Antigen Gp100 Research Articles

Synthesis and Immunological Evaluation of Pentamannose-Based HIV-1 Vaccine Candidates.

Dense glycosylation and the trimeric conformation of the human immunodeficiency virus-1 (HIV-1) envelope protein limit the accessibility of some cellular glycan processing enzymes and end up with high-mannose-type N-linked glycans on the envelope spike, among which the Man5GlcNAc2 structure occupies a certain proportion. The Man5GlcNAc2 glycan composes the binding sites of some potent broadly neutralizing antibodies, and some lectins that can bind Man5GlcNAc2 show HIV-neutralizing activity. Therefore, Man5GlcNAc2 is a potential target for HIV-1 vaccine development. Herein, a highly convergent and effective strategy was developed for the synthesis of Man5 and its monofluoro-modified, trifluoro-modified, and S-linked analogues. We coupled these haptens to carrier protein CRM197 and evaluated the immunogenicity of the glycoconjugates in mice. The serological assays showed that the native Man5 conjugates failed to induce Man5-specific antibodies in vivo, while the modified analogue conjugates induced stronger antibody responses. However, these antibodies could not bind the native gp120 antigen. These results demonstrated that the immune tolerance mechanism suppressed the immune responses to Man5-related structures and the conformation of glycan epitopes on the synthesized glycoconjugates was distinct from that of native glycan epitopes on gp120.

Dynamics of Antibodies to Ebolaviruses in an Eidolon helvum Bat Colony in Cameroon.

The ecology of ebolaviruses is still poorly understood and the role of bats in outbreaks needs to be further clarified. Straw-colored fruit bats (Eidolon helvum) are the most common fruit bats in Africa and antibodies to ebolaviruses have been documented in this species. Between December 2018 and November 2019, samples were collected at approximately monthly intervals in roosting and feeding sites from 820 bats from an Eidolon helvum colony. Dried blood spots (DBS) were tested for antibodies to Zaire, Sudan, and Bundibugyo ebolaviruses. The proportion of samples reactive with GP antigens increased significantly with age from 0–9/220 (0–4.1%) in juveniles to 26–158/225 (11.6–70.2%) in immature adults and 10–225/372 (2.7–60.5%) in adult bats. Antibody responses were lower in lactating females. Viral RNA was not detected in 456 swab samples collected from 152 juvenile and 214 immature adult bats. Overall, our study shows that antibody levels increase in young bats suggesting that seroconversion to Ebola or related viruses occurs in older juvenile and immature adult bats. Multiple year monitoring would be needed to confirm this trend. Knowledge of the periods of the year with the highest risk of Ebolavirus circulation can guide the implementation of strategies to mitigate spill-over events.

Phase I trial of adjuvant mature autologous dendritic cell/allogeneic tumor lysate vaccines in combination with temozolomide in newly diagnosed glioblastoma.

BackgroundGlioblastoma (GBM) has poor prognosis despite aggressive treatment. Dendritic cell (DC) vaccines are promising, but widespread clinical use has not been achieved, possibly reflecting manufacturing issues of antigen choice and DC potency. We previously optimized vaccine manufacture utilizing allogeneic human GBM tumor cell lysate and potent, mature autologous DCs. Here, we report a phase I study using this optimized DC vaccine in combination with standard therapy.MethodsFollowing surgical resection and radiation with concurrent temozolomide (TMZ), newly diagnosed adult GBM patients received intradermal DC vaccines plus TMZ. Primary endpoints were safety and feasibility. Immune and treatment responses were recorded.ResultsTwenty-one patients were enrolled in this study. One progressed between leukapheresis and vaccine manufacture. Twenty patients received treatment per protocol. Vaccine doses (≥15) were generated following a single leukapheresis for each patient. No dose-limiting vaccine toxicities were encountered. One patient had symptomatic, histologically proven pseudoprogression. Median progression-free survival was 9.7 months. Median overall survival was 19 months. Overall survival was 25% at 2 years and 10% at 4 years. One patient remains progression-free 5 years after enrollment. Specific CD8 T-cell responses for the tumor-associated antigen gp100 were seen post-vaccination. Patients entered the trial with a leukocyte deficit compared to healthy donors which partly normalized over the course of therapy.ConclusionsThis vaccine platform is safe and highly feasible in combination with standard therapy for newly diagnosed patients. Imaging, histological, survival, and immunological data suggest a positive biological response to therapy that warrants further investigation.

Gut Microbiota Tuning Promotes Tumor-Associated Antigen Cross Presentation and Enhances CAR T Antitumor Effects

Gut Microbiota Tuning Promotes Tumor-Associated Antigen Cross Presentation and Enhances CAR T Antitumor Effects

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

From Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Immune Response to Cancer Onset via Molecular Mimicry and Cross-Reactivity.

Background and Objectives Whether exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may predispose to the risk of cancer in individuals with no prior cancers is a crucial question that remains unclear. To confirm/refute possible relationships between exposure to the virus and ex novo insurgence of tumors, this study analyzed molecular mimicry and the related cross-reactive potential between SARS-CoV-2 spike glycoprotein (gp) antigen and human tumor-suppressor proteins. Materials and Methods Tumor-associated proteins were retrieved from UniProt database and analyzed for pentapeptide sharing with SARS-CoV-2 spike gp by using publicly available databases. Results An impressively high level of molecular mimicry exists between SARS-CoV-2 spike gp and tumor-associated proteins. Numerically, 294 tumor-suppressor proteins share 308 pentapeptides with the viral antigen. Crucially, the shared peptides have a relevant immunologic potential by repeatedly occurring in experimentally validated epitopes. Such immunologic potential is of further relevancy in that most of the shared peptides are also present in infectious pathogens to which, in general, human population has already been exposed, thus indicating the possibility of immunologic imprint phenomena. Conclusion This article described a vast peptide overlap between SARS-CoV-2 spike gp and tumor-suppressor proteins, and supports autoimmune cross-reactivity as a potential mechanism underlying prospective cancer insurgence following exposure to SARS-CoV-2. Clinically, the findings call for close surveillance of tumor sequelae that possibly could result from the current coronavirus pandemic.

Zoonotic risk factors associated with seroprevalence of Ebola virus GP antibodies in the absence of diagnosed Ebola virus disease in the Democratic Republic of Congo.

BackgroundEbola virus (EBOV) is a zoonotic filovirus spread through exposure to infected bodily fluids of a human or animal. Though EBOV is capable of causing severe disease, referred to as Ebola Virus Disease (EVD), individuals who have never been diagnosed with confirmed, probable or suspected EVD can have detectable EBOV antigen-specific antibodies in their blood. This study aims to identify risk factors associated with detectable antibody levels in the absence of an EVD diagnosis.MethodologyData was collected from September 2015 to August 2017 from 1,366 consenting individuals across four study sites in the DRC (Boende, Kabondo-Dianda, Kikwit, and Yambuku). Seroreactivity was determined to EBOV GP IgG using Zaire Ebola Virus Glycoprotein (EBOV GP antigen) ELISA kits (Alpha Diagnostic International, Inc.) in Kinshasa, DRC; any result above 4.7 units/mL was considered seroreactive. Among the respondents, 113 (8.3%) were considered seroreactive. Several zoonotic exposures were associated with EBOV seroreactivity after controlling for age, sex, healthcare worker status, location, and history of contact with an EVD case, namely: ever having contact with bats, ever having contact with rodents, and ever eating non-human primate meat. Contact with monkeys or non-human primates was not associated with seroreactivity.ConclusionsThis analysis suggests that some zoonotic exposures that have been linked to EVD outbreaks can also be associated with EBOV GP seroreactivity in the absence of diagnosed EVD. Future investigations should seek to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD.

Relation between anti-Porphyromonas gingivalis antibody titers and HLA-DRB1 neutral alleles in individuals with rheumatoid arthritis

Objective This study aimed to determine the relation between titres of anti-Porphyromonas gingivalis (P. gingivalis) antibody and rheumatoid arthritis (RA) HLA-DRB1 susceptibility region associated with shared epitope (SE) using the Gregersen’s and de Vries’s classification methods. Material and Methods In this cross-sectional study, results of immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) anti-P. gingivalis antibodies, anti-citrullinated protein antibodies (ACPA), diagnosis for RA, and periodontal disease (PD), and a genetic study of the HLA DRB1 region were obtained from 50 patients with RA and 50 control individuals. Results Anti-P. gingivalis antibody levels and PD parameters were similar in control and RA groups. Anti-P. gingivalis antibodies were not associated with SE or ACPA. There was no association between ACPA and SE. However, de Vries’ classification in RA patients revealed an association between the HLA DRB1 neutral alleles and higher titres of anti-P. gingivalis antibodies as follows: IgG1 anti-P. gingivalis ≥ 1:400 (p = .039); IgG2 anti-P. gingivalis ≥ 1:400 with neutral/neutral genotype (N/N), being exclusive for RA (p = .008); and IgG2 anti-P. gingivalis ≥ 1:200 and N/N (p = .016). Conclusions Although no association was found between SE and anti-P. gingivalis antibodies; according to the de Vries’ classification, there was an existing association between HLA DRB1 neutral alleles, with high titres of IgG anti-P.gingivalis antibodies for RA, focussing on novel associations between P.gingivalis and RA.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-β (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy

Lack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

Abstract 1661: Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models

Abstract Whilst mitogen-activated protein kinase (MAPK) pathway inhibitors are approved therapies in mutant-BRAF driven cancers and have demonstrated a high response rate in the clinic, the duration of response is often short-lived. Approaches targeting the immune system have elicited durable responses and led to the approval of checkpoint inhibitors such as the anti-PD1 therapy, pembrolizumab, in indications where MAPK pathway activation is often observed, such as melanoma. Activation of the MAPK pathway has been associated with an immunosuppressive tumor microenvironment (TME). Further, preclinical studies have demonstrated that in addition to inhibiting MAPK activity in tumor cells, MAPK pathway inhibitors such as the BRAFV600E inhibitor dabrafenib or MEK inhibitor trametinib have promoted a more proinflammatory TME leading to upregulated antigen presentation on tumor cells, increased CD8+ T cell infiltration and tumor cell killing. Similar preclinical results were recently reported for a KRASG12C inhibitor, AMG 510, where treatment of in vivo models led to an increase in tumor infiltrating lymphocytes, macrophages, dendritic cells and an increase in active immune gene signatures leading to tumor immunity. We have recently described the discovery of ASTX029, which is currently undergoing clinical development in advanced solid tumors (NCT03520075). ASTX029 is a dual-mechanism ERK1/2 (ERK) inhibitor, inhibiting both the catalytic activity and phosphorylation of ERK, and shows potent inhibition of MAPK-activated tumor growth in preclinical models. Previous studies have demonstrated a difference in regulation of genes involved in response to type I interferon following treatment with dual-mechanism compared to catalytic ERK inhibitors. We investigated whether treatment with ASTX029 modulates antigen presentation and the TME in MAPK-activated tumors. Following treatment of cell lines in vitro, we observed ASTX029-dependent changes consistent with increased antigen presentation, including an increase in cell surface expression of MHC class I and the increase in gene expression of tumor-specific antigens gp100 and MART-1 following treatment of melanoma cells. We have previously demonstrated that ASTX029 has good bioavailability following oral dosing in mice. To further investigate the effect of ASTX029 on the TME, syngeneic tumors grown in immunocompetent mice dosed with ASTX029 were characterized in terms of immune cell composition of the TME and gene expression. Our results were consistent with increased immune activation, including increased interferon signaling and a change in the immune cell composition of the TME. These data demonstrate that treatment with ASTX029 leads to modulation of the TME and we hypothesize that to optimize therapeutic activity, ASTX029 could be partnered either with an immunomodulatory or tumor-directed agent. Citation Format: Chris Hindley, Andrea Biondo, Kim-Hien Dao, Matthew Davis, Lynsey Fazal, Roberta Ferraldeschi, Keisha Hearn, Vanessa Martins, Harpreet Saini, Martin Sims, Nicola Wallis, Nicola Wilsher, Harold Keer, John Lyons, Joanne Munck. Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated 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 1661.

Abstract 517: Tebentafusp induces transient systemic inflammation and modifies the micro-environment to sensitize uveal melanoma tumors to cytotoxic CD8 cells

Abstract Background: Tebentafusp is a unique investigational TCR-anti-CD3 bispecific fusion protein that targets melanoma-expressed gp100 antigen and has shown monotherapy activity in advanced metastatic melanoma [1, 2, 3]. In a phase I/II trial (NCT02570308) that enrolled late stage metastatic uveal melanoma patients, we explored the effects of tebentafusp on serum cytokines and on the tumor and its micro-environment. Methods: NCT02570308 was conducted in HLA-A*02:01+ patients with advanced uveal melanoma: This analysis focuses on patients who went through weekly dosing at Cycle 1, Day1 (C1D1, 20mcg) and C1D8 (30mcg), followed by a dose of 68mcg administered weekly at C1D15 onwards. Serum samples and tumor biopsies were taken pre-treatment and at multiple time points post-treatment, and were profiled as follows: a multiplex panel of 11 immune markers (Luminex) in sera from 124 patients; immunohistochemistry (IHC) and genome-wide transcriptional RNAseq analysis of up to 57 paired baseline and on-treatment biopsies at C1D16. Whole slide digital image analysis was used to calculate positive IHC cells per mm2 in the tumor. Results: Tebentafusp caused transient immune activation as evidenced by significant increases in serum levels of IFNγ and IFNγ-inducible chemokines including CXCL9/10/11 reaching maximal changes at 8-24 hours after dosing (fold change FC: 8-10). Induction of the T cell cytokines IL-2 and IL-10 was accompanied by transient increases in the inflammatory cytokines IL-6, TNFα and the chemokine CCL2 (FC: 5-14).After 3 doses of tebentafusp, a significant increase in CD3 positive immune cells in the tumor was seen (p&amp;lt;0.001; FC=3), particularly cytotoxic CD8+ cells, (p=0.002; FC=2). RNAseq analysis revealed significant induction of IFNα (FC=2) and IFNγ (FC=2) signatures. mRNAs known to be induced by IFNγ and implicated in response to anti-PD1/L1 were significantly increased: CXCL9/10/11, GZMB (FC=3-5). Expression of antigen presentation genes including HLA class 1, TAP1, 2 and PSMB8 was significantly induced (FC=2-3). Homeostasis mechanisms characterized by IDO1 and PD-L1 were induced (FC=4 and 2 respectively); however, low baseline FOXP3 expression levels were unchanged. These immune responses were accompanied by significant reductions in expression levels of tumor-specific melanoma markers gp100 and TYRP1 (30-40% reduction) and tumor proliferation genes including MKI67, BIRC5, CDK2 (20-30% reduction). Conclusions: Tebentafusp caused transient increases in inflammatory cytokines and chemokines in serum and modified the micro-environment of metastatic uveal melanoma tumors to sensitize to cytotoxic CD8+ cells.[1] NCT01211262; ASCO 2019 [2] Clinical Cancer Research; 2020.[3] #195; ESMO IO; 2020 Citation Format: Marcus O. Butler, Sarah Stanhope, Revashnee Naidoo, Emma Leach, Sarnjeet Kaur, Laura Collins, Shaad Abdullah, Koustubh Ranade, Alexander N. Shoushtari. Tebentafusp induces transient systemic inflammation and modifies the micro-environment to sensitize uveal melanoma tumors to cytotoxic CD8 cells [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 517.

Abstract 63: Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, translating these results in solid tumors remains challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Herein, we established a fully immunocompetent syngeneic mouse model and evaluated the interaction of CAR-M with the tumor microenvironment (TME) and the endogenous adaptive immune system. Murine bone marrow-derived CAR-expressing macrophages (muCAR-M) were efficiently engineered to express an anti-huHER2 CAR using the chimeric adenoviral vector Ad5f35. MuCAR-M, but not control untransduced (UTD) macrophages, specifically phagocytosed HER2+ target cancer cell lines and killed HER2-expressing murine CT26 colorectal and human AU-565 (HER2+) breast cancer cells in a dose-dependent manner. Moreover, CAR-M induced MHC-I and MHC-II expression on tumor cells and cross-presented tumor-associated antigens (TAA) resulting in CD8 T cell activation. To evaluate muCAR-M in an immunocompetent in vivo setting, BALB/c mice were engrafted with subcutaneous CT26-HER2+ tumors and treated with HER2-CAR or UTD macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cells reactive to the CT26 MHC-I antigen gp70, indicating enhanced epitope spreading. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2- CT26 rechallenge model, indicating the induction of long-term T cell memory against TAA. To evaluate the systemic anti-tumor immune response, we simultaneously engrafted BALB/c mice with CT26-HER2+ and CT26-Wt tumors on opposite flanks and treated mice with local administration of CAR-M restricted to the HER2+ tumors. After CAR-M treatment, 75% of mice cleared their CT26-HER2+ tumors and the growth rate of the contralateral CT26-Wt tumors was significantly reduced, demonstrating an abscopal effect. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with PD1 checkpoint inhibitor therapy in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further improved tumor control and overall survival. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Linara Gabitova, Yumi Ohtani, Olga Shestova, Saar Gill, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid 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 63.

Vγ9Vδ2 T Cells Concurrently Kill Cancer Cells and Cross-Present Tumor Antigens.

The human Vγ9Vδ2 T cell is a unique cell type that holds great potential in immunotherapy of cancer. In particular, the therapeutic potential of this cell type in adoptive cell therapy (ACT) has gained interest. In this regard optimization of in vitro expansion methods and functional characterization is desirable. We show that Vγ9Vδ2 T cells, expanded in vitro with zoledronic acid (Zometa or ZOL) and Interleukin-2 (IL-2), are efficient cancer cell killers with a trend towards increased killing efficacy after prolonged expansion time. Thus, Vγ9Vδ2 T cells expanded for 25 days in vitro killed prostate cancer cells more efficiently than Vγ9Vδ2 T cells expanded for 9 days. These data are supported by phenotype characteristics, showing increased expression of CD56 and NKG2D over time, reaching above 90% positive cells after 25 days of expansion. At the early stage of expansion, we demonstrate that Vγ9Vδ2 T cells are capable of cross-presenting tumor antigens. In this regard, our data show that Vγ9Vδ2 T cells can take up tumor-associated antigens (TAA) gp100, MART-1 and MAGE-A3 - either as long peptide or recombinant protein – and then present TAA-derived peptides on the cell surface in the context of HLA class I molecules, demonstrated by their recognition as targets by peptide-specific CD8 T cells. Importantly, we show that cross-presentation is impaired by the proteasome inhibitor lactacystin. In conclusion, our data indicate that Vγ9Vδ2 T cells are broadly tumor-specific killers with the additional ability to cross-present MHC class I-restricted peptides, thereby inducing or supporting tumor-specific αβTCR CD8 T cell responses. The dual functionality is dynamic during in vitro expansion, yet, both functions are of interest to explore in ACT for cancer therapy.

The Protective HIV-1 Envelope gp41 Antigen P1 Acts as a Mucosal Adjuvant Stimulating the Innate Immunity.

Mucosal nasal vaccine development, although ideal to protect from pathogens invading mucosally, is limited by the lack of specific adjuvant. We recently used P1, a conserved region of HIV-1 gp41-envelope glycoprotein, as efficient antigen in a prophylactic HIV-1 mucosal vaccine applied nasally. Herein, P1 immunomodulation properties were assessed on human nasal mucosal models by measuring induction of cytokine and chemokine production, intracellular signaling pathways, mucosal dendritic cell (DC) activation, and T cell proliferation. P1 adjuvant properties were evaluated by quantification of antigen-specific B cell responses against a model antigen in an in vitro immunization model. We now demonstrated that P1 has additional immunological properties. P1 initiates immune responses by inducing nasal epithelial cells to secrete the Th2-cytokine thymic stromal lymphopoietin (TSLP), a described mucosal adjuvant. Secreted TSLP activates, in turn, intracellular calcium flux and PAR-2-associated NFAT signaling pathway regulated by microRNA-4485. Thereafter, P1 induces mucosal dendritic cell maturation, secretion of TSLP in a TSLP-receptor (R)-dependent autocrine loop, but also IL-6, IL-10, IL-8, CCL20, CCL22, and MMP-9, and proliferation of CD4+ T cells. Finally, P1 acts as an adjuvant to stimulate antigen-specific B cell responses in vitro. Overall, P1 is a multi-functional domain with various immuno-modulatory properties. In addition to being a protective vaccine antigen for HIV prevention, P1 acts as adjuvant for other mucosal vaccines able to stimulate humoral and cellular antigen-specific responses.

Designs and Characterization of Subunit Ebola GP Vaccine Candidates: Implications for Immunogenicity.

The humoral responses of Ebola virus (EBOV) survivors mainly target the surface glycoprotein GP, and anti-GP neutralizing antibodies have been associated with protection against EBOV infection. In order to elicit protective neutralizing antibodies through vaccination a native-like conformation of the antigen is required. We therefore engineered and expressed in CHO cells several GP variants from EBOV (species Zaire ebolavirus, Mayinga variant), including a soluble GP ΔTM, a mucin-like domain-deleted GP ΔTM-ΔMUC, as well as two GP ΔTM-ΔMUC variants with C-terminal trimerization motifs in order to favor their native trimeric conformation. Inclusion of the trimerization motifs resulted in proteins mimicking GP metastable trimer and showing increased stability. The mucin-like domain appeared not to be critical for the retention of the native conformation of the GP protein, and its removal unmasked several neutralizing epitopes, especially in the trimers. The soluble GP variants inhibited mAbs neutralizing activity in a pseudotype transduction assay, further confirming the proteins’ structural integrity. Interestingly, the trimeric GPs, a native-like GP complex, showed stronger affinity for antibodies raised by natural infection in EBOV disease survivors rather than for antibodies raised in volunteers that received the ChAd3-EBOZ vaccine. These results support our hypothesis that neutralizing antibodies are preferentially induced when using a native-like conformation of the GP antigen. The soluble trimeric recombinant GP proteins we developed represent a novel and promising strategy to develop prophylactic vaccines against EBOV and other filoviruses.

Heterologous Prime Boost Vaccination Induces Protective Melanoma-Specific CD8+ T Cell Responses

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Abstract 3295: Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma

Abstract Background Tebentafusp, a TCR-anti-CD3 bispecific fusion protein designed to target a peptide from melanocyte lineage antigen gp100, has shown monotherapy activity in advanced cutaneous and uveal melanoma (UM) [1]. gp100 is expressed in normal melanocytes in skin and hair follicles. We previously reported a possible association between rash and overall survival (OS) in a Ph 1/2 trial of HLA-A*0201 patients with UM [1]. Anti-PD1 therapy for melanoma can induce vitiligo in 10-25% of patients, with one study showing a median time to onset of 126 days (range 52 - 453) and a potential association with clinical benefit [2]. We now investigate the association of melanocyte-related adverse events (MRAEs) and OS following tebentafusp treatment of patients with UM. Methods To test the differential potency of tebentafusp, HLA-A2+ epidermal skin derived melanocytes (5 healthy donors) and gp100+ and gp100- melanoma cell lines were incubated with PBMCs and increasing tebentafusp concentrations, and tested in IFNγ (measure of T cell activation) and GrB (measure of T cell mediated killing) ELISPOT assays. The clinical trial database (NCT02570308; Ph1 n=19, Ph2 n=23) was also queried for AEs suggestive of melanocyte origin, including vitiligo, leukotrichia, and skin hyperpigmentation. Simon-Makuch estimates were derived to visualize the association between MRAEs and OS while adjusting for immortal time bias, and Mantel-Byar method was used to estimate odds of death associated with experiencing MRAEs. Results In vitro, tebentafusp redirected and activated T cells against gp100+ melanoma and skin-derived melanocytes but not gp100- melanoma; in all cases, potency for melanocytes was less than melanoma, potentially reflecting differential peptide presentation. 24 of 42 patients (57%) developed one or more MRAEs including vitiligo/skin hypopigmentation (28%), leukotrichia (33%), and hyperpigmentation (21%), with median time to onset of 67 days (range 24-221). The estimated probability of experiencing a MRAE within 6-months of continuous treatment is 70%. Among the 24 patients with MRAEs, 22 (92%) and 20 (83%) experienced rash and pruritus, respectively. Rash preceded MRAEs in all patients with both events, while pruritus preceded MRAEs in 77% of patients. In a post-hoc analysis, experiencing a MRAE was associated with a 72% lower odds of death compared to those who did not experience these AEs. Conclusion Over half of tebentafusp-treated patients with advanced UM experienced at least one MRAE, supporting the hypothesis that tebentafusp can redirect T cells against gp100+ cells in patients. This rate of MRAEs is higher than historically reported for anti-PD1 based therapies. MRAEs appear to be associated with prior rash, and even accounting for the time bias of onset, may be associated with OS. 1. Sato T. J Clin Oncol 36, 2018, suppl, 9521 2. Hua C. JAMA Dermatol 2016;152:45-51 Citation Format: Marlana Orloff, Joseph J. Sacco, Paul Nathan, Chris Holland, Chris Cohen, Jane Harper, Shaad E. Abdullah, Takami Sato, Richard D. Carvajal. Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3295.

Abstract 2198: The anti-tumor enhancement of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by IFNα and 5-Aza-2'-deoxycytidine treatments correlates with intratumoral CCL19 but not CCL21 expression

Abstract Background: The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated antigens gp100 and/or tyrosinase-related protein 2 (Trp2) have delayed tumor growth and increased survival time in the B16F10 mouse melanoma model system compared to vaccines lacking the chemokine. To further enhance the therapeutic effects of the vaccine, our laboratory has added type-I interferon (IFNα) and 5-Aza-2'-deoxycitidine (5Aza) to the therapy. Here, we report that the enhancement previously seen by the combination of IFNα, 5Aza, and a MIP-3α-Gp100-Trp2 (MGpTrp2) DNA vaccine correlates with increases of tumor-infiltrating CD8+T-cells (CD8 TILs) and intratumoral expression of CCL19 but not CCL21. Methods: Beginning on day five post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given two days later. 5Aza was given intraperitoneally at 1mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry with intracellular cytokine staining, respectively. Results: Previous work has shown that the combination of IFNα, 5Aza, and MGpTrp2 led to significantly reduced tumor burden and overall increases in mouse survival dependent upon all three components. The addition of 5Aza and IFNα to the vaccine affected T-cell tumor infiltration, increasing the proportion of CD3+CD8+ cells in gated TILs by 92% over vaccine alone (p&amp;lt;0.0001), which correlated with tumor size (r2: 0.507; p&amp;lt;0.0001). Interferon stimulated genes such as Mx1, MHC1, CXCL10, and GranzymeB were modestly upregulated in the tumor lysate. However, CCL19, normally expressed constitutively in lymphoid tissues, was upregulated 10-fold compared to vaccine alone (p&amp;lt;0.0001) and was correlated with tumor size (r2: 0.195; p=0.002). The CCL19 receptor, CCR7, was upregulated to a lesser degree (5-fold over vaccine; p&amp;lt;0.001), and interestingly its partner chemokine CCL21 did not have significantly different expression patterns across groups (p=0.14 to vaccine). Conclusions: Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This enhancement was correlated with CD8+ TIL recruitment and with the expression of the T-cell trafficking chemokine CCL19. Further potential therapy optimization currently undergoing investigation offers promise for this line of investigation to become a novel melanoma therapy. Citation Format: James T. Gordy, Avinaash K. Sandhu, Samuel K. Ayeh, Aakanksha Kapoor, Emily Kim, Petros C. Karakousis, Richard B. Markham. The anti-tumor enhancement of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by IFNα and 5-Aza-2'-deoxycytidine treatments correlates with intratumoral CCL19 but not CCL21 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2198.

A phase I/II first-in-human study of a novel anti-MAGE-A4 TCR/anti-CD3 bispecific (IMC-C103C) as monotherapy and in combination with atezolizumab in HLA-A*02:01-positive patients with MAGE-A4-positive advanced solid tumors (IMC-C103C-101).

TPS3165 Background: ImmTAC bispecifics are unique T cell receptor (TCR)/anti-CD3 bispecific molecules that are designed to redirect polyclonal T cells against intracellular antigens, in contrast to antibody-based therapies, which are limited to extracellular antigens. ImmTAC molecules recognize a specific peptide presented on defined Class I HLA molecules via an affinity enhanced, engineered, soluble TCR. Through the addition of an anti-CD3 scFv effector domain fused to the TCR targeting domain, ImmTAC molecules redirect T cell activity against cancer cells, regardless of the specificity of the T cell. IMC-C103C is an ImmTAC being investigated against MAGE-A4, which is among the most commonly expressed cancer testis antigens in solid malignancies, but with minimal to absent expression on normal tissues and/or hematopoietic cells. The most advanced ImmTAC in development, tebentafusp (IMCgp100), directed against melanocyte-associated lineage antigen gp100, has shown monotherapy activity in uveal melanoma and PD-1 refractory advanced cutaneous and uveal melanoma. Tebentafusp is being further evaluated in combination with durvalumab and tremelimumab. Methods: IMC-C103C-101 is a multi-center, open-label, Phase 1/2 first-in-human study of IMC-C103C as monotherapy and combination with atezolizumab in HLA-A*02:01-positive patients with MAGE-A4-positive advanced cancers. The study includes IMC-C103C monotherapy (Q1W) dose escalation, followed by expansion into indication specific arms to test for efficacy in defined patient cohorts. Concurrently, combinations with atezolizumab are planned. Primary objectives of dose escalation are to identify the MTD/RP2D, and characterize safety/tolerability. Secondary objectives include an assessment of efficacy (best overall response by RECIST v1.1), PK, PD, and ADA. IMC-C103C monotherapy dose escalation is in progress. Clinical trial information: NCT03973333 .