IFN-γ ELISPOT Research Articles

Acute exercise increases immune responses to SARS CoV-2 in a previously infected man

Evidence is emerging that exercise and physical activity provides protection against severe COVID-19 disease in patients infected with SARS-CoV-2, but it is not known how exercise affects immune responses to the virus. A healthy man completed a graded cycling ergometer test prior to and after SARS-CoV-2 infection, then again after receiving an adenovirus vector-based COVID-19 vaccine. Using whole blood SARS-CoV-2 peptide stimulation assays, IFN-γ ELISPOT assays, flow cytometry, ex vivo viral-specific T-cell expansion assays and deep T-cell receptor (TCR) β sequencing, we found that exercise robustly mobilized highly functional SARS-CoV-2 specific T-cells to the blood compartment that recognized spike protein, membrane protein, nucleocapsid antigen and the B.1.1.7 α-variant, and consisted mostly of CD3+/CD8+ T-cells and double-negative (CD4-/CD8-) CD3+ T-cells. The magnitude of SARS-CoV-2 T-cell mobilization with exercise was intensity dependent and robust when compared to T-cells recognizing other viruses (e.g. CMV, EBV, influenza). Vaccination enhanced the number of exercise-mobilized SARS-CoV-2 T-cells recognizing spike protein and the α-variant only. Exercise-mobilized SARS-CoV-2 specific T-cells proliferated more vigorously to ex vivo peptide stimulation and maintained broad TCR-β diversity against SARS-CoV-2 antigens both before and after ex vivo expansion. Neutralizing antibodies to SARS-CoV-2 were transiently elevated during exercise after both infection and vaccination. Finally, infection was associated with an increased metabolic demand to defined exercise workloads, which was restored to pre-infection levels after vaccination. This case study provides impetus for larger studies to determine if these immune responses to exercise can facilitate viral clearance, ameliorate symptoms of long COVID syndrome, and/or restore functional exercise capacity following SARS-CoV-2 infection.

Dynamic alterations of circulating T lymphocytes and the clinical response in patients with head and neck squamous cell carcinoma treated with nivolumab.

Cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). However, only approximately 20-30% of patients with recurrent/metastatic (R/M) HNSCC benefit. Moreover, the mechanisms underlying the response to ICIs remain unclear. We investigated the proportion, activation status, and expression level of immune checkpoint molecules in circulating T cell subsets in R/M HNSCC patients treated with nivolumab using flow cytometry and mass cytometry, and then determined whether treatment response was associated with these values. We also assessed the changes in the frequency of tumor-associated antigens, MAGE-A4 and p53, -specific T cells prior to and after nivolumab treatment using the IFN-γ ELISPOT assay. The proportion of activated CD4+ and CD8+ TEMRA cells significantly increased in the disease-controlled patients but not in disease-progressed patients. As expected, the expression of PD-1 in T cells markedly decreased regardless of the therapeutic response. Meanwhile, T cell immunoglobulin mucin-3 expression on CD8+ T cells was significantly higher in patients with disease progression than in disease-controlled patients after treatment. The frequency of the tumor-associated antigens, MAGE-A4- and p53-specific T cells, was not correlated with clinical responses; however, in the disease-controlled patients, the frequency of MAGE-A4-specific T cells was significantly augmented. We concluded that in R/M HNSCC patients treated with nivolumab, circulating T cells show dynamic alterations depending on treatment efficacy. An analysis of the immunokinetics of circulating T cells could thus provide new insights into rational therapeutic strategies in cancer immunotherapy for HNSCC.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Comparison of SARS-CoV-2- and HCoV-Specific T Cell Response Using IFN-γ ELISpot.

Herd immunity is essential to control severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), especially in immunocompromised patients. Convalescent individuals should be vaccinated later due to vaccine shortage, as studies show that neutralizing antibodies generated during infection are stable for at least 6 months. Cellular immunity is also detectable for months. However, there is evidence of cross-reactivity of T cells with human endemic coronaviruses (HCoVs). Here, we show that cross-reactivity—which may prevent the specific detection of SARS-CoV-2-specific T cell responses—can be avoided if cells are stimulated with the N-terminus of the spike protein in IFN-γ ELISpot. In contrast to previous studies, we examined T-cell responses against all four known HCoVs using IFN-γ ELISpot in 19 convalescent volunteers and 10 fully vaccinated volunteers. In addition, we performed Spearman analyses to detect cross-reactivity of T cells. We observed no correlation between T-cell responses against SARS-CoV-2 and human endemic coronaviruses, either in the whole cohort or in the individual groups. The use of the respective stimuli could lead to a more accurate assessment of cellular immunity in recovered individuals. This testing procedure could help to define the best time point at which convalescents should receive SARS-CoV-2 vaccination.

Dose Dependent Antimicrobial Cellular Cytotoxicity-Implications for ex vivo Diagnostics.

Introduction:Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity.Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32–100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Weak immunogenicity of SARS-CoV-2 vaccine in patients with hematologic malignancies

This study evaluated the safety and immunogenicity of BNT162b2 vaccine in patients with hematological malignancies. Antibodies blocking spike binding to immobilized ACE-2 (NAb) correlated with anti-Spike (S) IgG d42 titers (Spearman r = 0.865, p < 0.0001), and an anti-S IgG d42 level ≥3100 UA/mL was predictive of NAb ≥ 30%, the positivity cutoff for NAb (p < 0.0001). Only 47% of the patients achieved an anti-S IgG d42 level ≥3100 UA/mL after the two BNT162b2 inocula, compared to 87% of healthy controls. In multivariable analysis, male patients, use of B-cell targeting treatment within the last 12 months prior to vaccination, and CD19+ B-cell level <120/uL, were associated with a significantly decreased probability of achieving a protective anti-S IgG level after the second BNT162b2 inoculum. Finally, using the IFN-γ ELISPOT assay, we found a significant increase in T-cell response against the S protein, with 53% of patients having an anti-S IgG-positive ELISPOT after the second BNT162b2 inoculum. There was a correlation between the anti-S ELISPOT response and IgG d42 level (Spearman r = 0.3026, p = 0.012). These findings suggest that vaccination with two BNT162b2 inocula translates into a significant increase in humoral and cellular response in patients with hematological malignancies, but only around half of the patients can likely achieve effective immune protection against COVID-19.

IL-7 expands lymphocyte populations and enhances immune responses to sipuleucel-T in patients with metastatic castration-resistant prostate cancer (mCRPC)

BackgroundSipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that...

Preformed T cell alloimmunity and HLA eplet mismatch to guide immunosuppression minimization with tacrolimus monotherapy in kidney transplantation: Results of the CELLIMIN trial

Personalizing immunosuppression is a major objective in transplantation. Transplant recipients are heterogeneous regarding their immunological memory and primary alloimmune susceptibility. This biomarker-guided trial investigated whether in low immunological-risk kidney transplants without pretransplant DSA and donor-specific T cells assessed by a standardized IFN-γ ELISPOT, low immunosuppression (LI) with tacrolimus monotherapy would be non-inferior regarding 6-month BPAR than tacrolimus-based standard of care (SOC). Due to low recruitment rates, the trial was terminated when 167 patients were enrolled. ELISPOT negatives (E-) were randomized to LI (n=48) or SOC (n=53), E+ received the same SOC. Six- and 12-month BPAR rates were higher among LI than SOC/E- (4/35 [13%] vs. 1/43 [2%], p=.15 and 12/48 [25%] vs. 6/53 [11.3%], p=.073, respectively). E+ patients showed similarly high BPAR rates than LI at 6 and 12months (12/55 [22%] and 13/66 [20%], respectively). These differences were stronger in per-protocol analyses. Post-hoc analysis revealed that poor class-II eplet matching, especially DQ, discriminated E- patients, notably E-/LI, developing BPAR (4/28 [14%] low risk vs. 8/20 [40%] high risk, p=.043). Eplet mismatch also predicted anti-class-I (p=.05) and anti-DQ (p < .001) de novo DSA. Adverse events were similar, but E-/LI developed fewer viral infections, particularly polyoma-virus-associated nephropathy (p=.021). Preformed T cell alloreactivity and HLA eplet mismatch assessment may refine current baseline immune-risk stratification and guide immunosuppression decision-making in kidney transplantation.

Melioidosis Patient Survival Correlates With Strong IFN-γ Secreting T Cell Responses Against Hcp1 and TssM.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Refining “Long-COVID” by a Prospective Multimodal Evaluation of Patients with Long-Term Symptoms Attributed to SARS-CoV-2 Infection

IntroductionCOVID-19 long-haulers, also decribed as having “long-COVID” or post-acute COVID-19 syndrome, represent 10% of COVID-19 patients and remain understudied.MethodsIn this prospective study, we recruited 30 consecutive patients seeking medical help for persistent symptoms (> 30 days) attributed to COVID-19. All reported a viral illness compatible with COVID-19. The patients underwent a multi-modal evaluation, including clinical, psychologic, virologic and specific immunologic assays and were followed longitudinally. A group of 17 convalescent COVID-19 individuals without persistent symptoms were included as a comparison group.ResultsThe median age was 40 [interquartile range: 35–54] years and 18 (60%) were female. At a median time of 152 [102–164] days after symptom onset, fever, cough and dyspnea were less frequently reported compared with the initial presentation, but paresthesia and burning pain emerged in 18 (60%) and 13 (43%) patients, respectively. The clinical examination was unremarkable in all patients, although the median fatigue and pain visual analog scales were 7 [5–8] and 5 [2–6], respectively. Extensive biologic studies were unremarkable, and multiplex cytokines and ultra-sensitive interferon-α2 measurements were similar between long-haulers and convalescent COVID-19 individuals without persistent symptoms. Using SARS-CoV-2 serology and IFN-γ ELISPOT, we found evidence of a previous SARS-CoV-2 infection in 50% (15/30) of patients, with evidence of a lack of immune response, or a waning immune response, in two patients. Finally, psychiatric evaluation showed that 11 (36.7%), 13 (43.3%) and 9 (30%) patients had a positive screening for anxiety, depression and post-traumatic stress disorder, respectively.ConclusionsHalf of patients seeking medical help for post-acute COVID-19 syndrome lack SARS-CoV-2 immunity. The presence of SARS-CoV-2 immunity, or not, had no consequence on the clinical or biologic characteristics of post-acute COVID-19 syndrome patients, all of whom reported severe fatigue, altered quality of life and psychologic distress.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00484-w.

Abstract LB027: Tracking and decoding the antigen specificity of peripherally derived T cells that infiltrate into solid tumors in patients treated with an autologous T cell therapy, PRIME IL-15 (RPTR-147)

Abstract PRIME IL-15 (RPTR-147) is a novel non-genetically modified, autologous, multi-clonal T cell product loaded with an IL-15Fc nanogel. The product is derived from peripheral T cells that are primed and expanded against five tumor associated antigens (TAAs) known to be overexpressed in multiple different tumor types: PRAME, NY-ESO, SSX-2, WT-1 and Survivin. 17 patients with advanced or metastatic solid-tumor cancers were dosed in a Phase I dose escalation study. Where samples were available, we characterized the reactivity and specificity of the cellular product, and tracked its persistence and localization post administration in patients. We report cellular composition of the product, along with phenotype and TAA-specificity of the T cells. Antigen specificity of patient-derived product samples was determined by Repertoire Immune Medicines' proprietary DNA-barcoded pMHC tetramer platform (CIPHERTM), as well as by functional immunological methods including IFNg ELISpot and flow cytometry assessment of activation induced markers (AIM). The CIPHER platform was used to identify TAA-specific CD8 T cell clonotypes, allowing decoding of T cell receptor (TCR) sequences and their cognate epitope/MHC. Distinct peptides binding from all five TAAs were observed across patients, and were presented across multiple HLA alleles. The phenotype of antigen-specific T cells identified via CIPHER was also assessed via single cell gene expression. Consistent with our previous studies on healthy donors, reactivity via IFNg ELISpot and AIM was also observed to all targeted TAAs. TCR CDR3 sequences were used as molecular signatures of antigen-specific cells to track the product post administration. Bulk sequencing of TCRVβ was performed on tumors pre- and post-treatment. Several product-derived, TAA-specific CD8+ T cell clonotypes were observed in the post-, but not pre-treatment biopsies. We identified additional CD4+ and CD8+ clones that were enriched in the post-treatment biopsies, and some of these clonotypes were also enriched in the product. To de-orphan these tumor infiltrating lymphocytes (TIL) of interest, we cloned selected TIL TCRs to assess their reactivity using Repertoire Immune Medicines' proprietary MCR epitope screening platform. In summary, characterization of antigen-specific T cells in the PRIME IL-15 product was performed by a variety of functional and molecular immune-based methods. Antigen specific T cell reactivities and clonotypes were identified in the peripheral repertoire. We confirmed that T cells were successfully expanded against TAAs and that these T cells do infiltrate the tumors as evidenced by bulk TCR sequencing of post-treatment tumor biopsies. Citation Format: John David Pajerowski, William Gordon, Phil Bardwell, Christine McInnis, Ji Young Hwang, Tabasum Huseni, Parul Agnihotri, Josh Francis, Christina Tarr, Florian Renoux, Sebastian Heer, Nastassja Cerreghetti-Terraneo, Marisa Loi, Erika Hamilton, Sarah Nikiforow, George Blumenschein, Mihaela Christea, Keren Osman, Anthony Shields, Harriet Kluger, Franz-Josef Obermair, Daniel Pregibon, Jan Kisielow, Anthony Coyle, Mojca Skoberne. Tracking and decoding the antigen specificity of peripherally derived T cells that infiltrate into solid tumors in patients treated with an autologous T cell therapy, PRIME IL-15 (RPTR-147) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB027.

Abstract 73: A personal neoantigen vaccine NEO-PV-01 in combination with chemotherapy and pembrolizumab induces broad de novo immune responses in first-line non-squamous NSCLC: Associations with clinical outcomes

Abstract Background: Neoantigens arising from mutations in cancer cell DNA are important targets for T cell-mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides (14-35 amino acids) based on a patient's HLA profile and bioinformatic analysis of tumor neoantigens. We report here relationships between baseline tumor characteristics, immune response, and clinical outcomes from NT-002, a Phase 1b study of NEO-PV-01 with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous NSCLC (NCT03380871). The primary objective of this study was to evaluate the safety of the combination. Materials: Serial blood and tumor biopsies were collected at: i) prior to treatment, ii) after 12 weeks of chemotherapy-pembrolizumab treatment, and iii) after completion of NEO-PV-01 vaccination. Tumor biopsies were characterized by immunohistochemistry for immune and tumor markers, gene expression, whole-exome and TCR sequencing, and single-cell analysis. Antigen-specific responses were measured in blood samples by IFNγ ELISpot, intracellular cytokine staining and functional phenotyping by FACS. Results: A total of 38 patients initiated study treatment (ITT); 21 patients received at least 1 dose of NEO-PV-01 (VAX). The regimen was well-tolerated and consistent with the pembrolizumab plus pemetrexed/carboplatin safety profile. The overall response rate (ORR)/clinical benefit rate (CBR) for the ITT and VAX were 37%/69% and 57%/95%, respectively. Median PFS was 7.2 months (95% CI: 5.6,16.8) for both the ITT and VAX, and median OS 16.8 months (95% CI: 11.6, NR) for both groups. Immune analysis on 12 patients with available samples revealed neoantigen-specific CD4+ and CD8+ T cell responses in all patients tested with an average of 55% of vaccine peptides generating an immune response post-vaccination. Vaccine-induced immune responses were mutant-specific and durable at 52-week treatment timepoint. T cell responses were polyfunctional, as evident by secretion of multiple cytokines (TNFα, IL2, IFNγ), and were activated memory cells with a cytotoxic phenotype. Epitope spread was observed in 7 of 11 patients analyzed thus far. Comprehensive analysis by gene expression, ctDNA and TCR repertoire analysis demonstrated correlations to extended PFS. Additional data on single-cell sequencing of neoantigen-specific T cells and tumor biopsies and correlates to clinical outcomes will be presented. Conclusions: NEO-PV-01 in combination with pembrolizumab and carboplatin/pemetrexed has a good safety profile and induces de novo immune responses in first-line non-squamous NSCLC. The association of baseline disease characteristics to prolonged PFS suggests future patient enrichment strategies for evaluation of this novel regimen in a phase 2 trial. Citation Format: Mark M. Awad, Ramaswamy Govindan, David R. Spigel, Edward B. Garon, Victoria Kohler, Rohit Vyasamneni, Suchitra Ramesh, Tracey E. Sciuto, Melissa A. Moles, Jennifer Tepper, Amy Wanamaker, Zakaria S. Khondkar, John Srouji, Jesse Z. Dong, Kristen N. Balogh, Asaf Poran, Meghan E. Bushway, Mark DeMario, Richard B. Gaynor, Lakshmi Srinivasan. A personal neoantigen vaccine NEO-PV-01 in combination with chemotherapy and pembrolizumab induces broad de novo immune responses in first-line non-squamous NSCLC: Associations with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 73.

Abstract 1586: Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models

Abstract Oncolytic virotherapy is an emerging treatment modality that may hold promise as a therapeutic option for cancer patients who do not respond to checkpoint inhibitors. Oncolytic viruses preferentially replicate in tumor cells, offer the ability to express transgenes and have been shown to modify the local and distant tumor microenvironments from immunosuppressive to immunostimulatory. We generated HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12, an oncolytic virus based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce murine FMS-like tyrosine kinase 3 ligand (mFLT3L) to enhance dendritic cell expansion and T cell priming, and murine interleukin-12 (mIL12) to potentiate type I helper and cytotoxic T cell responses. We confirmed the expression and bioactivity of mFLT3L and mIL12 in vitro and evaluated the efficacy of HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 alone and in combination with anti-PD-1 or anti-4-1BB treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used and only one tumor was treated via intratumoral injection with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12. Anti-tumor responses were evaluated in the injected tumor (local response), the uninjected tumor (abscopal) and via splenic immune profiling (systemic). HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 treatment alone led to tumor growth inhibition in both treated and contralateral tumors, and a significant increase in overall survival in the A20, Neuro2a and MC38 models. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with anti-PD-1 blocking antibody further improved tumor growth inhibition in both treated and contralateral tumors in the MC38 syngeneic model, and significantly increased median overall survival compared to either agent as a monotherapy. In addition, HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with an agonistic antibody targeting 4-1BB significantly increased overall survival compared to either agent as a monotherapy in the MC38 model. To understand antigen-specific anti-tumor T cell responses, MC38 tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell responses were measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 induced and/or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that treatment with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 can cause direct tumor lysis and induce an adaptive, systemic T cell-mediated anti-tumor immune response that can be further enhanced by concurrent PD-1 blockade or 4-1BB agonism in multiple syngeneic mouse tumor models. Citation Format: Keegan Cooke, Walter H. Meisen, Petia Mitchell, Juan Estrada, Jinghui Zhan, Jessica Orf, Peng Li, Christina de Zafra, Jing Qing, Jason DeVoss. Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1586.

Abstract 1635: Sirt2 blockade promotes T cell metabolism and restores the anti-tumor immunity

Abstract Background: The majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive. Sirt2 is an NAD+ dependent histone deacetylase and conflicting evidences suggest its tumor-suppressor and oncogenic roles. In inflammatory response, Sirt2 suppresses inflammation via negative regulation of NF-κB subunit; however, the role of Sirt2 in tumor immunity has not been described. Method: Human TILs from non-small cell lung cancer (NSCLC) tumor samples and matched peripheral blood T cells were analyzed for Sirt2 expression by flow cytometry. The role of Sirt2 in anti-tumor immunity was studied by in vivo B16F10 tumor challenge in Wild-type (Wt) and Sirt2 knockout (Sirt2KO) mice. The role of Sirt2 in T cell effector functions was investigated ex vivo by CFSE proliferation assay, IFN-γ ELISpot assay, intracellular staining of effector markers and LDH cytotoxicity assay on Wt versus Sirt2KO T cells. Sirt2 targets were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. T cells metabolic changes were investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. Sirt2 blockade in human T cells was performed using AGK2, a Sirt2 selective inhibitor. Result: We show that Sirt2 expression is upregulated within the TME, and its upregulation in human TILs is associated with a poor clinical response to immunotherapy in a phase I clinical trial in advanced NSCLC. We also show that, Sirt2 deficiency in mice boosts T cell effector functions and tumor rejection in vivo. Our molecular and metabolomic studies revealed multiple metabolic pathways as Sirt2 targets including glycolysis, TCA-cycle, FAO and glutaminolysis. We found that Sirt2 deacetylase deficiency increased acetylation and enzymatic activity of key metabolic enzymes leading to a hyper metabolic status of T cells. Finally pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhances their metabolic fitness and effector functions. Conclusion Our findings indicate Sirt2 as a master suppressor of T cell metabolism amenable to therapeutic targeting and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the metabolically challenging TME, thus, leading to an effective anti-tumor immune response. Citation Format: Imene Hamaidi, Lin Zhang, Nayoung Kim, Min Hsuan Wang, Cristina Iclozan, Bin Fang, Min Liu, John M. Koomen, Anders E. Berglund, Sean J. Yoder, Jiqiang Yao, Robert W. Engelman, Ben C. Creelan, Jose R. Conejo-Garcia, James J. Mulé, Scott J. Antonia, Sungjune Kim. Sirt2 blockade promotes T cell metabolism and restores the anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1635.

Abstract 1826: Comprehensive preclinical study on GI-101, a novel CD80-IgG4-IL2 variant protein, as a therapeutic antibody candidate with bispecific immuno-oncology target

Abstract Introduction: GI-101 was designed to address the significant unmet needs in immunotherapy for noninflamed tumor. The harmonized mechanisms of action consist of the extracellular domain of CD80 acting as a CTLA-4 inhibitor, together with a long-acting IL-2 variant that preferentially binds to the IL2Rβ. Therefore, GI-101 can play a role in the activation of cytotoxic immune cells and inhibition of CTLA-4-B7.1 axis-based immune suppression. Methods: The binding affinity of GI-101 to IL-2Rs, CTLA-4, and CD28 was performed by SPR and immune cell proliferation was analyzed by CFSE assay in vitro. GI-101 induced dose-dependent pharmacodynamics effects with consistent magnitude following repeat administration in monkeys. Direct anti-tumor effect of GI-101 was tested by single or combination treatment manner in multiple syngeneic and humanized models. Immune profiling in TME was analyzed by flow cytometry, IHC and IFN-γ ELISPOT assay. To mimic the standard care (SOC) in clinic, TC1 lung cancer model was involved in evaluating the efficacy of both 1st line and maintenance therapy of GI-101 with or without immuno-chemotherapy (Cisplatin, Pemetrexed, and anti-PD-1). Results: CD80 of GI-101 highly binds to CTLA-4 (Kd, 2.9nM), acting as a decoy ligand. IL-2 variant induces CD8+ T and NK cell proliferation. However, GI-101 had no evidence of toxicity related to IL-2 activity in the non-GLP monkey study, including vascular leakage syndrome and cytokine storm. GI-101 elicits improved restoration of immune functions in human PBMCs co-cultured setting with PD-L1/CTLA-4 co-expressed tumor cells. A dose-dependent (3 to 12 mg/kg) single inhibition of tumor growth was observed in CT26 syngeneic model. Immune profiling revealed a robust increase of M1 macrophages, CD8+ central memory T (Tcm) and NK cells but not Tregs in TME. Splenocyte tumor-specific immune cells were strongly proliferated when stimulated with CT26 neoantigens (gp70). IFN-γ+ T cells were significantly increased in draining lymph nodes from GI-101 treated mice. Furthermore, GI-101 was superior at inhibiting tumor growth when co-treated with anti-PD-1 in syngeneic (MC38, TC1, and B16F10) and MDA-MB-231 humanized mice models. Finally, the combination of GI-101 and immuno-chemotherapy showed not only suppressed tumor growth but also improved survival compared to immuno-chemotherapy alone. Conclusion: The complementary modes of action of GI-101 via checkpoint blockade and IL-2 activity to enhance the proliferation and activation of Tcm and NK cells are projected to translate into superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI-101 has promising potential to replace the first-generation ICBs as a monotherapy or in combination with other immunotherapies. Our findings provide a rationale for further clinical investigations. Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Hwan Kim, Youngseon Byeon, Ha Ni Jo, Young Seob Kim, Wongeun Lee, Do Hee Kim, Seul Lee, Dong Kwon Kim, Eun ji Lee, Beung-Chul Ahn, Min Hee Hong, Myoung Ho Jang, Sun Min Lim, Hye Ryun Kim, Su Youn Nam, Byoung Chul Cho. Comprehensive preclinical study on GI-101, a novel CD80-IgG4-IL2 variant protein, as a therapeutic antibody candidate with bispecific immuno-oncology target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1826.

Safety and Efficacy of Adoptively Transferred Donor-Derived Tumor-Directed T Cells with Relapsed AML Post-Allogeneic HSCT

Acute myeloid leukemia (AML) relapse post-allogeneic transplant (HSCT) has a dismal prognosis with a median survival of just 4.5 months. Conventional chemotherapy has limited efficacy in treating relapsed AML and is also poorly tolerated by allogeneic HSCT recipients. Similarly, the adoptive transfer of unmanipulated donor lymphocytes (DLIs) to provide a graft versus leukemia (GvL) effect has demonstrated clinical benefit in only a minority (~10%) of patients but frequently (>50%) induces debilitating graft versus host disease (GVHD). Thus, the goal of this project was to enhance both the safety and efficacy associated with adoptively-transferred donor-derived T cells by selectively enriching and infusing leukemia-directed populations that lack alloreactive potential.To date we have generated 18 clinical grade T cell lines by stimulating donor PBMCs with dendritic cells loaded with overlapping peptide libraries spanning the leukemia-associated antigens (LAA) - WT1, PRAME, NY-ESO1 and Survivin. The lines were polyclonal, comprising both CD4+ (26.7±3.5%) and CD8+ (44±2.9%) cells and expressed memory markers CD45RO+/CD62L-/CCR7- (29.3±4%) associated with long term in vivo persistence. The lines were specific for the targeted antigens WT1, PRAME, NY-ESO1 and Survivin by IFNg ELIspot, and did not exhibit any activity against non-malignant patient-derived cells (3±3.5%, E:T 20:1) - a study release criterion.To test the safety and clinical effects of these donor-derived multiLAA T cells, we developed a phase I clinical trial - ADSPAM (IND #16700) to infuse escalating doses of cells (ranging from 0.5 to 2x107 cells/m2) asadjuvant therapy for those with adverse prognosis AML (Arm A) or to treat active, relapsed disease (Arm B). To date, 12 patients (prior histories summarized in Table 1) have been infused with multiLAA T cells (7 on Arm A and 5 on Arm B). Of note, pt1, was treated on both Arms A and B and except for this patient, who had a transient elevation of liver enzymes (AST and ALT) 2 weeks after the infusion of T cells (on Arm A at 0.5 x 107 cells/m2), there were no other infusion-related adverse events.Of the 7 patients who were infused on the adjuvant arm (Arm A), 2 (pts 1 and 4) developed relapsed disease. Pt4 developed brain parenchymal and leptomeningeal relapse 8 months post-multiLAA T cells and 10 months post-allogeneic HSCT, while pt1 relapsed in the bone 4 months post-multiLAA T cells (9 months post-allogeneic-HSCT) while receiving prednisone. Given the potential impact of the steroids on circulating T cells this patient was subsequently re-enrolled and treated on our active disease arm.Among 5 patients infused with multiLAA T cells to treat relapsed disease, 1 is still too early, 2 developed disease progression (months 1 and 3 post-multiLAA T cells, respectively) and 2 (pts 1 and 10) had clinical responses. Pt10, with a history of chemotherapy- and DLI-refractory AML and 30-40% bone marrow blasts at the time of infusion had a 50% reduction in disease burden within 1 month of infusion and remains in active follow-up (currently month 2 post-infusion) with additional multiLAA infusions planned. Pt1, who developed an extramedullary AML relapse at multiple sites within the axial skeleton, had a complete resolution of all disease-related symptoms (back and hip pain and limited range of motion at the shoulder) and a near complete resolution of all bony lesions, based on imaging studies, within 1 month of infusion. Currently, this patient remains disease-free 5 months post-reinfusion on Arm B and 1.5 years from transplant. Interestingly, in this patient whose blasts expressed WT1, we detected a marked and sustained increase in the circulating frequency of WT1-directed T cells in both peripheral blood and marrow that were confirmed of multiLAA origin based on TCR deep sequencing. In summary, infusion of donor multi-LAA-specific T cells to patients with AML post allogeneic HSCT is safe and associated with clinical benefit in individuals with highly treatment-resistant leukemias. [Display omitted] DisclosuresLulla:Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; ASH Scholar Award: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees. Tzannou:ViraCyte LLC: Consultancy. Heslop:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties: EBV specific T cells, Research Funding; Marker Therapeutics: Equity Ownership; Celgene: Research Funding. Vera:ViraCyte LLC: Equity Ownership; Wilson Wolf Manufacturing: Consultancy; Marker Therapeutics: Equity Ownership. Leen:ViraCyte LLC: Equity Ownership.

Neoantigen Synthetic Peptide Vaccine for Multiple Myeloma Elicits T Cell Immunity in a Pre-Clinical Model

Cancer immunotherapies such as immune checkpoint inhibitors (CI) are approved for a broad range of solid tumors and selected hematologic malignancies. Clinical response to CIs has been shown to be associated with mutation burden and is predicated on the presence of T cells specific to tumor-associated antigens. Therefore, increasing the frequency of such T cell responses should increase the efficacy of anti-tumor immunity. Individualized neoantigen peptide vaccines are a promising strategy to activate tumor-specific T cell immunity. Next generation sequencing of tumor specimens is now widely available and novel computational algorithms provide the means for the prediction and selection of neoantigens.We are conducting a preclinical study of a personalized vaccine in an animal model of multiple myeloma (MM). MM is an incurable cancer of plasma cells that resides in the bone marrow (BM) and causes lytic bone disease and other end-organ damage. MM is characterized by an intermediate level of somatic mutation that may yield mutational neoantigens for vaccine design. This investigation pursued two goals: first, to determine if neoantigens are immunogenic, and second, if they induce protective immunity against tumors. To address the immunogenicity of neoantigens, we used the MOPC.315.BM myeloma cell line in the immunocompetent BALB/c mouse background. MOPC.315.BM cells mimic human disease by homing to the BM and causing osteolytic lesions. We performed whole exome sequencing and RNAseq on MOPC.315.BM cells to identify expressed somatic mutations. Neoantigens were then selected based on predicted MHC class I and II binding and expression levels, and corresponding 27-mer peptides were synthesized and tested in vivo for their immunogenicity. Mice were immunized 3 times at day 0, 14 & 21 with pools of peptides in the presence of poly (I:C) adjuvant, and splenocytes were harvested at day 28 to measure peptide-specific T cell activation by IFNg ELISpot. Of the selected peptides, 25 of 66 induce high IFNg T cell responses in the spleen. Interestingly, 88% of these responses were CD4 T cells and 12% were CD8 T cells, consistent with other preclinical and clinical studies where neoantigens predicted to bind MHC-I molecules induced mainly CD4 T cell activation. Humoral immune response specific to the peptides were analyzed by ELISA and showed that 8 of the 25 peptides induce peptide-specific IgG antibodies in the serum.To determine whether neoantigens generate tumor protective immunity, we challenged vaccinated mice with the MOPC.315.BM cell line. We used a well-characterized antigen lambda 2315 idiotype (Id) produced by MOPC cells and known to activate CD4 T cells. Vaccines using only the Id were found to be weakly immunogenic and failed to elicit protective responses. We developed a new strategy of vaccination by adding a proprietary amino acid sequence comprising a high affinity HSP70 binding motif to the idiotype peptide. Mice vaccinated at days 0, 14 & 21 with the so-modified peptide and poly-(I:C) adjuvant had 4 times higher Id-specific IFNg CD4 T cell response in the spleen compared to mice vaccinated with the unmodified Id-peptide. When challenged with MOPC.315.BM cells after vaccination with the modified Id peptide, 60% of these mice maintained non-detectable Id level in their blood and resisted tumor growth for more than 70 days while unvaccinated mice had very high levels of circulating Id and died around 30 days after tumor injection. Id-mediated cytotoxicity is being investigated in vitro to complement these results. We are conducting tumor protection experiments with the 25 immunogenic peptides identified in the vaccination experiments described in the previous section.In summary, we showed that neoantigens derived from bioinformatic analysis of tumor genome sequencing are immunogenic and that they mainly activate CD4 T cells in this setting. Additionally, our vaccination strategy using the idiotype peptide showed that CD4 T cell immunity can be protective against tumor growth. We are currently investigating the potential of the neoantigens identified in the immunogenicity study to protect mice against tumor growth and eradicate pre-existing tumors. Our findings indicate that neoantigen vaccination is very promising and that future investigations combining this strategy with CIs and/or immunomodulatory agents may lead to the efficient generation of protective anti-tumor immunity. DisclosuresBekri:Agenus Inc: Research Funding. Cho:Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Agenus, Inc.: Research Funding.

Evaluating Cyclin A1 As an Immunotherapeutic Target in Acute Myeloid Leukemia (AML)

T cell based immunotherapy targeting leukemia associated antigens (LAAs) such as Wilm's tumor 1 antigen (WT1) has proven both safe and clinically effective, with the induction of durable responses in patients with relapsed AML. However, because of heterogeneous LAA expression among leukemic blasts, targeting a single LAA has led to tumor immune escape and relapse, highlighting the need to identify and simultaneously target an array of tumor-expressed LAAs. To expand the target antigen spectrum we sought to immunologically characterize cyclin A1 (CCNA1), a meiosis-specific cell cycle antigen that is selectively expressed in AML blasts with limited normal tissue expression and which has been implicated as playing a role in leukemogenesis based on preclinical animal models.To assess the suitability of CCNA1 as an immunotherapeutic target we first sought to expand reactive populations ex vivo using an overlapping peptide library (15mers overlapping by 11 amino acids) spanning the entire antigen as a source of stimulation. In 12 donors tested we achieved a mean 5.2±0.65 fold expansion of T cells, of which 11 proved to recognize CCN1 as measured by an IFNg ELISpot [mean 377.3 spot forming cells (SFCs)/1x105 T cells (range 38 to 1188 SFCs) ]. The antigen specific lines were polyclonal (23±4% CD4+ and 64±5% CD8+ T cells) that expressed markers for both central and effector memory (29±7% CD45RO+CD62L+ and 28±6% CD45RO+CD62L-), required for long term in vivo persistence. Importantly both helper (CD4+) and cytotoxic (CD8+) subsets contained CCNA1-directed T cell populations, which secreted multiple cytokines (1.47±0.07% CD4+IFNg+TNFa+ and 9.52±0.25% CD8+IFNg+TNFa+) as measured by intracellular cytokine staining, confirming their polyfunctionality. Moreover, CCNA1-specific T cells were able to selectively kill pepmix-loaded autologous target cells and a partially HLA-matched cell line that endogenously-expresses cyclin A1 (44±7% and 55±1%, respectively; E:T 20:1). without non-specific activity against control (unloaded) targets (9±2%; E:T 20:1). This killing was confirmed to be HLA-restricted since the addition of an MHC class I blocking antibody diminished specific lysis (decrease from 44% to 26%). Finally, using individual peptides organized into minipools, we were able to identify 4 novel HLA class I-restricted epitope peptides.To assess the clinical relevance of CCNA1-directed T cells we assessed whether patients with AML had detectable circulating T cells and in 5 of 9 patients who had undergone an allogeneic hematopoietic stem cell transplant as disease treatment we could detect CCNA1-specific T cells (mean 117±52.8 SFC/2x105 expanded PBMCs), and detection of reactive cells correlated with a lower risk for relapse.In summary, we have identified CCNA1 as a suitable candidate antigen to target using immunotherapeutic approaches for future clinical trials of adoptively transferred T cells. DisclosuresLulla:Leukemia Texas: Membership on an entity's Board of Directors or advisory committees; ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding. Tzannou:ViraCyte LLC: Consultancy. Heslop:Celgene: Research Funding; Cell Medica: Patents & Royalties: EBV specific T cells, Research Funding; Viracyte: Equity Ownership; Marker Therapeutics: Equity Ownership. Vera:Wilson Wolf Manufacturing: Consultancy; ViraCyte LLC: Equity Ownership; Marker Therapeutics: Equity Ownership. Leen:ViraCyte LLC: Equity Ownership.

T Cell Responses to Tumor Associated Antigens in Multiple Myeloma Patients Treated with MV-NIS, an Oncolytic Measles Virus

Introduction: Oncolytic virus therapy leads to immunogenic death of virus infected tumor cells. Evidence from mouse studies shows that this mode of tumor cell killing can boost the cytotoxic T lymphocyte response against tumor associated antigens (TAA), leading to ‘bystander’ killing of uninfected tumor cells.Methods: To investigate whether oncolytic virotherapy can boost immune responses to tumor antigens in human subjects we studied T cell responses to 10 different TAA reported to have high expression in patients with multiple myeloma (n=10) before and after intravenous administration of an oncolytic measles virus (MV-NIS). Baseline and 6 week post-therapy PBMC samples were frozen at the time of collection from each enrolled patient throughout the course of the clinical trial. After thawing, 2 x 105 PBMC were stimulated with peptide pools specific to each of the selected TAA and their responses to this antigenic stimulation were measured using IFNγ ELISPOT. Spots more than 50 were counted as positive response.Results: Despite their prior exposure to multiple immunosuppressive anti-myeloma treatment regimens, some of the tested patients had higher T cell responses to some of the TAA even prior to Measles virotherapy. Measurable baseline T cell responses against MAGEc1 were present in 50% of the patients, and against hTERT and NYESO-1 in 30% of the patients for each target. Furthermore, MV-NIS treatment significantly boosted T cell responses against NYESO-1, MAGEc1, MAGEA1 and MUC1 in 40% of the patients. Interestingly, in one outlier patient who achieved a stringent and durable complete disease remission after MV-NIS therapy, strong baseline T cell responses were present against 8 of the 10 tested TAA but did not change significantly after virotherapy. Based on the T cell responses to the TAA, three distinct phenotypes can be identified. Phenotype 1 was characterized by positive T cell responses to TAA prior and post therapy, Phenotype 2 was characterized by positive T cell responses to TAA only post therapy and phenotype 3 was characterized by negative T cell responses to TAA prior and post therapy (Figure 1). Additionally, these observed phenotypes in T cell responses correlated with anti-tumor response.Conclusion: Our data suggests oncolytic measles virotherapy boosted the cytotoxic T lymphocyte response against tumor associated antigens in some of the patients. Identifying patient's phenotype based on responsiveness to the TAA could be useful marker for identifying the immune responsive state in multiple myeloma patients and identify patients who can be most responsive to oncolytic measles virotherapy. Additionally, the boosted T cell responses against TAA post measles virotherapy provide a good rationale for combining oncolytic virotherapy with checkpoint antibody therapy to increase the efficacy of the oncolytic measles virotherapy.Figure 1: T cell response phenotypes against 10 different TAAs. Representative IFNγ ELISPOT data shown below. PRE: T cell responses prior to virotherapy; Post 6 wks: T cell responses 6 weeks post virotherapy [Display omitted] DisclosuresDispenzieri:Celgene, Millenium, Pfizer, Janssen: Research Funding. Peng:Imanis Life Sciences: Employment, Equity Ownership; Vyriad: Equity Ownership. Russell:Vyriad: Equity Ownership; Imanis Life Sciences: Equity Ownership.

Interim Analysis of Immunogenicity to the Vector Capsid and Transgene-Expressed Human FVIII in a Phase-1/2 Clinical Study of Bmn 270, an AAV5-Mediated Gene Therapy for Hemophilia a

▪When assessing the immunogenicity of gene therapy drug products, two components need to be considered: immunogenicity toward the vector delivery system and immunogenicity specific for the transgene product. BMN 270 is an AAV5-mediated gene therapy indicated for the treatment of Hemophilia A, and encodes for a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver specific promoter. In study BMN 270-201, the first-in-human clinical trial, prospective patients with severe Hemophilia A were screened for both pre-existing antibodies directed against the AAV5 vector and inhibitors of transduction using a cell-based in vitro transduction inhibition (TI) assay. Patients testing positive in either assay were excluded from the trial, as were patients who had a history of FVIII inhibitors. Following infusion of BMN 270, patient plasma was analyzed for total antibody (TAb) responses specific for the AAV5 capsid and TAb responses directed against FVIII, using bridging ECLA immunoassays. Development of FVIII inhibitors was monitored using the Nijmegen-modified Bethesda assay. Additionally, peripheral blood mononuclear cells were collected for analysis by IFN-γ and TNF-α ELISpot assays for detection of capsid-specific and hFVIII-SQ specific cellular immune responses. The available data indicate that, as expected, all patients develop anti-AAV5 TAb by Week 8 post BMN 270 infusion, the first immunogenicity time-point assessed. One patient screened and confirmed positive at a single time point in the anti-FVIII TAb assay. This response was below the minimum required dilution to determine a titer, and was negative at all subsequent time points. No patients have tested positive in the Bethesda assay for FVIII inhibitors.We investigated the possibility that cell mediated immunity may explain the observed elevation in hepatic enzymes. To date, several patients dosed with BMN 270 experienced asymptomatic elevation of alanine aminotransferase (ALT) laboratory values ranging from 44 to 141 U/L (normal, 6-43 U/L) that resolved without sequelae. Analysis of cellular immune response in these patients by IFN-γ ELISpot assay against peptides spanning AAV5 capsid or the hFVIII-SQ protein was negative, as it was for all other subjects in the study across all time points tested to date. Unlike the IFN-γ assay, intermittent responses were detected across several patients in response to hFVIII-SQ peptide pools in the TNF-α ELISpot assay. In general, these responses were self-limiting and were not temporally associated with increases in ALT or a decline in FVIII activity measures, and did not correlate with results from the IFN-γ assay. Similar TNF-α responses to hFVIII-SQ peptides were also observed in a subset of healthy donors. Overall, immune responses to BMN 270 were characterized by the production of anti-AAV5 antibodies, which is an expected finding following viral vector administration. In conclusion, no consistent association could be made with increases in ALT and cellular immune responses. Our data to date indicate that although antibodies to the AAV5 capsid develop in all patients, concomitant cellular immunity is not readily detectable, and has not been associated with liver function abnormalities nor changes in transgene expression levels. DisclosuresLong:BioMarin: Employment. Kim:BioMarin: Employment. Wong:BioMarin: Employment. Yang:BioMarin: Employment. Vettermann:BioMarin: Employment. Pryer:BioMarin: Employment. Hardet:Genethon: Employment. Kuranda:Genethon: Employment. Mingozzi:Genethon: Employment. Pierce:Roche: Consultancy; BioMarin: Consultancy. Schweighardt:BioMarin: Employment.