, it has restricted expression in only a subset of B cells among normal tissues. We previously identified a TCL1-derived HLA-A2-binding epitope (TCL170-79 SLLPIMWQLY) that can be used to generate TCL1-specific CD8+ T cells from peripheral blood mononuclear cells of both HLA-A2+ normal donors and lymphoma patients. More importantly, we showed that the TCL1-specific CD8+ T cells lysed autologous primary lymphoma cells but not normal B cells (Weng et al. Blood 2012).To translate the above discovery into clinic, we cloned the T-cell receptor (TCR) alpha and beta chains from a TCL1-specific CD8+ T-cell clone and showed that this TCL1-TCR could be transduced into polyclonal donor T cells using a lentiviral system with a transduction efficiency of >40% as determined by TCL170-79 tetramer positive T cells. Furthermore, we demonstrated that the TCL1-TCR-transduced T cells recognized T2 cells pulsed with TCL170-79 peptide producing IFN- γ >8 ng/ml and IL-2 >350 ng/ml but were not reactive to control HIV-Gag peptide (IFN- γ <0.1 ng/ml and IL-2 <0.2 ng/ml). The TCL1-TCR-transduced T cells recognized TCL170-79 peptide pulsed onto T2 cells at a concentration of 1-10 nM (IL-2 >10 ng/ml) suggesting it has moderate to high avidity. Importantly, TCL1-TCR-transduced T cells lysed HLA-A2+ (up to 43% lysis of Mino and 25% lysis of Jeko-1 at 40:1 Effector:Target ratio) but not HLA-A2- lymphoma cell lines (5.5% lysis of HLA A2- Raji and 2.3% lysis of Daudi at 40:1 Effector:Target ratio). TCL1-TCR-transduced T cells were also cytotoxic to HLA-A2+ primary lymphoma tumor cells (up to 48% lysis of CLL, 43% lysis of FL, 41% lysis of DLBCL, 46% lysis of splenic marginal zone lymphoma, and 11% lysis of MCL at 40:1 Effector:Target ratio) but not normal B cells derived from the same patients. Lastly, TCL1-TCR transduced T cells showed high efficacy in in vivo models. Adoptive transfer of the TCL1-TCR-tranduced T cells significantly reduced lymphoma tumor growth and extended survival in Mino mantle cell lymphoma cell line xenograft model (48% survival in TCL1-TCR-T treated group vs. 12.5% survival in control group at 10 weeks n=7-8 mice/group; P=0.02).Collectively, our data suggest that the high expression in B-cell tumors, restricted expression in normal tissues, and presence of an immunogenic CD8 T-cell epitope, make TCL1 a target for T cell-based therapeutic approaches in multiple B-cell malignancies. Our results also demonstrate that the TCL1-specific TCR-transduced T cells may serve as a novel adoptive immunotherapy approach for the treatment of patients with various B-cell malignancies (including FL, MCL, DLBCL, CLL).Acknowledgments: This study is supported by MD Anderson Moon Shot Program and CPRIT and the National Natural Science Foundation of China Grant (No. 81570189) DisclosuresNeelapu:Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Analysis of the Multiple Myeloma HLA Peptidome Identifies a Naturally Presented Bcma-Derived Peptide As an Immunogenic T-Cell Epitope for Immunotherapeutic Approaches

The B-cell maturation antigen (BCMA) is selectively expressed by cells of the B-lineage, including multiple myeloma (MM) cells, and constitutes a promising target for immunotherapeutic approaches. At present, BCMA is being evaluated as target for immunotherapeutic approaches, such as CAR T cells and bispecific antibodies, which have demonstrated promising results in phase I clinical trials. The utilization of cytotoxic T cells bearing T-cell receptors against BCMA constitutes an alternative promising approach to target MM cells. Therefore, the identification of BCMA-derived peptides that are naturally presented by human leukocyte antigens (HLA) and thus can serve as target structures for CD8+ T cells, is indispensable.In a previous study, we characterized the immunopeptidomic landscape of MM by mass spectrometry-based analysis of naturally presented HLA ligands from primary MM samples and MM cell lines (Walz et al., Blood, 2015). Comparative HLA peptidome profiling of the MM-derived HLA ligands versus the immunopeptidome of numerous benign samples from different tissues identified several strictly MM-associated antigens. Here, we evaluated this dataset for the presence of BCMA-derived MM-exclusive antigens and identified two HLA class I-restricted, BCMA-derived peptides in the immunopeptidome of our cohort comprising 15 primary MM samples and MM cell lines. Notably, one of these peptides showes strictly MM-associated presentation and was never detected on any benign tissues according to our extensive immunopeptidome database (135,354 HLA ligands originating from 16,626 source proteins detected in 337 samples from various benign tissues including blood, bone marrow, lung, kidney, liver, and spleen). This HLA-B*18-restricted ligand P(BCMA)B*18 is represented in 20% (3/15) of the analyzed MM immunopeptidomes.For immunological characterization of the P(BCMA)B*18 peptide, we performed in vitro artificial antigen-presenting cell-based priming experiments engaging naïve CD8+ T cells obtained from healthy volunteers (HV). Induction of tetramer-positive T-cell populations with frequencies ranging from 0.1-2.9% of viable CD8+ T cells was observed for all analyzed healthy whole blood donors, which demonstrates the immunogenicity of P(BCMA)B*18. Subsequently, we functionally characterized the induced P(BCMA)B*18-specific CD8+ T cells using intracellular cytokine staining. Upon stimulation with P(BCMA)B*18, we observed an increased IFNγ and TNF production specifically in the peptide-specific CD8+ T cells. In addition, the degranulation marker CD107a was found to be upregulated in the analyzed tetramer-positive T cells, confirming the activity of CD8+ T cells upon peptide-stimulation. Priming experiments using naïve CD8+ T cells obtained from MM patients as well as in vitro cytotoxicity assays with polyclonal peptide-specific effector T cells are presently ongoing in order to assess the capacity of P(BCMA)B*18-specific CD8+ T cells to induce antigen-specific cell lysis.Taken together, we identified a naturally presented and MM-associated, BCMA-derived peptide, which constitutes a promising target for tailored T cell-based immunotherapeutic approaches. DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Weisel:Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen, Celgene, Janssen, and Sanofi: Research Funding.

High Therapeutic Efficacy of a New Survivin LSP-Cancer Vaccine Containing CD4+ and CD8+ T-Cell Epitopes

The efficacy of an antitumoral vaccine relies both on the choice of the antigen targeted and on its design. The tumor antigen survivin is an attractive target to develop therapeutic cancer vaccines because of its restricted over-expression and vital functions in most human tumors. Accordingly, several clinical trials targeting survivin in various cancer indications have been conducted. Most of them relied on short peptide-based vaccines and showed promising, but limited clinical results. In this study, we investigated the immunogenicity and therapeutic efficacy of a new long synthetic peptide (LSP)-based cancer vaccine targeting the tumor antigen survivin (SVX). This SVX vaccine is composed of three long synthetic peptides containing several CD4+ and CD8+ T-cell epitopes, which bind to various HLA class II and class I molecules. Studies in healthy individuals showed CD4+ and CD8+ T-cell immunogenicity of SVX peptides in human, irrespective of the individual's HLA types. Importantly, high frequencies of spontaneous T-cell precursors specific to SVX peptides were also detected in the blood of various cancer patients, demonstrating the absence of tolerance against these peptides. We then demonstrated SVX vaccine's high therapeutic efficacy against four different established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses. When tumors were eradicated, generated memory T-cell responses protected against rechallenge allowing long-term protection against relapses. Treatment with SVX vaccine was also found to reshape the tumor microenvironment by increasing the tumor infiltration of both CD4+ and CD8+ T cells but not Treg cells therefore tipping the balance toward a highly efficient immune response. These results highlight that this LSP-based SVX vaccine appears as a promising cancer vaccine and warrants its further clinical development.

Systematically benchmarking peptide-MHC binding predictors: From synthetic to naturally processed epitopes.

A number of machine learning-based predictors have been developed for identifying immunogenic T-cell epitopes based on major histocompatibility complex (MHC) class I and II binding affinities. Rationally selecting the most appropriate tool has been complicated by the evolving training data and machine learning methods. Despite the recent advances made in generating high-quality MHC-eluted, naturally processed ligandome, the reliability of new predictors on these epitopes has yet to be evaluated. This study reports the latest benchmarking on an extensive set of MHC-binding predictors by using newly available, untested data of both synthetic and naturally processed epitopes. 32 human leukocyte antigen (HLA) class I and 24 HLA class II alleles are included in the blind test set. Artificial neural network (ANN)-based approaches demonstrated better performance than regression-based machine learning and structural modeling. Among the 18 predictors benchmarked, ANN-based mhcflurry and nn_align perform the best for MHC class I 9-mer and class II 15-mer predictions, respectively, on binding/non-binding classification (Area Under Curves = 0.911). NetMHCpan4 also demonstrated comparable predictive power. Our customization of mhcflurry to a pan-HLA predictor has achieved similar accuracy to NetMHCpan. The overall accuracy of these methods are comparable between 9-mer and 10-mer testing data. However, the top methods deliver low correlations between the predicted versus the experimental affinities for strong MHC binders. When used on naturally processed MHC-ligands, tools that have been trained on elution data (NetMHCpan4 and MixMHCpred) shows better accuracy than pure binding affinity predictor. The variability of false prediction rate is considerable among HLA types and datasets. Finally, structure-based predictor of Rosetta FlexPepDock is less optimal compared to the machine learning approaches. With our benchmarking of MHC-binding and MHC-elution predictors using a comprehensive metrics, a unbiased view for establishing best practice of T-cell epitope predictions is presented, facilitating future development of methods in immunogenomics.

Human Papilloma Virus Specific Immunogenicity and Dysfunction of CD8+ T Cells in Head and Neck Cancer.

Human papillomavirus subtype 16 (HPV16) is the primary cause of an increasing number of head and neck squamous cell carcinomas (HNSCC), providing strong rationale for T-cell immune therapies against HPV+ HNSCC. Here we assess immunogenicity of HPV16-specific CD8+ T cells (CTL) and characterize HPV-specific mechanisms of T-cell dysfunction. We identified 16 strong and 29 moderately immunogenic CTL-epitopes from HPV16 E2, E6, and E7 antigens restricted by 12 common HLA class I alleles. E2-specific CTL-reactivity was higher in patients with HPV+ HNSCC than in healthy controls (>3-fold; P = 0.026). Patient-derived E2, E6, and E7 peripheral CTLs exhibited heterogeneity in dysfunctional phenotypes. Immunogenomic analyses of 119 HNSCC transcriptomes revealed high T-cell infiltration and dysfunction in HPV+ HNSCC and correlation of HPV antigen expression with T-cell exhaustion gene signatures. Indoleamine 2,3-dioxygenase (IDO-1) was strongly expressed in HPV+ HNSCC versus HPV- HNSCC (P = 0.001) and correlated with E7 expression (R 2 = 0.84; P = 0.033). Combination treatment with PD-1 blockade and IDO-1 inhibition overcame profound CTL-dysfunction, enhancing HPV+ HNSCC sensitivity to CTL-cytotoxicity in vitro (up to 10-fold in E7-CTLs, P = 0.011). Our findings implicate mechanisms of T-cell escape in HPV+ HNSCC, wherein high tumoral HPV-antigen load results in high expression of immune dysfunction genes on tumor cells (e.g., IDO-1), and dysfunction of HPV-specific CTLs (e.g., E7, E2-CTLs). The HPV16 CTL-epitopes identified in this study, in combination with blockade of HPV+ HNSCC-specific PD-1/IDO-1 checkpoints, may be useful for targeted immunotherapy.Significance: This study evaluates the HPV antigen T-cell immunogenicity role of inhibitory receptors and other exhaustion markers in the cytotoxic function of HPV antigen-specific CTLs and identifies combined inhibition of PD-1/IDO-1 as a strategy to enhance CTL targeting of HPV+ HNSCC. Cancer Res; 78(21); 6159-70. ©2018 AACR.

Prediction of cross-clade HIV-1 T-cell epitopes using immunoinformatics analysis.

Epitope mapping has emerged as a powerful tool to develop peptide vaccines against hypervariable viruses such as HIV. This method has led to stimulate a specific immune response and achieve advanced vaccine formulations. In this study, we identified peptides that were potentially immunostimulatory and highly conserved in HIV-1 main group. The analyses included were CTL assay, Tap transport, and the potential allergenicity. The highest population coverage rate was also found for all potential T-cell epitopes in 16 specified geographic regions of the world. The current study is the first attempt to explore peptide-protein flexible docking across all the major epitopes of HIV-1. Our data indicated that REV54-63 and VPU58-66 with the highest epitope identification scores, GP16037-46 and VPR38-47 with the highest conservation (98.89%), and NEF134-144 and GP16037-46 epitopes with a higher quality of peptide-protein interaction models in docking procedure were chosen as putative epitopes among all HLA class I epitopes. TAT40-67 , VPR65-82 , and VPU30-44 with the highest score of binding affinity, VPR65-82 with the highest conservation (97.55%), and GP160481-498 epitope with a higher quality of peptide-protein interaction models in docking procedure were determined as putative epitopes among all HLA-DR epitopes. Furthermore, two epitopes of GP160481-498 and VIF144-159 were predicted to bind 22 and 21 HLA-II alleles, respectively. Accumulative population coverage of potential helper T-cell epitopes and CTL epitopes varied between 90.82% and 100%. Generally, these predicted highly immunogenic T-cell epitopes can contribute to design HIV-1 peptide vaccine candidates. Combination of bioinformatics tools with in vivo methods will be necessary for HIV-1 vaccine development.

Evaluation of emerging biomarkers for major organ toxicities and disease states

Zika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens.T-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice–compliant protocol.We successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo–expanded T cells comprised both CD4+ and CD8+ T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential “off-the-shelf” therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II–restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion.Our findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naïve donors for future clinical use in an “off-the-shelf” setting.

Methods for Measuring T-Cell Memory to Vaccination: From Mouse to Man.

The development of effective vaccines continues to be a key goal for public health bodies, governments, funding bodies and pharmaceutical companies. With new vaccines such as Shingrix targeting Shingles and Bexsero for Meningitis B, licensed in recent years, today’s population can be protected from more infectious diseases than ever before. Despite this, we are yet to license vaccines for some of the deadliest endemic diseases affecting children, such as malaria. In addition, the threat of epidemics caused by emerging pathogens is very real as exemplified by the 2014–2016 Ebola outbreak. Most licensed vaccines provide efficacy through humoral immunity and correlates of protection often quantify neutralising antibody titre. The role of T-cells in vaccine efficacy is less well understood and more complex to quantify. Defining T-cell responses which afford protection also remains a challenge, although more sophisticated assays for assessing cell-mediated immunity with the potential for higher throughput and scalability are now available and warrant review. Here we discuss the benefits of multiparameter cytokine analysis and omics approaches compared with flow cytometric and ELISpot assays. We also review technical challenges unique to clinical trial studies, including assay validation across laboratories and availability of sample type. Measuring T-cell immunogenicity alongside humoral responses provides information on the breadth of immune responses induced by vaccination. Accurately enumerating and phenotyping T-cell immunogenicity to vaccination is key for the determination of immune correlates of protection. However, identifying such T-cell parameters remains challenging without a clear understanding of the immunological mechanisms by which a T-cell-mediated response induces protection.

Improving the In Vivo Efficacy of an Anti-Tac (CD25) Immunotoxin by Pseudomonas Exotoxin A Domain II Engineering

Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38-kDa fragment of Pseudomonas exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II-deleted toxin variants. The domain II-deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy in vivo We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies. Mol Cancer Ther; 17(7); 1486-93. ©2018 AACR.

Tumor lysate-loaded Bacterial Ghosts as a tool for optimized production of therapeutic dendritic cell-based cancer vaccines

Cancer immunotherapy with dendritic cell (DC)-based vaccines has been used to treat various malignancies for more than two decades, however generally showed a limited clinical success. Among various factors responsible for their modest clinical activity is the lack of universally applied, standardized protocols for the generation of clinical-grade DC vaccines, capable of inducing effective anti-tumor immune responses. We investigated Bacterial Ghosts (BGs) – empty envelopes of Gram-negative bacteria – as a tool for optimized production of DC vaccines. BGs possess various intact cell surface structures, exhibiting strong adjuvant properties required for the induction of DC maturation, whereas their empty internal space can be easily filled with a source tumor antigens, e.g. tumor lysate. Hence BGs emerge as an excellent platform for both the induction of immunogenic DC maturation and loading with tumor antigens in a single-step procedure. We compared the phenotype, cytokine secretion profile, functional activity and ability to induce immunogenic T-cell responses in vitro of human monocyte-derived DCs generated using BG platform and DCs matured with widely used lipopolysaccharide (LPS) plus interferon-γ cocktail and loaded with tumor lysate. Both approaches induced DC maturation, however BG-based protocol was superior to LPS-based protocol in terms of the ability to induce DCs with a lower tolerogenic potential, resulting in a more robust CD8+ T cell activation and their functional activity as well as significantly lower induction of regulatory T cells. These superior parameters are attributed, at least in part, to the ability of BG-matured DCs to resist potential immunosuppressive and pro-tolerogenic activity of various tumor cell lysates, including melanoma, renal carcinoma and glioblastoma.