Expansion Of Antigen-specific CD8 Research Articles

Genetically engineered CD80-pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement.

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide-MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols.

Human CD34+-derived complete plasmacytoid and conventional dendritic cell vaccine effectively induces antigen-specific CD8+ T cell and NK cell responses in vitro and in vivo

Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34+ stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8+ T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-α production. Additionally, antigen-specific CD8+ T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT.

Abstract 2853: Co-delivery of antigen-encoding mRNA and signal 2/3 mRNAs to PBMCs by Cell Squeeze® technology generates SQZ™ eAPCs that prime CD8+T cells in a humanized mouse model

Abstract Antigen-specific CD8+ T cells are critical for mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells. To engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNAs encoding for non-self-antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membrane-bound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines at the immune synapse and limit off-target effects. Flow cytometry confirmed translation of delivered signal 2/3 mRNAs by all major subsets within PBMCs: T cells, B cells, NK cells, and monocytes. The potency of these SQZ™ eAPCs was assessed in vitro by culturing the eAPCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA. Using this approach, we demonstrate that Cell Squeeze® co-delivery of antigen mRNA and signal 2/3 mRNAs significantly enhances CD8+ T cell responses to a variety of antigens, including CMV pp65, Influenza M1, HPV16 E6, and HPV16 E7. Furthermore, we demonstrate that SQZ™ eAPCs drive significant expansion of antigen-specific CD8+ T cells in a humanized mouse model. Thus, we demonstrate that Cell Squeeze® can deliver multiple mRNAs encoding for signals 1, 2, and 3 to human PBMCs and has the potential to generate enhanced APCs that drive strong CD8+ T cell responses against multiple antigens. The versatility of this approach has the potential to enable rapid exchange of mRNA to encode for other antigens or T cell activation signals. Citation Format: Michael F. Maloney, Emrah Ilker Ozay, Katarina Blagovic, Carolyne Smith, Andrea A. Silva, Amber Martin, Sanjana Manja, Madhav Upadhyay, Lindsay J. Moore, Ryan Stagg, Henry Mack, Christine Trumpfheller, Pablo Umana, Armon Sharei, Howard Bernstein, Scott M. Loughhead. Co-delivery of antigen-encoding mRNA and signal 2/3 mRNAs to PBMCs by Cell Squeeze® technology generates SQZ™ eAPCs that prime CD8+T cells in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2853.

Development and Characterization of a Novel Non-Lytic Cancer Immunotherapy Using a Recombinant Arenavirus Vector Platform.

Engineered viral vectors represent a promising strategy to trigger antigen-specific antitumor T cell responses. Arenaviruses have been widely studied because of their ability to elicit potent and protective T cell responses. Here, we provide an overview of a novel intravenously administered, replication-competent, non-lytic arenavirus-based vector technology that delivers tumor antigens to induce antigen-specific anti-cancer T cell responses. Preclinical studies in mice and cell culture experiments with human peripheral blood mononuclear cells demonstrate that arenavirus vectors preferentially infect antigen-presenting cells. This, in conjunction with a non-lytic functional activation of the infected antigen-presenting cells, leads to a robust antigen-specific CD8+ T cell response. T cell migration to, and infiltration of, the tumor microenvironment has been demonstrated in various preclinical tumor models with vectors encoding self- and non–self-antigens. The available data also suggest that arenavirus–based vector therapy can induce immunological memory protecting from tumor rechallenge. Based on promising preclinical data, a phase 1/2 clinical trial was initiated and is currently ongoing to test the activity and safety of arenavirus vectors, HB-201 and HB-202, created using lymphocytic choriomeningitis virus and Pichinde virus, respectively. Both vectors have been engineered to deliver non-oncogenic versions of the human papilloma virus 16 (HPV16) antigens E7 and E6 and will be injected intravenously with or without an initial intratumoral dose. This dose escalation/expansion study is being conducted in patients with recurrent or metastatic HPV16+ cancers. Promising preliminary data from this ongoing clinical study have been reported. Immunogenicity data from several patients demonstrate that a single injection of HB-201 or HB-202 monotherapy is highly immunogenic, as evidenced by an increase in inflammatory cytokines/chemokines and the expansion of antigen-specific CD8+ T cell responses. This response can be further enhanced by alternating injections of HB-202 and HB-201, which has resulted in frequencies of circulating HPV16 E7/E6-specific CD8+ T cells of up to 40% of the total CD8+ T cell compartment in peripheral blood in analyses to date. Treatment with intravenous administration also resulted in a disease control rate of 73% among 11 evaluable patients with head and neck cancer dosed every three weeks, including 2 patients with a partial response.

LTBK-01. INO-5401 AND INO-9012 DELIVERED INTRAMUSCULARLY (IM) WITH ELECTROPORATION (EP) IN COMBINATION WITH CEMIPLIMAB (REGN2810) IN NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND Novel T cell-enabling therapies, in combination with checkpoint inhibition, may improve OS in GBM. INO-5401 (synthetic DNA plasmids encoding hTERT, WT-1, PSMA) plus INO-9012 (synthetic DNA plasmid encoding IL-12), and the PD-1 immune checkpoint inhibitor cemiplimab, is given to patients with newly diagnosed GBM to evaluate tolerability, efficacy and immunogenicity. METHODS Phase I/II, single arm, 2 cohort study (A: MGMT unmethylated, B: MGMT methylated). Primary endpoint is safety; efficacy and immunogenicity are secondary. Nine mg INO-5401 plus 1 mg INO-9012 (every 3 weeks x 4 doses, then Q9W) is given IM with EP by CELLECTRA® 2000 with cemiplimab (350 mg IV Q3W). RT is given as 40 Gy over 3 weeks. TMZ is given with radiation (all patients), and adjuvantly (Cohort B only). RESULTS Fifty-two subjects enrolled: 32 in Cohort A; 20 in Cohort B. 35% women; median age 60 years (19–78 years). The adverse event profile is consistent with single-agent (INO-5401, INO-9012, EP and cemiplimab) reported events. OS at 12 months was 84.4% (Cohort A) and 85% (Cohort B). OS at 18 months in Cohort A is 50% (95% CI 31.9 - 68.1); median OS is 17.9 months (14.5 - NR); Cohort B OS18 and median OS will be presented. Tumor gene transcripts at diagnosis confirmed expression of INO-5401 antigens. Peripheral immune responses following INO-5401 revealed antigen-specific T cell responses by Interferon gamma ELISpot and flow cytometry, including cytokine production and expansion of antigen specific CD8+T cells with lytic potential. CONCLUSIONS INO-5401 + INO-9012, a novel DNA plasmid immunotherapy, demonstrates acceptable risk/benefit and generates robust systemic immune responses to encoded tumor antigens when administered with cemiplimab and RT/TMZ in newly diagnosed GBM patients. Overall survival is encouraging. Clinical trial information: NCT03491683.

Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses.

Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells.

290-OR: Diabetes Progression in IFG-Receptor Deficient NOD Mice Is Associated with Increased CD8+ T Lymphocyte Sensitivity to Common Gamma Chain Cytokines

IFN-γ is a potential drug target in type 1 diabetes and this is part of the rationale for trialing JAK inhibitors (JAKi) for treatment of new onset T1D. It regulates migration and adhesion of leucocytes, as well as expression of MHC class I on islet beta cells. However, neither genetic deficiency of IFN-γ nor its receptor in NOD mice modifies disease progression. To understand this paradox we characterised islet antigen-specific T cells in NOD mice lacking the IFN-γ receptor (Ifngr) generated directly in NOD mouse embryos using CRISPR/Cas9 gene editing. Islet antigen-specific CD8+ T cells (proinsulin B15-23 and IGRP206-214) and CD4+ T cells (proinsulin B10-23and BDC2.5 mimotope) were quantified using tetramer enrichment and flow cytometry. At 125 days of age, insulin-specific (177±41 cells in wild type and 1799±1123 cells in Ifngr mutant, p=0.036) and IGRP-specific CD8+ T cells (2524±735 cells in wild type and 30598±10426 cells in Ifngr mutant, p<0.0001) were present in significantly greater numbers in Ifngr mutant NOD mice. There were no differences in antigen-specific CD4+ T cells. Islet infiltrating T cells from Ifngr mutant mice had increased responses to common gamma chain (γc) cytokines, prevented by a JAKi, and reduced expression of suppressor of cytokine signalling-1 (Socs1) mRNA. Our data suggest that as well as its pro-inflammatory effects, IFN-γ homeostatically controls the expansion of antigen-specific CD8+ T cells by regulating their response to γc cytokines, by mechanisms that include regulation of SOCS-1 expression. Expansion of antigen-specific CD8+ T cells is likely to explain the lack of protection from diabetes in Ifngr mutant NOD mice. JAKi therapy experimentally controlled this increased responsiveness to γc cytokines. Our data show mechanisms underlying protection from diabetes in NOD mice by JAKi and the lack of protection by blocking IFN-γ alone. Disclosure T. Kay: None. G. Jhala: None. B. Krishnamurthy: None. H.E. Thomas: None.

Novel HPV-E7 immune modulators to activate HPV-specific T cells to eliminate cervical cancer.

e18027 Background: Human papilloma virus (HPV) infection can lead to several types of cancers in both men and women. HPV+ tumor cells constitutively express the HPV-E7 antigen which can act as an oncogene to promote tumor growth and malignant transformation. Here, we report the application of novel Tavo Immune Modulator (TIM) biologics molecules which are consisted of a pMHC complex with an epitope peptide derived from HPV-E7 and co-stimulatory modulators of T cell activity. The HPV-E7 TIM molecules can specifically recognize and activate HPV-E7-specific T cells for the elimination of HPV affected cells. Methods: HPV-E7 TIM molecules were engineered as fusion molecules with HLA-A*02:01 MHC complexed with an HPV-E7 (11-20) epitope peptide at the N-termini, and various T cell costimulatory modulators at the C-termini of IgG heavy and light chains. TIM molecules were expressed in Expi293 cells and purified by Protein A affinity chromatography. Specific binding of TIM with HPV-E7 specific T cells was confirmed by immunostaining and flow cytometry. The activation and expansion of antigen specific CD8+ T cells were elucidated in T cell activation and recall assays. Results: HPV-E7 TIM molecules with various T cell co-stimulator molecules were engineered to specifically recognize HPV-specific T cells. Activation of T cells was antigen-specific and depended on the presence of an engineered T cell modulatory component on the TIM framework. The effects of various costimulatory molecules in different combinations on T cell activation were explored and an optimal combination was identified which facilitated high potency antigen-specific T cell activation. Such molecular combinations could facilitate T cell expansion and activation in T cell recall assays. Efficacy of HPV-E7 TIM molecules by inhibiting tumor growth in a syngeneic tumor model is ongoing. Conclusions: This study demonstrates that HPV-E7 TIM molecules selectively recognize and activate HPV-specific CD8+ T cells in the presence of a combination of two T cell costimulatory factors. Such novel biologics provide distinctive approaches in the treatment of HPV-related cancers and warrant further investigation. Additional in vitro and in vivo studies are ongoing to demonstrate the utility in eliminating HPV-infected tumor cells. Full data will be presented at the meeting.

TCR engaging antigen-scaffolds for targeted expansion of functionally improved T cells for adoptive cell therapy

Abstract Background Precise targeting of patient’s tumor can be facilitated through T cells recognizing specific pMHC complexes on these tumors. Once such antigen landscape is defined, an additional challenge relates to the ability to mount a functional T cell response specific to such antigens. Current T Cell expansion techniques provide no efficient strategy for antigen-specific stimulation and results largely in exhausted T cells. Here we present a new technology for ex-vivo peptide-MHC directed expansion and functional enhancement of T cells, using artificial antigen-presenting scaffolds (Ag-scaffolds). Methods Dextran backbone conjugated with streptavidin was used for Ag-scaffold assembly. Such backbone was incubated together with biotinylated MHC class I molecules holding a given peptide (pMHC) and biotinylated human cytokines in various ratios. PBMCs from healthy donors with known virus epitopes and from melanoma patients with known epitopes were expanded for 2 weeks with Ag-scaffold, added when the cell cultures were initiated and twice per week for two weeks. The expansion of antigen-specific CD8 T cells was monitored and analyzed by flow cytometry. Results Here we demonstrate the value of the antigen-scaffold based expansion, by the expansion of virus-CD8 T cells from peripheral blood, and tumor-specific T cells from both peripheral blood and tumor site. We can favorably expand both neo- and shared antigen specific T cell populations. The resulting T cell product exhibit a multifunctional cytokine profile upon antigen challenge, high levels of CD28 expression, and reduced levels of PD-1 expression. Importantly, numerous different pMHC-specific T-cell populations can be stimulated in a single culture, and we can obtain better tumor killing properties than conventional TIL-ACT products. Conclusion This study presents a new technology for expanding functionally enhanced antigen-specific CD8 T cells suited for implementation to ACT strategies. T cell phenotype and functionality are known to be important parameters in successful adoptive T cell transfer, and hence it´s plausible that antigen scaffold based expanded T cell product may improve treatment outcome compared to conventional expansion strategies. Legal entity responsible for the study The authors. Funding Innovation Fund Denmark. Disclosure All authors have declared no conflicts of interest.

Regulation of PD-L1 expression in stimulated antigen specific CD8+ T cells by immunoglobulin 4 (IgG4)

Abstract Background Many studies have focused on finding a correlation between the responsiveness to immune checkpoint (IC) therapies and the expression level of IC markers. Here, we report the studies on the expression of co-inhibitory molecules on healthy donor peripheral mononuclear cells (PBMC). We co-cultured PBMCs from different donors with recall antigen peptide to analyze the responses to PD-1 blockade antibody treatment. We have used our previously reported optimized recall antigen assay in which the expanded antigen specific CD8+ T cells (TET+) were measured as a readout after the peptide stimulation. Interestingly, the IgG4 appears to reduce the TET+ cell population. To further study this potential effect of IgG4, we conducted a series of immune-profiling studies by comparing TET+ gated and TET− CD8+ gated T cells. Results and conclusion Healthy donor PBMCs were stimulated with recall peptide alone, or peptide plus either PD-1 blockade, or IgG4 and evaluated the PD-1, PD-L1, Tim-3, and Lag-3 expressions in either T cells or in monocytes. We then compared such expressions in TET+ and TET− CD8+ T cells. Unexpectedly, IgG4 isotype control treatment was associated with upregulated PD-L1 expression only in the TET+ CD8+ T cells. To confirm whether the decrease of TET+ population in our assay was caused by the up regulation of PD-L1, we blocked the PD-1 signal transduction pathway by treating the cells with a validated PD-1 blocking antibody. As a result of PD-1 blockade, the expansion of TET+ cell population was recovered by anti-PD-1 blocking treatment. Taken together, these results suggest that IgG4 treatment with recall peptide up regulates PD-L1 expression and therefore reduces the expansion of antigen specific CD8+ T cells in some donors.

Lymphocytes are a major source of circulating soluble dipeptidyl peptidase 4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease that is expressed constitutively by many haematopoietic and non-haematopoietic tissues. It exists as a membrane-associated protein, as well as in an active, soluble form (herein called sDPP4), present at high concentrations in bodily fluids. Despite the proposed use of sDPP4 as a biomarker for multiple diseases, its cellular sources are not well defined. Here, we report that individuals with congenital lymphocyte immunodeficiency had markedly lower serum concentrations of sDPP4, which were restored upon successful treatment and restoration of lymphocyte haematopoiesis. Using irradiated lymphopenic mice and wild-type to Dpp4-/- reciprocal bone marrow chimeric animals, we found that haematopoietic cells were a major source of circulating sDPP4. Furthermore, activation of human and mouse T lymphocytes resulted in increased sDPP4, providing a mechanistic link between immune system activation and sDPP4 concentration. Finally, we observed that acute viral infection induced a transient increase in sDPP4, which correlated with the expansion of antigen-specific CD8+ T cell responses. Our study demonstrates that sDPP4 concentrations are determined by the frequency and activation state of lymphocyte populations. Insights from these studies will support the use of sDPP4 concentration as a biomarker for inflammatory and infectious diseases.

Abstract 5559: Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy

Abstract Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely due to the fact that these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the fact that the ECDs are in opposite orientation. Here we report the generation of a two-sided fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain. Initial confirmation of the structural integrity of TIM3-Fc-OX40L was performed using functional ELISA assays, wherein simultaneous binding to GAL9 and OX40 was observed. A potential advantage of utilizing the intact ECD of TIM3 is the ability to bind all candidate TIM3 ligands, and the TIM3 end of the fusion protein binds both GAL9 and phosphatidylserine (PS) on the surface of human tumor cells. The OX40L end of the fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in cells engineered to overexpress OX40 and an NFκB-luciferase reporter in the absence of Fc receptor cross-linking. Additionally, the TIM3-Fc-OX40L ARC added to primary human PBMCs along with the super-antigen Staphylococcal enterotoxin B, induced secretion of the cytokines IL2 and TNFα. In vitro human T cell/tumor cell co-culture studies indicated that TIM3-Fc-OX40L stimulated enhanced T cell mediated killing of GAL9 positive human tumor cells as compared to OX40L-Fc controls. In vivo, TIM3-Fc-OX40L stimulated significant expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum. Time-lapse high content live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L functionally tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was significantly superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. Importantly, a pharmacodynamic biomarker of tumor rejection was identified by the elevation in serum cytokines post-treatment. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the same microenvironmental context, at the time in which T cells are engaging cognate tumor antigen. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5559.

Abstract 4690: Hexavalent CD27 agonists show single agent anti-tumor activity and enhanced memory formation in mouse syngeneic tumor models

Abstract Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins comprise a perfect molecular mimic of the TNFSF cytokine structure and are based on dimerization of trivalent single-chain TNFSF receptor-binding domains (scTNFSF-RBD) via a Fc-γ receptor (FcγR) binding deficient immunoglobulin Fc domain. As a result of this molecular design, HERA proteins are capable of clustering six receptors in a spatially well-defined manner. Signaling following treatment with the Apogenix HERA “scTNFSF-RBD-Fc fusion proteins” is entirely independent of secondary crosslinking through FcγRs that is required for many agonistic anti-TNFRSF antibodies. The HERA engineering concept has been successfully translated to TRAIL, GITRL, CD40L, LIGHT and CD27L resulting in agonists that are currently in development. CD27L is a potent co-stimulatory molecule that drives T cell activation and survival through interaction with its receptor (CD27). HERA-CD27L is expressed in CHO suspension cells followed by a lab-scale purification process that results in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor with high affinity. In vitro, HERA-CD27L was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Binding significantly increased T cell expansion following αCD3/αCD28 stimulation and leads to increased expression of OX40 on CD4+ T cells and 4-1BB on CD8+ T cells, respectively. In vivo, a single dose of 10mg/kg HERA-CD27L increases clonal expansion of antigen-specific CD8+ T cells upon immunization with Ovalbumin (Ova) in the mouse OT-1 model with a kinetics leading to peak levels of >25% Ova-specific CD8+ T cells at day 6 after treatment. Anti-tumor efficacy of HERA-CD27 was evaluated in MC38-CEA and CT26 colorectal syngeneic murine tumor models. In both models treatment with HERA-CD27L resulted in a dose dependent inhibition of tumor growth. CT26 tumor bearing mice treated with 1mg/kg HERA-CD27L, twice weekly showed an 85% tumor-growth inhibition (TGI) compared to the control group. A significant TGI of 48% could be observed in the MC38-CEA model upon treatment with 10mg/kg, twice weekly. Analysis of peripheral lymphoid tissues in the MC38-CEA bearing mice could furthermore show that HERA-CD27L treatment is accompanied with enhanced memory formation in both CD4+ & CD8+ T cells. In summary, the data on the hexavalent HERA-CD27L indicate a potent immune cell driven anti tumor efficacy. Therefore, HERA-CD27 agonists could be applied for the treatment of cancer as a single agent or in combination with check-point Inhibitors. Citation Format: Christian Gieffers, David Richards, Jaromir Sykora, Mauricio Redondo-Müller, Meinolf Thiemann, Christian Merz, Karl Heinonen Heinonen, Viola Marschall, Harald Fricke, Oliver Hill. Hexavalent CD27 agonists show single agent anti-tumor activity and enhanced memory formation in mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4690. doi:10.1158/1538-7445.AM2017-4690

Abstract 567: In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens

Abstract Cancer immunotherapy relies on presentation of shared- and neo- antigens from a patient's tumor cells for recognition and clearance by the immune system. However, the tumor microenvironment deploys multiple strategies to evade immune recognition and often remains non-immunogenic, which is one of the challenges that need to be addressed when designing new therapies. Among the strategies to increase a tumor's immunogenicity is their genetic manipulation in situ via expression of molecular chaperones, T cell costimulators and/or pro-inflammatory genes using DNA/RNA vectors packaged in oncolytic viruses, lipid based components or through electroporation. In vivo electroporation-mediated gene transfer of IL-12 triggers tumor regression and systemic anti-tumor immune responses in experimental mouse models and in patients, demonstrating the feasibility of this intratumoral (IT) gene-transfer technology. We set out to test whether intratumoral electroporation of Gp96-Ig/Fc-OX40L, a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression, would generate a robust anti-neoantigen CD8+ T cell response. Gp96-Ig/Fc-OX40L is a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression. To assess antigen-specific CD8+ expansion, mice were adoptively transferred with OT-I cells after B16.F10-ovalbumin cells were injected to generate primary and contralateral melanotic tumors. Contralateral tumors were monitored to assess whether a systemic CD8+ T cell response could be elicited following primary tumor electroporation. IT electroporation of DNA expressing Gp96-Ig/Fc-OX40L in the primary tumor triggered a significant expansion of antigen-specific OT-I cells, which was absent in control mice. Remarkably, increases in antigen-specific OT-I cells correlated with regression of both the treated primary and untreated contralateral tumors. We further validated our findings in a CT26 mouse colorectal cancer tumor model, in which the expression of Gp96-Ig/Fc-OX40L from electroporated DNA stimulated an expansion of antigen-specific CD8+ T cells and again led to regression of both the treated primary and untreated contralateral tumor. Our findings demonstrate that in situ manipulation of intratumoral cells to express Gp96-Ig/Fc-OX40L stimulates potent antigen-specific cross priming to tumor specific neoantigens that culminates in robust systemic anti-tumor response. These findings provide exciting proof-of-principal and warrant further investigation into the direct delivery of molecular chaperones such as Gp96-Ig/Fc-OX40L and/or pro-inflammatory molecules for elevating the immunogenicity of tumors for a potent anti-tumor CD8+ T cell response. Citation Format: Suresh de Silva, George Fromm, Jamil Haque, Jean S. Campbell, Robert H. Pierce, Taylor H. Schreiber. In vivo intra-tumoral electroporation of Gp96-Ig/Fc-OX40L stimulates CD8+ T cell cross-priming to tumor specific neoantigens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 567.

Systemic DC Activation Modulates the Tumor Microenvironment and Shapes the Long-Lived Tumor-Specific Memory Mediated by CD8+ T Cells.

Strategies to reprogram the tumor microenvironment are being explored to improve cancer immunotherapy. In one approach, we have targeted dendritic cells (DC) to improve their function with adjuvant vector cells (aAVC) that are engineered from NKT ligand-loaded CD1d(+) allogeneic cells transfected with tumor antigen mRNAs. Here, we report the finding that this approach also programs local immune responses by establishing tertiary lymphoid structures (TLS), which include expanded antigen-specific CD8(+) T-cell clones, mobilized DCs, and normalized tumor vasculature. aAVC therapy also expanded specific Vβ-expressing antitumor T-cell clones, leading to the formation of long-term memory T cells. When combined with PD-1 blockade, aAVC infusion triggered regression of poorly immunogenic tumor cells that did not respond to PD-1 blockade alone, as well as expansion of antigen-specific CD8(+) T-cell clones in the tumor. The findings of this study help to inform a next-generation platform for the generation of efficacious cancer vaccines. Cancer Res; 76(13); 3756-66. ©2016 AACR.

Exhaustion of Human Dendritic Cells Results in a Switch from the IL-15R/IL-15- to IL-2R/IL-2-driven Expansion of Antigen-specific CD8+ T Cells

Abstract Maturation of dendritic cell (DC) is considered to be crucial for their ability to promote clonal expansion of antigen-specific T cells. Here, we show that the ability to expand human antigen-specific CD8+ T cells is a feature of specialized “effector” DCs at early stages of maturation, and it is suppressed in “exhausted” DCs at late stages of maturation and in DCs matured in the conditions of chronic inflammation. Effector DCs, at early stages of maturation induced by TLR ligands and interferons, express membrane IL-15Rα/IL-15 complex, which promotes highly efficient and IL-2/IL-2Rα-independent expansion of MART-1-specific IL-15Rαhigh CD8+ T cells and cooperates with DC-produced IL-12p70 in inducing poly-functional CTL phenotype. In sharp contrast, exhausted DCs which lack membrane IL-15Rα/IL-15 complex, utilize a significantly less efficient IL-2Rα/IL-2 trans-presentation pathway to co-stimulate CD8+ T cells. The currently-reported switch from the IL-15Rα/IL-15- to IL-2Rα/IL-2 costimulatory pathway in exhausted DCs helps to understand the role of inflammation in clonal expansion and contraction of CD8+ T cells at different stages of immune responses and helps to design improved modes of immunotherapy of cancer and chronic infections.

FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3)ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

Abstract LB-227: Novel synthetic vesicle for rapid in vivo expansion of CD8 T cells can significantly improve checkpoint inhibitor therapy

Abstract Harnessing the immune system for cancer treatment offers many advantages over traditional methods as the immune system can generate tumor-specific killer cells and long-term memory cells to prevent recurrence. The success of recent clinical trials using T cell therapies has proven the potential of immunotherapy, but the ex vivo generation of T cells in these trials is costly and labor-intensive. Here, we developed wholly synthetic, pathogen-mimicking nanocapsules, Interbilayer-Crosslinked Multilamellar Vesicles (ICMVs), to deliver antigens to the lymphoid system, which prime large tumor-specific CD8 T cell populations in vivo. ICMVs use bilayer-to-bilayer crosslinks in the walls of multi-lamellar lipid vesicles to create an easily manufacturable, highly stable virus-like particle to present antigens and/or adjuvants to immune cells. ICMV carrying HPV16 E6 and E7 full-length proteins were administered alone or co-encapsulated with TLR agonists to mice. CD8 T cell responses were assessed by tetramer staining and intracellular cytokine staining (ICS) was done to assay functionality. Efficacy of ICMV treatment was demonstrated by monitoring tumor size and survival in C57BL/6 mice bearing a lethal dose of TC-1 tumor cells. ICMVs induced unprecedented antigen-specific CD8 T cell frequencies against HPV whole proteins even without added adjuvant. Repeat doses of ICMVs boosted CD8 responses resulting in 30% of the total CD8 T cell population in blood being E7-specific while treatment with free protein elicited <1% E7-specific cells, indicating that ICMVs promote strong cross-presentation of antigens. When ICMVs were combined with anti-PD-1 to treat tumor-bearing mice, 10-15% of CD8 T cells produced IFNγ+ in response to either E6 or E7 antigens, while treatment with anti-PD-1 alone elicited <1% IFNγ+ T cells. Significant tumor regression was seen with ICMV+anti-PD-1 treatment, doubling overall survival time while anti-PD-1 treatment alone delayed tumor growth only slightly. Similar results were seen when ICMV were combined with various immunomodulators including anti-PD-L1, anti-CTLA-4 and anti-CD40. These results indicate that the ICMV-induced expansion of antigen-specific CD8 T cells can significantly improve the therapeutic efficacy of checkpoint inhibitors. A triple therapy of ICMV + checkpoint inhibitor + Treg inhibiting drugs is now being assessed and eradication of >85% of established tumors was detected in preliminary experiments. These findings demonstrate the potential of ICMVs as a novel cancer immunotherapy platform for the in vivo generation of CD8 T cells. ICMV are a cost effective, easily manufactured and versatile strategy to generate T cells for immunotherapy. Citation Format: Adrienne V. Li, Jackson K. Eby, Peter C. DeMuth, Darrell J. Irvine. Novel synthetic vesicle for rapid in vivo expansion of CD8 T cells can significantly improve checkpoint inhibitor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-227. doi:10.1158/1538-7445.AM2015-LB-227

CD1d Expression and Invariant NKT Cell Responses in Herpesvirus Infections.

Invariant natural killer T (iNKT) cells are a highly conserved subset of unconventional T lymphocytes that express a canonical, semi-invariant T cell receptor and surface markers shared with the natural killer cell lineage. iNKT cells recognize exogenous and endogenous glycolipid antigens restricted by non-polymorphic CD1d molecules, and are highly responsive to the prototypical agonist, α-galactosylceramide. Upon activation, iNKT cells rapidly coordinate signaling between innate and adaptive immune cells through the secretion of proinflammatory cytokines, leading to the maturation of antigen-presenting cells, and expansion of antigen-specific CD4+ and CD8+ T cells. Because of their potent immunoregulatory properties, iNKT cells have been extensively studied and are known to play a pivotal role in mediating immune responses against microbial pathogens including viruses. Here, we review evidence that herpesviruses manipulate CD1d expression to escape iNKT cell surveillance and establish lifelong latency in humans. Collectively, published findings suggest that iNKT cells play critical roles in anti-herpesvirus immune responses and could be harnessed therapeutically to limit viral infection and viral-associated disease.

Combination OX40 agonism/CTLA-4 blockade with vaccination reverses anergy and primes tumor-specific CD8 T cells in mice with spontaneous prostate cancer

Targeted immunotherapy, such as anti-CTLA-4 and anti-PD-1, has proven effective in treating cancer patients. However, despite these advances, cancer remains the second leading cause of death in the US. More effective strategies designed to maximize anti-tumor CD8 T cell responses are necessary to sustain long-term immunity. Agonist anti-OX40 and antagonist anti-CTLA-4 mAb augment the CD8 T cell response through different mechanisms. Therefore, we investigated the additive effects of these modalities on CD8 T cell responses. Combination anti-OX40/anti-CTLA-4 therapy more effectively primed antigen-specific CD8 T cells through enhanced expansion (82% of circulating CD8 T cells) compared to anti-OX40 (61%; P<0.05) or anti-CTLA-4 (41%; P<0.01) alone. Dual therapy also drove expansion of the polyclonal CD8 population. Moreover, combination therapy induced more proliferation (Ki-67) and differentiation (granzyme B, CD127, KLRG-1) following priming, suggesting potent additive effects of these modalities. However, it is known that tumors can induce T cell anergy, thereby limiting an effective anti-tumor response. We tested whether combination therapy could overcome anergy using a mouse model in which mice are tolerant to membrane-bound OVA. Combination anti-OX40/anti-CTLA-4 therapy was uniquely capable of driving robust expansion (12% of total CD8 in the spleen, vs. 2.5% anti-OX40, P<0.05, or 1% anti-CTLA-4, P<0.01) of anergic CD8 T cells, along with enhanced Ki-67 and granzyme B. To further drive expansion of antigen-specific CD8 T cells following therapy, we examined several methods of vaccination using nanoparticles, a Listeria monocytogenes vector, or anti-DEC-205 mAb (all conjugated to OVA) in the presence of anti-CD40. In anergic mice given combination therapy, vaccination with anti-DEC-205-OVA/anti-CD40 produced a 9-fold expansion of OT-I CD8 T cells compared to soluble OVA alone, greater than all other vectors combined. We next tested the efficacy of combination therapy with vaccination on anergy in a spontaneous model of prostate cancer. Combination therapy with vaccination drove expansion (2-fold over monotherapy, P<0.05) and differentiation (granzyme B, KLRG-1, CD25) of both anergic tumor-specific and polyclonal CD8 T cells. Importantly, dual therapy with vaccination enhanced the frequency and number of IFN-γ, TNF-α, and IL-2 producing CD8 T cells. These data indicate that anti-OX40/anti-CTLA-4 combination therapy with vaccination can uniquely augment and drive a robust CD8 T cell response capable of overcoming anergy.