Tumor-directed Immune Responses Research Articles

Preclinical Evaluation of 177Lu-OncoFAP-23, a Multivalent FAP-Targeted Radiopharmaceutical Therapeutic for Solid Tumors.

Fibroblast activation protein (FAP) is abundantly expressed in the stroma of most human solid tumors. Clinical-stage radiolabeled FAP ligands are increasingly used as tools for the detection of various cancer lesions. To unleash the full therapeutic potential of FAP-targeting agents, ligands need to remain at the tumor site for several days after administration. We recently described the discovery of OncoFAP, a high-affinity small organic ligand of FAP with a rapid accumulation in tumors and low uptake in healthy tissues in cancer patients. Trimerization of OncoFAP provided a derivative (named TriOncoFAP, or OncoFAP-23) with improved FAP affinity. In this work, we evaluated the tissue biodistribution profile and the therapeutic performance of OncoFAP-23 in tumor-bearing mice. Methods: OncoFAP-23 was radiolabeled with the theranostic radionuclide 177Lu. Preclinical experiments were conducted on mice bearing SK-RC-52.hFAP (BALB/c nude mice) or CT-26.hFAP (BALB/c mice) tumors. 177Lu-OncoFAP and 177Lu-FAP-2286 were included in the biodistribution study as controls. Toxicologic evaluation was performed on Wistar rats and CD1 mice by injecting high doses of OncoFAP-23 or its cold-labeled counterpart, respectively. Results: 177Lu-OncoFAP-23 emerged for its best-in-class biodistribution profile, high and prolonged tumor uptake (i.e., ∼16 percentage injected dose/g at 96 h), and low accumulation in healthy organs, which correlates well with its potent single-agent anticancer activity at low levels of administered radioactivity. Combination treatment with the tumor-targeted interleukin 2 (L19-IL2, a clinical-stage immunocytokine) further expands the therapeutic window of 177Lu-OncoFAP-23 by potentiating its invivo antitumor activity. Proteomics studies revealed a potent tumor-directed immune response on treatment with the combination. OncoFAP-23 and natLu-OncoFAP-23 exhibited a favorable toxicologic profile, without showing any side effects or signs of toxicity. Conclusion: OncoFAP-23 presents enhanced tumor uptake and tumor retention and low accumulation in healthy organs, findings that correspond to a strongly improved invivo antitumor efficacy. The data presented in this work support the clinical development of 177Lu-OncoFAP-23 for the treatment of FAP-positive solid tumors.

50P Vididencel, a cell-based cancer vaccine, induces tumor-directed immune responses in high-grade serous ovarian carcinoma patients

50P Vididencel, a cell-based cancer vaccine, induces tumor-directed immune responses in high-grade serous ovarian carcinoma patients

Abstract LB400: Evolution of anti-viral and anti-tumor immunity in tumors in response to oncolytic virus therapy

Abstract Preclinical and clinical studies have shown that intratumoral oncolytic viruses (OVs) can convert “cold” tumors to “hot” and overcome resistance to immune checkpoint blockade. However, their modest clinical efficacy to date highlights major gaps in our understanding of how OVs interact with the immune system, and very limited information is available regarding the specificity of the T cell repertoires that emerge within tumors in response to OV therapy. To address these gaps, we interrogated the evolution of immune response to OV therapy in mice using oncolytic Newcastle Disease Virus (NDV). Leveraging a bilateral flank melanoma mouse model, we identified and phenotypically characterized anti-viral and anti-tumor T cells in local and abscopal tumors with single cell (sc) RNA and T cell receptor (TCR) sequencing. Intratumoral NDV therapy to a single flank tumor resulted in increased infiltration of CD4+ and CD8+ T cells in both the injected and distant tumors, higher TCR overlap between treated and distant tumors, and higher frequency of convergent TCR clonotypes, suggesting their non-random expansion, albeit without specificity information. To identify the specificity of the emergent TCR clones in an unbiased fashion, we developed an approach based on adoptive transfer of T cells from tumor-bearing control or NDV-treated donor mice into congenically marked recipients with either B16 or NDV antigen stimulation. This allowed the isolation and identification of expanded B16- or virus-specific TCR clones that were then linked to donor tumors to define their relative frequencies, their distribution across the treated and distant tumors, and their associated phenotypic states. We observed significantly greater expansion of B16-reactive T cells adoptively transferred from NDV-treated donors (compared to control donors), suggesting that NDV therapy leads to potentiation of tumor-specific immune response. Intriguingly, virus-specific and tumor-specific T cells appeared to distribute to distinct phenotypic clusters, with virus-specific and tumor-specific T cells found predominantly in the activated-dysfunctional and proliferating clusters, respectively. When looking at the distribution of the TCR clones, virus-specific clones dominated the majority of the response in both treated and distant tumors, despite the lack of virus injection in distant tumors. These results provide insights into understanding the balance between virus- and tumor-directed immune responses elicited by OV therapy and highlight that even in the presence of significant abscopal immune effect, the significant proportion of T cells in “hot” tumors represent anti-viral rather than anti-tumor response. These findings provide tools for tracking of immune response specificity and generate rationale for development of OV engineering strategies and combinations that favor anti-tumor rather than anti-viral immunity. Citation Format: Olga Lyudovyk, Bharat Burman, Nicholas Ceglia, Benjamin Greenbaum, Yuval Elhanati, Dmitriy Zamarin. Evolution of anti-viral and anti-tumor immunity in tumors in response to oncolytic virus therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB400.

Abstract 1174: Incorporation of multisector analysis into the design of personalized DNA vaccines for patients with newly diagnosed glioblastoma

Abstract Glioblastoma (GBM) is the most common malignant central nervous system (CNS) tumor in adults. Despite multimodality treatment including surgery, radiation, and chemotherapy, patients with GBM have a median survival of less than 2 years. Recent clinical trials have reported promising results using cancer vaccines to stimulate a tumor-directed immune response in several histologies. The majority of trials targeted tumor neoantigens derived from a single tumor sample, which limits the antigen pool in spatially heterogeneous cancers like GBM. Our group previously implemented multisector sequencing into the design of personalized peptide vaccines to increase the candidate pool of targetable neoantigens (NCT03422094). Although the peptide vaccine treatment was successful in stimulating an effector T cell response, it was limited by a long turnaround time from vaccine design to administration and a low peptide production rate. Therefore, we incorporated multisector sampling into the design of a personalized DNA-based GBM vaccine (NCT04015700). An average of 33% of the candidate targetable neoantigens were spatially restricted to a single sampled region, which would have been missed without multisector sequencing. DNA-based vaccines are potentially advantageous over peptide-based vaccines because they enable a higher neoantigen payload and faster manufacturing time with potent immunogenicity. Here, we report the results from 9 subjects enrolled onto the study. Spatially distinct tumor regions were subjected to whole exome sequencing (WES) and RNA-sequencing (RNA-seq), data from which were used to identify neoantigens through the pVACseq algorithm (http://pvactools.org). An average of 32 neoantigens were included in each DNA vaccine, compared to an average of only 9 for the peptide vaccine, and the turnaround time from date of surgery to administration of the DNA vaccine was about half the time. Three subjects had radiographic evidence of tumor progression prior to vaccination, highlighting the aggressive nature of GBM and the need for short manufacturing timeframes. PBMCs pre- and post-DNA vaccination were collected from 7/9 subjects. All subjects presented detectable responses and 6/7 tested revealed a sustained increment in T cell reactivity against tumor neoantigens post-treatment by IFN-γ ELISpot. Intracellular staining showed a neoantigen-specific antitumor CD8/CD4 T cell cytolytic (CD69+, CD107a+) and proliferative (Ki67+) profile. Post-vaccination tumor resections were performed for 2 subjects who also were treated with PD1 blockade upon progression, providing an opportunity to explore vaccine-induced changes in the tumor microenvironment. Herein, we demonstrate the advantages of incorporating multisector sequencing into the development of DNA-based GBM vaccines and provide insights into the resulting immune responses. Citation Format: Elizabeth A. Garfinkle, Katherine E. Miller, Alexandra J. Livingstone, Renzo Perales-Linares, Neil Cooch, Alfredo Perales-Puchalt, Sarah Rochestie, Joann Peters, Niranjan Y. Sardesai, William E. Gillanders, Elaine R. Mardis, Gavin P. Dunn, Tanner M. Johanns. Incorporation of multisector analysis into the design of personalized DNA vaccines for patients with newly diagnosed glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1174.

Tolerability and efficacy of the cancer vaccine UV1 in patients with recurrent or metastatic PD-L1 positive head and neck squamous cell carcinoma planned for first-line treatment with pembrolizumab - the randomized phase 2 FOCUS trial.

Globally, head and neck squamous cell carcinoma (HNSCC) is the seventh most common malignancy. Despite aggressive multimodal treatment approaches, recurrent and/or metastatic (R/M) disease develops in >50% of patients. In this setting, pembrolizumab was approved for patients with PD-L1 expression. However, response rates with checkpoint inhibitor monotherapy remain limited and strategies to strengthen tumor-directed immune responses are needed. The FOCUS trial is designed to estimate the effectiveness of UV1 vaccination in combination with pembrolizumab versus pembrolizumab as a single agent in patients with R/M HNSCC. The FOCUS trial is a two-armed, randomized, multicenter phase II study which was designed to evaluate the efficacy and feasibility of the hTERT-targeted cancer vaccine UV1 as add-on to pembrolizumab in the 1st line treatment of patients with R/M PD-L1 positive (combined positive score ≥1) HNSCC. Secondary objectives are the exploration of patient subgroups most likely deriving benefit from this novel combination and the establishment of liquid biopsy tumor monitoring in HNSCC. This clinical study was designed and will be conducted in compliance with Good Clinical Practice and in accordance with the Declaration of Helsinki. It is intended to publish the results of this study in peer-reviewed scientific journals and to present its content at academic conferences. A significant number of patients with R/M HNSCC are frail and may not tolerate chemotherapy, these patients may only be suitable for pembrolizumab monotherapy. However, long term disease stabilizations remain the exception and there is a need for the development of efficacious combination regimens for this patient population. The FOCUS study aims to optimize treatment of R/M HNSCC patients with this promising new treatment approach. https://clinicaltrials.gov/study/NCT05075122, identifier NCT05075122.

Abstract B045: EV-mediated immune modulation of CD8+ T cells in renal cell carcinoma

Abstract Introduction: Treatment regimens for advanced renal cell carcinoma (RCC) involve immune checkpoint inhibition targeting the PD-(L)1 pathway. PD-L1 is often overexpressed on tumor cells in advanced disease and plays a crucial role in regulating tumor-directed immune responses. It can also be expressed on tumor-secreted extracellular vesicles (EVs), which are an essential part of intercellular communication. PD-L1 carrying EVs have been shown to have immunomodulatory effects in several tumor entities, however their role in RCC is still unknown. The aim of this study was the evaluation of putative immunomodulatory effects of RCC-derived small EVs on CD8+ T cells. Materials and Methods: EVs of 5 untreated as well as interferon γ (IFNγ)-treated RCC cell lines were isolated through serial ultracentrifugation. Quality and quantity of the EVs were verified using Western Blotting (WB), Nano Particle Tracking Analysis and Transmission Electron microscopy, their PD-L1 expression was analyzed semi quantitatively through WB. CD8+ T cells were isolated from the blood of healthy donors, activated and subsequently co-cultured with RCC-derived EVs for a duration of 3 days. Proliferation and PD-1 expression of the T cells was assessed using flow cytometry. ELISAs were performed to analyze cyto- and chemokine secretion. Results: All RCC cell lines expressed PD-L1, albeit at varying low levels. Four out of 5 treated cell lines responded to IFNγ treatment with an upregulation of PD-L1 expression on both cells and secreted EVs. Co-cultures of CD8+ T cells and RCC-derived EVs, particularly PD-L1+ EVs isolated from IFNγ-stimulated cells, lead to a reduction in T cell PD-1 expression. Additionally, PD-L1-expressing EVs of RCC cell line RCC53 had an antiproliferative effect on CD8+ T cells. EVs also increased the secretion of IL-2, IL-10, CXCL10 and CCL5 by the CD8+ T cells. Conclusions: PD-L1 is expressed at low levels in RCC cell lines. However, repetitive IFNγ stimulation can lead to upregulation of PD-L1 on both cells and EVs enabling us to use this model to further investigate EV-mediated, PD-L1 dependent effects. In co-cultures, RCC-derived EVs impede early T cell activation and proliferation patterns of CD8+ T cells. Furthermore, early results indicate a shift towards a more pro-inflammatory cyto- and chemokine production by the T cells induced by RCC-derived EVs. The results support the hypothesis of a tumor-EV-mediated immune modulation in RCC. Citation Format: Greta Jaschkowitz, Elfriede Noessner, Angela Zaccagnino, Michael Stöckle, Kerstin Junker, Philip Zeuschner. EV-mediated immune modulation of CD8+ T cells in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B045.

Contrasting Features of T-Cell Expansion in Classical Hodgkin Lymphoma: Tumor Microenvironment Vs. Peripheral Blood and before Vs. during Anti-PD1 Treatment

Introduction The Tumor Microenvironment (TME) in classical Hodgkin Lymphoma (HL) contains abundant CD4+ and CD8+ T-cells and only few Hodgkin-Reed-Sternberg cells (HRSC). Despite their low abundance, HRSC comprise the neoplastic cell population that intensively interacts with cells of the TME. Understanding these interactions is crucial to the further development of immune checkpoint blockade (ICB) based treatment options. Clinical trials have shown high efficacy of ICB with anti-PD1 antibodies in relapsed HL and more recently promising results of anti-PD1 antibodies in combination with conventional chemotherapy were reported in the first-line setting. (NIVAHL trial, NCT03004833) In solid cancers, anti-PD1 ICB was shown to revert tumor-induced exhaustion of CD4+ and CD8+ T-cells, thereby enabling T-cell activation and a tumor-directed immune response. Since HL differs in many aspects from non-lymphoid tumors due to e.g. loss of HLA-expression, the exact mechanisms of action of ICB in HL is not fully understood and T-cell expansion after anti-PD1 first-line treatment not yet studied. Methods To characterize T-cell activation at different timepoints and to investigate a possible T-cell mediated immune response in HL, we analyzed T-cell Receptor (TCR) repertoires of the NIVAHL study cohort before (tissue: n=90; blood: n=9) and during anti-PD1 treatment (tissue: n=4; blood: n=9). The final cohort comprised additional TCR repertoires of in-house tissue biopsies of treatment-naïve HL (n=18), relapsed HL after chemotherapy (HL-R; n=18)) and publicly available TCR repertoires of the blood of treatment-naïve HL (n=11), HL-R (n=20) and HL-R patients during anti-PD1 treatment (n=20). (Cader et al. Nat Med. 2020 Sep;26(9):1468-1479) TCR repertoires of in-house reactive lymph nodes (n=8, healthy control) and breast cancer (BC; n=6, positive control) patients served as controls for T-cell activation in the tissue, publicly available TCR repertoires of CMV- (n=22, healthy control) and CMV+ otherwise healthy (n=9, positive control) people as controls in the blood. (De Neuter et al. Genes Immun. 2019 Mar;20(3):255-260) TCR repertoires were sequenced by Adaptive Biotechnologies. For each sample, TCR sequences with the same amino acid sequence were aggregated by the sum of their counts. We computed three measures to describe T-cell expansion: debiased Simpson's Clonality (dSC), Percentage of Singletons (PoS), and clonal expansion. Singletons have recently been defined as TCR sequences detected only once in a given sample. Clonal expansion is computed as the percentage of TCR sequences that increase their frequency in a patient from one timepoint to a following by ≥ 2. Results In tissue biopsies, treatment-naïve HL showed a significantly lower dSC compared to treatment-naïve BC and significantly higher dSC compared to RLN from healthy controls. No significant differences could be observed comparing HL with different treatments (during anti-PD1 vs. after chemotherapy) and treatment-naïve HL vs. HL during anti-PD1 treatment. HL and HL-R biopsies showed a significantly lower clonal expansion of Non-Singletons than BC tumors. In peripheral blood, healthy controls, treatment-naïve HL and HL patients during anti-PD1 treatment showed significantly lower dSC in their CD4+ T-cells compared to CMV+ controls and also compared to HL-R patients. We identified significantly higher dSC in CD8+ T-cells compared to CD4+ T-cells in the blood of HL patients before and during any treatment and in relapsed HL. During anti-PD1 treatment, CD8+ T-cells showed a significantly higher clonal expansion of Non-Singletons than CD4+ T-cells in HL-R and a similar but not significant trend in HL. All observed differences in dSC were significant for the PoS too, but as expected by their respective definitions with an opposing pattern (i.e. dSC high = PoS low). Discussion We did not observe features of intratumoral T-cell expansion in primary HL samples, early during anti-PD1 treatment or at relapse after chemotherapy, suggesting that within the HL tissue, T-cell expansion is hampered. However, patterns of T-cell expansion differed between TME and the peripheral blood, where we observed features of CD8+ T-cell clonal expansion, already prior to any treatment. In summary, our findings suggest a possible anti-tumor immune response of CD8+ T-cells in the peripheral blood of HL patients, that is not found in the TME.

A phase 1b/2 trial of dupilumab given in conjunction with PD-(L)1 blockade in the treatment of relapsed/refractory metastatic NSCLC.

TPS9139 Background: Although tumor microenvironments may contain chronic inflammatory cells, this milieu can lead to immune evasion and may contribute to resistance to immunotherapy. Dendritic cells are one of the most potent cross presenters of tumor antigen, and their function is crucial to tumor-directed adaptive immune responses. In the KP mouse model of lung adenocarcinoma (KrasG12D; Tp53-/-), we recently identified interleukin-4 (IL-4)-driven suppression of tumor-antigen charged dendritic cells, which was similarly seen in human lung cancer lesions (Maier, B., et al., A conserved dendritic-cell regulatory program limits antitumour immunity. Nature, 2020.580[7802]:257-262). In the mouse model IL-4 blockade resulted in an increase in IL-12, IFNg and TNF in CD8+ T cells and decreased tumor burden, and further antitumor activity was seen when combined with PD-L1 blockade. Dupilumab is a fully human monoclonal antibody that blocks the IL-4 receptor alpha subunit which disrupts signaling through receptors for both IL-4 and IL-13; it is FDA approved for patients with multiple atopic conditions, in whom series adverse effects are rare. Based on this pre-clinical data, we hypothesize that the addition of dupilumab to anti PD-(L)1 therapy will be well tolerated, and will rescue the anti-tumor effect of immune checkpoint blockade. Methods: This is a phase Ib/II trial. Patients with relapsed/refractory NSCLC who have received prior anti PD-(L)1 treatment are eligible for enrollment. In a phase Ib safety run-in, six patients will be enrolled in a modified set-dose 3+3 design. If no more than 1 DLT is observed during this phase, the trial will proceed to phase II. Phase II will enroll a further 15 patients in a minimax design with early stopping for futility to a maximum total of 21 patients. Patients will undergo pre-treatment biopsies and peripheral blood sampling prior to receiving 3 doses of dupilumab, administered every three weeks, in conjunction with continuing standard-of-care anti-PD-(L)1 therapy. Patients will undergo repeat biopsies 4 weeks after starting therapy. After completion of dupilumab (at 9 weeks) patients will undergo repeat staging. The primary endpoint of phase 1b is safety as measured by frequency and severity of adverse effects. The primary endpoint of phase II is overall response rate as assessed using RECIST 1.1 criteria at the time of post-dupilumab imaging. Exploratory endpoints include analysis of peripheral blood by CyTOF and O-link, and tissue biopsies will be analyzed by multiplex-IHC and bulk-RNA-sequencing. T cell receptor sequencing will be performed on tumor and matched peripheral blood samples, and circulating tumor DNA will be assessed at multiple time points. Phase 1b is currently enrolling as planned. Clinical trial information: NCT05013450.

A study of neo-adjuvant and adjuvant ofra-vec (VB-111) for treatment of surgically accessible recurrent GBM.

TPS2075 Background: Ofranergene obadenovec (ofra-vec, VB-111) is an anti-cancer gene based immune activator and targeted vascular disruptor. The dual mechanism of action triggers a broad antiangiogenic effect and induces of a tumor directed immune response. A previous study demonstrated a survival benefit for patients with recurrent glioblastoma (rGBM) treated with ofra-vec monotherapy, that was continued after progression in combination with bevacizumab. Glioblastoma is an immunologically “cold” microenvironment which fosters immunosuppression and antagonizes anti-tumor immune responses. The role of T-cell infiltration in combating cancer has been increasingly recognized and associated with improved participant outcomes. Based on these observations, this study will assess the hypothesis that neoadjuvant use of ofra-vec will lead to a statistically significant increase in tumor infiltrating T lymphocyte (TIL) density within the tumor and enhanced systemic tumor-specific T cell responses. Methods: Study NCT04406272 is a multicenter, randomized, blinded, placebo-controlled, phase 2 surgical trial to evaluate early immunologic pharmacodynamic parameters for the viral cancer therapy ofra-vec in rGBM. 45 participants with rGBM indicated for resection will randomized to one of three treatment arms: Neoadjuvant Arm: intravenous ofra-vec prior to resection, and ofra-vec every 6 weeks after resection. Adjuvant Arm: placebo prior to resection, and ofra-vec every 6 weeks afterwards. The control arm will receive placebo prior to resection followed by standard of care. Upon evidence of contrast-enhancing progression, bevacizumab may be initiated as needed for supportive care; however, ofra-vec will continue until progression is supported at two consecutive time points. Tumor samples will be obtained and archived at the time of surgery, and blood samples will be obtained as pharmacodynamic markers throughout the study to allow DNA sequencing of T cells. The primary endpoint is to evaluate the influence of neoadjuvant ofra-vec on TIL density. Other endpoints include safety and tolerability, peripheral T cell response, 6mPFS and OS. Study is open for enrolment. Clinical trial information: NCT04406272.

Pivotal study of ofra-vec (VB-111) combined with paclitaxel versus paclitaxel for treatment of platinum-resistant ovarian cancer (OVAL, VB-111-701/GOG-3018).

TPS5606 Background: Ofranergene obadenovec (Ofra-vec, VB-111) is an anti-cancer gene based immune activator and targeted vascular disruptor. The dual mechanism of action triggers a broad antiangiogenic effect and induces of a tumor directed immune response. A phase II trial in patients with platinum resistant ovarian cancer (PROC) demonstrated that ofra-vec in combination with weekly paclitaxel was well tolerated and associated with a CA-125 Objective Response Rate (ORR) of 58%, a trend for improved survival and induction of an immunotherapeutic effect of tumor infiltration with CD-8 T cells. Based on these observations, a pivotal phase III study was initiated in collaboration with the GOG Foundation, Inc. Methods: Study NCT03398655 is an international, randomized, double-blind, placebo-controlled, phase III study. Eligible patients have recurrent PROC and may have been previously treated with up to 5 prior lines of therapy (but not >2 for PROC). Patient are randomized 1:1 to receive ofra-vec (1x1013 Viral Particles) with weekly paclitaxel (80mg/m2), or weekly paclitaxel with placebo. Randomization is stratified by number of prior treatment lines, prior antiangiogenic therapy and platinum refractory disease status. The dual primary endpoints are OS and PFS. A pre-planned interim analysis of CA-125 response (GCIG) performed by the DSMC met the pre-defined criteria showing that CA-125 ORR in the treatment arm was at least 10% higher than in the control arm. Study is enrolling in the US, EU, Japan and Israel, with 90% enrollment to date. Completion of accrual is anticipated in Q1 2022. Clinical trial information: NCT03398655.

Advances in Chimeric Antigen Receptor (CAR) T-Cell Therapies for the Treatment of Primary Brain Tumors.

Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a patient’s own immune cells to generate directed anti-tumor activity, are emerging technologies that hold promise to improve the treatment of primary brain tumors in children and adults. Here, we review recent advances in chimeric antigen receptor (CAR) T-cell therapies for the treatment of aggressive primary brain tumors, including glioblastoma and diffuse midline glioma, H3 K27M-mutant. We highlight current approaches, discuss encouraging investigational data, and describe key challenges in the development and implementation of these types of therapies in the neuro-oncology setting.

GOBLET: A phase 1/2 multiple-indication biomarker, safety, and efficacy study in advanced or metastatic gastrointestinal cancers exploring treatment combinations with pelareorep and atezolizumab.

TPS216 Background: Checkpoint blockade therapy only benefits a small subset of GI cancer patients (approximately 4%) with microsatellite instability-high (MSI-H) tumors, which are characterized as immunologically ‘hot’ (Bonneville et al., 2017). Most GI cancers, however, have microsatellite stable (MSS) tumors, which have an immunologically “cold” phenotype with fewer genetic mutations, reduced immune cell infiltration, and downregulated immune checkpoint proteins. These attributes make MSS tumors resistant to conventional immunotherapy including checkpoint blockade therapy (Ooki et al., 2021). Pelareorep is a naturally occurring, non-genetically modified reovirus. Upon intravenous administration, pelareorep selectively kills tumor cells and promotes several immunologic effects that prime tumors to respond to checkpoint blockade. These include the stimulation of tumor-directed innate and adaptive immune responses, increased T cell infiltration, expansion of new T cell clones, and increased PD-L1 expression in tumors (Samson et al., 2018, Manso et al. 2021 AACR). Given its expected synergy with checkpoint blockade, as well as its encouraging efficacy in prior GI cancer studies (Mahalingam et al. 2020), the GOBLET study is designed to evaluate pelareorep plus atezolizumab in multiple GI cancer indications. Methods: GOBLET is an open-label, non-randomized, multiple-cohort, phase 1/2 study in patients with advanced or metastatic GI cancers. This study employs a Simon two-stage design. Stage 1 comprises four treatment groups: Cohort 1 – First-line pancreatic cancer treated with pelareorep plus atezolizumab and chemotherapy (gemcitabine and nab-paclitaxel) (N = 12); Cohort 2 – First-line MSI-H colorectal cancer (CRC) treated with pelareorep plus atezolizumab (N = 19); Cohort 3 – Third-line CRC treated with pelareorep plus atezolizumab and chemotherapy (trifluridine/tipiracil) (N = 14); and Cohort 4 – Second-line or later squamous cell carcinoma of the anal canal treated with pelareorep plus atezolizumab (N = 10). The first 3-6 patients enrolled into the chemotherapy-containing cohorts (Cohorts 1 and 3) comprise a safety run-in that must be successfully concluded prior to enrolling additional patients into these cohorts. The primary objectives are safety and efficacy based on objective response rate (ORR) at week 16. Any cohort showing a promising ORR in Stage 1, based on pre-specified criteria, may be advanced to Stage 2 and enroll additional patients. Clinical trial information: 2020-003996-16.

Interleukins in the treatment of melanoma.

Interleukins (ILs) and associated cytokines serve as the means of communication for immune cells and non-immune cells. The use of ILs in harnessing the immune system to cancer treatment has been a promising approach. ILs not only nurture an environment enabling cancer growth but also simultaneously trigger a productive tumor-directed immune response. These properties of ILs are increasingly being explored as a strategy to improve the outcomes of cancer. Here, we describe recently innovative technological approaches that have been developed to improve the pharmacokinetics, safety, and efficacies of IL-2, 15, 10, and 18 in the treatment of melanoma. Furthermore, the combination of ILs and immune checkpoint inhibition may synergize to reshape the tumor environment, thus yielding better clinical benefits in the future.

582 Immune correlates from QuEST1 in men with castration-resistant prostate cancer

BackgroundImmune checkpoint inhibitors have limited efficacy in unselected metastatic castration-resistant prostate cancer (mCRPC) patients. Combination immunotherapy approaches to generate a tumor-directed immune response (vaccine) and facilitate the resulting anti-tumor immune activity (checkpoint inhibitors, cytokines) have shown synergy preclinically. In the phase I/II Quick Efficacy Seeking Trial (QuEST1, NCT03493945), combination of a brachyury-targeting vaccine (BN-brachyury), TGF-β/anti-PD-L1 blocking bifunctional fusion protein (bintrafusp alfa), and IL-15 superagonist (N-803) have produced preliminary evidence of efficacy in CRPC, with 5/12 patients having a sustained prostate-specific antigen (PSA) response that included 2 radiographic partial responses, compared to 1/13 patients who received BN-brachyury plus bintrafusp alfa. Here, we present immune correlates from patients enrolled in Arm 2.1A (BN-brachyury + bintrafusp alfa) and Arm 2.2A (BN-brachyury + bintrafusp alfa + N-803).MethodsPeripheral blood mononuclear cells (PBMC) and serum were obtained from 25 patients pre and multiple time points post treatment. PBMCs were assessed for antigen specific T cells targeting brachyury and MUC-1 by intracellular cytokine staining, 158 peripheral immune cell subsets by multicolor flow cytometry, and TCRVβ sequencing. Patients were also evaluated for complete blood counts, and serum cytokines/soluble factors using ELISA assays and OLINK’s immuno-oncology panel. Immune parameters were compared between Arm 2.1A and Arm 2.2A and evaluated for associations with clinical response in Arm 2.2A.ResultsBrachyury and MUC-1 specific T cells were increased in most patients post treatment in both arms. A greater increase in total NK cells, refined NK subsets expressing markers of activation/adhesion, and TCR diversity was observed after 2 weeks of therapy in Arm 2.2A than Arm 2.1A. Absolute lymphocyte counts and serum levels of granzyme B, sCD27, and sCD40L were also increased after 2 weeks in Arm 2.2A compared to Arm 2.1A. Serum proteomic analyses revealed a greater increase in analytes related to NK cell signaling in Arm 2.2A than Arm 2.1A. Specific immune parameters at baseline associated with development of clinical response in patients treated in Arm 2.2A; responders had trends of higher frequencies of CD4+ and CD8+ T cells, lower frequencies of MDSCs and monocytes, and lower levels of serum IL-6 and sCD40 than non-responders.ConclusionsThese findings demonstrate enhanced immune activation of both NK and T cells with the addition of N-803 in Arm 2.2A, where more clinical activity was observed than in Arm 2.1A. These findings support the continued evaluation of the combination of BN-brachyury, bintrafusp alfa, and N-803 in patients with mCRPC.AcknowledgementsThis research was supported in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health, and via Cooperative Research and Development Agreements (CRADAs) between the NCI and EMD Serono, the NCI and Bavarian Nordic, and the NCI and ImmunityBio.Trial RegistrationNCT03493945Ethics ApprovalThe trial was approved by the Institutional Review Board of the Center for Cancer Research, National Cancer Institute (ClinicalTrials.gov identifier: NCT03493945).

Clinical trial in progress: Pivotal study of VB-111 combined with paclitaxel versus paclitaxel for treatment of platinum-resistant ovarian cancer (OVAL, VB-111-701/GOG-3018).

TPS5599 Background: Ofranergene obadenovec (VB-111) is a targeted anti-cancer gene therapy with a dual mechanism of action that includes a broad antiangiogenic effect and induction of a tumor directed immune response. A phase II trial in patients with platinum resistant ovarian cancer showed that VB-111 in combination with weekly paclitaxel was well tolerated and associated with a CA-125 Objective Response Rate (ORR) of 58% with a trend for improved survival. The favorable outcomes were associated with induction of an immunotherapeutic effect of tumor infiltration with CD-8 T cells. Based on these observations, a phase III study was initiated in collaboration with the GOG Foundation, Inc. Methods: Study NCT03398655 is an international, randomized, double-blind, placebo-controlled, phase III study. Eligible patients have recurrent platinum-resistant epithelial ovarian cancer with measurable disease (RECIST 1.1), and may have been previously treated with up to 5 prior lines of therapy. Patient are randomized 1:1 to receive VB-111 (1x1013 VPs) with weekly paclitaxel (80mg/m2), or weekly paclitaxel with placebo. Randomization is stratified by number of prior treatment lines, prior antiangiogenic therapy and platinum refractory disease status. The efficacy endpoints are OS, PFS and ORR by RECIST 1.1 and by CA-125 (GCIG criteria). A pre-planned interim analysis was performed by the DSMC in the first 60 patients evaluable for CA-125 response. The analysis met the pre-defined criteria of a CA-125 ORR (GCIG) in the treatment arm at least 10% higher than in the control arm. Study enrolment is ongoing and over 220 patients were enrolled in the US, EU, and Israel. Enrolment of the full sample size of 400 patients is expected to complete by the end of 2021. Clinical trial information: NCT03398655.

Immune Modulation Plus Tumor Ablation: Adjuvants and Antibodies to Prime and Boost Anti-Tumor Immunity In Situ.

In situ tumor ablation techniques, like radiotherapy, cryo- and heat-based thermal ablation are successfully applied in oncology for local destruction of tumor masses. Although diverse in technology and mechanism of inducing cell death, ablative techniques share one key feature: they generate tumor debris which remains in situ. This tumor debris functions as an unbiased source of tumor antigens available to the immune system and has led to the concept of in situ cancer vaccination. Most studies, however, report generally modest tumor-directed immune responses following local tumor ablation as stand-alone treatment. Tumors have evolved mechanisms to create an immunosuppressive tumor microenvironment (TME), parts of which may admix with the antigen depot. Provision of immune stimuli, as well as approaches that counteract the immunosuppressive TME, have shown to be key to boost ablation-induced anti-tumor immunity. Recent advances in protein engineering have yielded novel multifunctional antibody formats. These multifunctional antibodies can provide a combination of distinct effector functions or allow for delivery of immunomodulators specifically to the relevant locations, thereby mitigating potential toxic side effects. This review provides an update on immune activation strategies that have been tested to act in concert with tumor debris to achieve in situ cancer vaccination. We further provide a rationale for multifunctional antibody formats to be applied together with in situ ablation to boost anti-tumor immunity for local and systemic tumor control.

Tumor-infiltrating plasmacytoid dendritic cells are associated with survival in human colon cancer

BackgroundPlasmacytoid dendritic cells (pDCs) play a key role in the induction and maintenance of antitumor immunity. Conversely, they can act as tolerogenic DCs by inhibiting tumor-directed immune responses. Therefore, pDCs...

Abstract IA07: The QuEST for an effective immunotherapy for prostate cancer

Abstract Background: Immune checkpoint inhibitors have minimal activity in unselected CRPC patients (pts). Combination regimens that generate a tumor-directed immune response (vaccine) and facilitate the resulting anti-tumor immune activity (checkpoint blockade, cytokines) have shown synergy in preclinical models. BNVax is a therapeutic poxviral vaccine targeting brachyury, a transcription factor involved in invasion and metastasis. BA is a bifunctional fusion protein: anti-PD-L1 monoclonal antibody fused to the TGFb-β-RII receptor extracellular domain (a TGF-β trap). N-803 is an IL-15 superagonist complex. Here we report an interim efficacy analysis of the QuEST1 study, aimed to rapidly interrogate safety and efficacy of immunotherapy combinations in CRPC. Methods: Asymptomatic/minimally symptomatic CRPC pts received BNVax + BA (Arm 2.1) or BNVax + BA + N-803 (Arm 2.2). BNVax is given as 2 prime doses followed by boosts. BA 1,200 mg intravenously and N-803 15 µg/kg subcutaneously are given every 2 weeks. Efficacy is defined as objective response by RECIST v1.1 or PSA decrease ≥ 30% sustained for ≥ 21 days. Safety was a secondary endpoint. Results: As of 10/11/20, 26 CRPC pts enrolled and had 1 to 23 months follow up. 1/13 pts (Arm 2.1) had a PSA response sustained for 13 months. 5/12 (41.6%) pts on treatment for at least 12 weeks (Arm 2.2) had sustained PSA responses, including 2 pts who progressed on enzalutamide. Of 3 patients with measurable disease, 2 had confirmed PR by RECIST. Grade 3 TRAEs: 1 pancreatitis (Arm 2.1), 1 secondary adrenal insufficiency (Arm 2.1), 1 tumor hemorrhage (Arm 2.1), 1 hyperglycemia due to new onset diabetes mellitus (Arm 2.2), 1 cystitis (Arm 2.2), 1 Rash with eosinophilia (Arm 2.2). One patient had grade 4 neutropenia that resolved with growth factor support and oral corticosteroids. Conclusions: BVax + BA + N-803 demonstrated a manageable safety profile and preliminary evidence of efficacy in CRPC. Addition of N-803 in Arm 2.2 was associated with more activity in this small sample. Arm 2.2 met the predetermined threshold for efficacy and will expand (n=25). Citation Format: James L. Gulley. The QuEST for an effective immunotherapy for prostate cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA07.

Abstract 6257: DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX

Abstract Small molecule drugs and biological macromolecules are currently being investigated as therapies for various types of disorders such as cancer and infectious disease. Generally, these agents are systemically administered. However, many immunomodulatory agents are active primarily on specific immune cells such as dendritic cells or T cells. The purpose of this study was to evaluate the efficacy of the novel delivery platform, DPX, for targeted delivery of immunomodulatory agents to the lymph node. As the lymph node is the primary site for priming and activation of immune cells, this approach may improve delivery over systemic administration resulting in increased drug efficacy, the use of less drug, and reduced off-target toxicity. To evaluate the efficacy of the DPX platform for lymph node-targeted drug delivery, we used the C57Bl/6 murine model. DPX formulation of small molecule dyes allowed us to visualize the kinetics of lymph node-specific delivery by visual assessments and by flow cytometry. DPX-dye formulations demonstrated a controlled and sustained uptake of molecules from the site of injection, travelling to the regional lymph node, without significant systemic exposure. To test impacts on tumor directed immune responses, the HPV16 E7 expressing C3 tumor model was used. Novel formulations were tested in conjunction with DPX-FP (a C3 tumor-specific antigen HPV16 E749-57 conjugated to a T helper epitope). DPX-mediated delivery of low doses of cyclophosphamide (CPA) were delivered subcutaneously as a single injection. This low dose of CPA has shown to act as an immunomodulatory agent when delivered systemically over a week. Using DPX to deliver CPA resulted in a significant reduction in the number of lymph node cells. This was evident after a single administration, showing equivalent therapeutic benefit was achieved with significantly less active agent. This was observed irrespective of whether CPA was administered alone or with DPX-FP. We also evaluated DPX delivery of the checkpoint inhibitor anti-CTLA-4. We found that administration in DPX was equivalent to systemic administration by intraperitoneal injection in terms of overall survival of tumor-bearing mice and in the proportion of CD3+ T cells bound by anti-CTLA-4, with fewer administrations of antibody. This demonstrates maintenance of biological functionality of macromolecules in the DPX platform. In conclusion, DPX holds promise in terms of lymph node-targeted delivery of functional agents. This delivery is facilitated by a sustained uptake from the site of injection and can offer reduced dosing and off-target toxicity as compared to systemic delivery. Many approaches to targeted therapies have failed to provide a therapeutic index commensurate with their promise through systemic delivery, with a challenge in achieving selectivity for the target cell type. Here, we show the DPX drug delivery platform has the potential to overcome this challenge and thereby increase the efficacy of immune and cancer therapies. Citation Format: Heather Torrey, Ava Vila-Leahey, Yuchen Cen, Danielle MacKay, Alecia MacKay, Leanne Lucas, Brittany Myers, Arthvan Sharma, Douglas Vieira, Andrea Penwell, Rajkannan Rajagopalan, Marianne Stanford. DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX [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 6257.

Clinical trial in progress: Pivotal study of VB-111 combined with paclitaxel versus paclitaxel for treatment of platinum-resistant ovarian cancer (OVAL, VB-111-701/GOG-3018).

TPS6097 Background: Ofranergene obadenovec (VB-111) is a targeted anti-cancer gene therapy with a dual mechanism: a broad antiangiogenic effect and induction of a tumor directed viral immune response. In a phase II trial in platinum resistant ovarian cancer VB-111 in combination with weekly paclitaxel showed a CA-125 response rate (RR) of 58% and median overall survival (OS) of 498 days compared to 172.5 days in the sub-therapeutic dose (p = 0.028). The combination treatment was well tolerated. Favorable outcomes were associated with induction of an immunotherapeutic effect of tumor infiltration with CD-8 T cells. Based on these observations, a phase III randomized controlled trial, VB-111-701/GOG-3018 (OVAL) was initiated in collaboration with the GOG Foundation, Inc. Methods: The OVAL study, NCT03398655, is an international, randomized, double-blind, placebo-controlled, phase III study. Patients with recurrent platinum-resistant epithelial ovarian cancer, who have measurable disease (RECIST 1.1) and were previously treated with up to 5 lines are randomized 1:1 to receive VB-111 (1x1013 VPs) with weekly paclitaxel (80mg/m2), or weekly paclitaxel with placebo. Randomization is stratified by number of prior treatment lines, prior antiangiogenic therapy and platinum refractory disease status. Treatment beyond asymptomatic RECIST progression may continue until progression is confirmed by follow up imaging. The primary endpoints are OS, safety and tolerability. Secondary endpoints include progression free survival, and objective RR by CA-125 (per GCIG criteria) and RECIST 1.1. The sample size calculation of 400 patients (event driven) provides 92% power to detect a difference in survival at the two-sided 5% significance level using the logrank test. A pre-planned interim analysis will take place in Q1 2020 to assess whether the CA-125 RR per GCIG criteria in the treatment arm is sufficiently larger than in the control arm and is comparable to the positive results of the phase II study. Study enrolment is ongoing and over 80 patients were enrolled in the US and Israel. Enrollment expansion to Europe is planned in 2020. Clinical trial information: NCT03398655.