Tumor-specific Immune Responses Research Articles

Emerging role of immunogenic cell death in cancer immunotherapy

Cancer immunotherapy, such as immune checkpoint blockade (ICB), has emerged as a groundbreaking approach for effective cancer treatment. Despite its considerable potential, clinical studies have indicated that the current response rate to cancer immunotherapy is suboptimal, primarily attributed to low immunogenicity in certain types of malignant tumors. Immunogenic cell death (ICD) represents a form of regulated cell death (RCD) capable of enhancing tumor immunogenicity and activating tumor-specific innate and adaptive immune responses in immunocompetent hosts. Therefore, gaining a deeper understanding of ICD and its evolution is crucial for developing more effective cancer therapeutic strategies. This review focuses exclusively on both historical and recent discoveries related to ICD modes and their mechanistic insights, particularly within the context of cancer immunotherapy. Our recent findings are also highlighted, revealing a mode of ICD induction facilitated by atypical interferon (IFN)-stimulated genes (ISGs), including polo-like kinase 2 (PLK2), during hyperactive type I IFN signaling. The review concludes by discussing the therapeutic potential of ICD, with special attention to its relevance in both preclinical and clinical settings within the field of cancer immunotherapy.

Abstract PO5-19-07: A Single Arm Phase 2 Study of Peri-Operative Immune Checkpoint Inhibition and Cryoablation in Women With Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer

Abstract Background: Triple negative breast cancer (TNBC) is a biologically distinct subtype with a high risk of early relapse. Patients not achieving a pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) have a 3-year event free survival (EFS) of < 60%. Approximately 1/3 of patients receiving NAC with pembrolizumab-mediated immune therapy will not achieve a pCR and of those, approximately 1/3 will experience a recurrence within 3 years. Physical disruption of tumors with local strategies such as cryoablation (cryo) induces inflammation and releases antigens that can activate tumor-specific immune responses. The combination of cryo with immune checkpoint inhibition (ICI) has been shown to induce tumor regression and prevented tumor re-challenge in pre-clinical models. Clinical studies have also demonstrated that the combination of pre-operative cryo with ICI is safe in women with operable, early-stage breast cancer and generates intra-tumoral and systemic immune responses (NCT01502592, NCT02833233). This multi-center, single arm, phase 2 study is currently evaluating the impact of cryo with standard-of-care pembrolizumab (pembro) in women with residual TNBC after taxane-based NAC, a group at high risk of early relapse (NCT03546686). Methods: Eligible women are ≥18y, with ER < 10%, PR < 10%, HER2 negative (per ASCO/CAP definition), ≥ 1.0 cm, residual operable disease after taxane-based NAC. Pts undergo percutaneous, image-guided cryo with concurrent research core biopsy 7-10 days prior to surgery and receive peri-operative pembro per standard-of-care. Adjuvant capecitabine or olaparib is recommended for all patients per local standard-of-care. Patients are stratified by NAC platinum administration, NAC anthracycline administration, and clinical nodal status (positive versus negative). The primary endpoint is 3-year Event Free Survival (EFS). Secondary endpoints include Invasive Disease-Free Survival (IDFS), Distant Disease-Free Survival (DDFS), overall survival (OS) and safety. Exploratory correlative studies will be performed on tumor and serum to characterize the immunologic impact of the intervention and to explore predictors of efficacy and toxicity. Funding sources: Conquer Cancer Foundation, Breast Cancer Research Foundation, Boston Scientific Citation Format: Heather McArthur, Elizabeth Comen, Yolanda Bryce, Stephen Solomon, Sangeetha Reddy, Isaac Chan, Baṣak Dogan, Dawn Klemow, Nisha Unni, Jorge Henrique Santos Leal, Cristal Martinez, Reva Basho, Dorothy Park, Philomena McAndrew, Brigid Larkin, David Page, William Mills, Staci Mellinger, Nicole Fredrich, Nicole Moxon, Lynette Currie, Meredith Carter, Melissa Ramos, Stephanie Rice, Sujata Patil, Margaret Gatti-Mays, Larry Norton. A Single Arm Phase 2 Study of Peri-Operative Immune Checkpoint Inhibition and Cryoablation in Women With Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-19-07.

Expanding the Potential of Neoantigen Vaccines: Harnessing Bacille Calmette-Guérin Cell-Wall-Based Nanoscale Adjuvants for Enhanced Cancer Immunotherapy.

Personalized antitumor immunotherapy utilizing neoantigen vaccines holds great promise. However, the limited immunogenicity of existing recognized neoantigens and the inadequate stimulation of antitumor immune responses by conventional adjuvants pose significant challenges. To address these limitations, we developed a nanovaccine that combines a BCG bacterial cell wall skeleton (BCG-CWS) based nanoscale adjuvant (BCNA) with peptide neoantigens (M27 and M30). This integrated approach provides an efficient translational strategy for cancer immunotherapy. The BCNA nanovaccine, formulated with PLGA as an emulsifier, exhibits excellent biocompatibility and superior antigen presentation compared with conventional BCG-CWS adjuvants. Subcutaneous immunization with the BCNA-based nanovaccine effectively targets lymph nodes, eliciting robust innate and tumor-specific immune responses. Importantly, our findings demonstrate that BCNAs significantly enhance neoantigen immunogenicity while minimizing acute systemic toxicity. Furthermore, when combined with a mouse PD-L1 antibody, our strategy achieves complete tumor elimination in 60% of cases and prevents 25% of tumor growth in a melanoma mouse model. In conclusion, our BCNA-based nanovaccine represents a promising avenue for advancing personalized therapeutic neoantigen vaccines and holds significant implications for enhancing personalized immunotherapy and improving patient outcomes in the field of cancer treatment.

Novel Personalized Cancer Vaccine Using Tumor Extracellular Vesicles with Attenuated Tumorigenicity and Enhanced Immunogenicity.

Cancer vaccines offer a promising avenue in cancer immunotherapy by inducing systemic, tumor-specific immune responses. Tumor extracellular vesicles (TEVs) are nanoparticles naturally laden with tumor antigens, making them appealing for vaccine development. However, their inherent malignant properties from the original tumor cells limit their direct therapeutic use. This study introduces a novel approach to repurpose TEVs as potent personalized cancer vaccines. The study shows that inhibition of both YAP and autophagy not only diminishes the malignancy-associated traits of TEVs but also enhances their immunogenic attributes by enriching their load of tumor antigens and adjuvants. These revamped TEVs, termed attenuated yet immunogenically potentiated TEVs (AI-TEVs), showcase potential in inhibiting tumor growth, both as a preventive measure and a possible treatment for recurrent cancers. They prompt a tumor-specific and enduring immune memory. In addition, by showing that AI-TEVs can counteract cancer growth in a personalized vaccine approach, a potential strategy is presented for developing postoperative cancer immunotherapy that's enduring and tailored to individual patients.

Single-cell T-cell receptor repertoire profiling in dogs

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

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.

Combining multi-mode thermal therapy with IL-6 and IL-17A neutralization amplifies antitumor immunity to facilitate long-term survival in LLC1-bearing mice

Non-small cell lung cancer (NSCLC) is known for rapid development and chronic inflammation-induced immunosuppression. IL-6 and IL-17A are the essential cytokines that facilitate NSCLC progression and myeloid-derived suppressive cell (MDSC)-mediated evasion. IL-6 or IL-17A targeting, especially IL-6, shown outstanding efficacy in patient NSCLC controlling, but failed to completely eradicate tumor. The local tumor multi-mode thermal therapy developed in our prior research was demonstrated to stimulate systemic and durable tumor-specific immune response thereby promoting long-term tumor-free survival of mice and prolong the progression-free survival of patients, although the therapeutic efficacy was still affected by high-level preoperative MDSCs. To further improve the efficacy, in this study, IL-6 and IL-17A neutralization were combined with multi-mode thermal therapy in mouse LLC1 NSCLC model. Study revealed that combined with single cytokine neutralization only prolonged the survival time while triple combination therapy efficiently improved the survival rate. Additionally, triple combination therapy reduced the accumulation of MDSCs but promoted their maturation with strengthened activation and function of myeloid cells, thereby triggering a Th1-dominant-CD4+ T cell-response and enhancing the malignant cell-killing capacity of immune cells. Our study highlights the extraordinary efficacy of combining multi-mode thermal therapy with IL-6 and IL-17A neutralization, revealing a new strategy for refractory NSCLC patients.Graphical

Pan-cancer analyses of immunogenic cell death-derived gene signatures: Potential biomarkers for prognosis and immunotherapy.

Immunogenic cell death (ICD) is a type of regulated cell death that is capable of initiating an adaptive immune response. Induction of ICD may be a potential treatment strategy, as it has been demonstrated to activate the tumor-specific immune response. The biomarkers of ICD and their relationships with the tumor microenvironment, clinical features, and immunotherapy response are not fully understood in a clinical context. Therefore, we conducted pan-cancer analyses of ICD gene signatures across 33 cancer types from The Cancer Genome Atlas database. We identified key genes that had strong relationships with survival and the tumor microenvironment, contributing to a better understanding of the role of ICD genes in cancer therapy. In addition, we predicted therapeutic agents that target ICD genes and explored the potential mechanisms by which gemcitabine induce ICD. Moreover, we developed an ICD score based on the ICD genes and found it to be associated with patient prognosis, clinical features, tumor microenvironment, radiotherapy access, and immunotherapy response. A high ICD score was linked to the immune-hot phenotype, while a low ICD score was linked to the immune-cold phenotype. We uncovered the potential of ICD gene signatures as comprehensive biomarkers for ICD in pan-cancer. Our research provides novel insights into immuno-phenotypic assessment and cancer therapeutic strategies, which could help to broaden the application of immunotherapy to benefit more patients.

Blocking senescence and tolerogenic function of dendritic cells induced by γδ Treg cells enhances tumor-specific immunity for cancer immunotherapy

BackgroundRegulatory T (Treg) cells are a key component in maintaining the suppressive tumor microenvironment and immune suppression in different types of cancers. A precise understanding of the molecular mechanisms used...

Abstract 5310: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC

Abstract CD40 is a key molecule expressed on antigen presenting cells (APCs), whose signaling activates APCs and generates antitumor immune responses. Although CD40 agonists have been investigated in clinical trials, systemic toxicities have limited their full potential in providing adequate antitumor effects. To overcome this limitation, we developed KK2269, a bispecific antibody, using REGULGENT™ technology. KK2269 binds to CD40 and tumor-expressed epithelial cell adhesion molecule (EpCAM) to allow activation of CD40+ APCs only at EpCAM+ tumor sites, thus avoiding systemic toxicity. EpCAM is overexpressed in many types of epithelial cancers, including non-small cell lung cancer (NSCLC). KK2269 exerts synergistic antitumor activity and tumor-specific immune responses in combination with docetaxel, a common chemotherapy for NSCLC, in an EpCAM-expressing tumor mouse model. Hence, we aimed to evaluate KK2269 for the treatment of NSCLC. Docetaxel is used as a combination therapy with ramucirumab, an anti-VEGF receptor 2 (VEGFR2) antibody, for previously treated advanced NSCLC. In addition, many clinical studies are evaluating the add-on effects of PD-1/PD-L1 blockade combined with standard chemotherapies (including docetaxel) in NSCLC, which may become the standard of care for advanced NSCLC in the future. Therefore, we investigated the antitumor effect of KK2269 in combination with docetaxel + anti-mouse VEGFR2 (study 1) and with docetaxel + anti-mouse PD-1 (study 2) using a mouse EpCAM-expressing B16F10 tumor-bearing human CD40 transgenic mouse model. The prolongation of TTP5X (the day on which the tumor volume is observed to be ≥5-fold of that observed on day 0) was evaluated. In study 1, the median TTP5X (95% confidence interval [CI]) with the control, docetaxel + anti-VEGFR2, and triple combination (KK2269, docetaxel, anti-VEGFR2) was 10.0 days (7.0, 10.0), 26.0 days (17.0, 28.0), and 52.0 days (42.0, not calculable), respectively. The TTP5X was significantly prolonged with the triple combination compared with the control (p=0.000007, log-rank test) and docetaxel + anti-VEGFR2 (p=0.000004, log-rank test). In study 2, the median TTP5X (95% CI) was 7.0 days (not calculable), 9.0 days (6.0, 11.0), 16.0 days (7.0, 18.0), and 26.5 days (7.0, 32.0) with the control, KK2269, docetaxel + anti-PD-1, and triple combination (KK2269, docetaxel, anti-PD-1), respectively. TTP5X was significantly prolonged with the triple combination compared with the control, KK2269, and docetaxel + anti-PD-1 (p<0.0001, p=0.0003, and p=0.0023, respectively, log-rank test). Thus, a significant antitumor effect was demonstrated when KK2269 was added to the standard treatment of NSCLC (docetaxel + anti-VEGFR2 antibody) and to docetaxel + anti-PD-1 antibody. Our findings strongly suggest that KK2269 provides improved treatment options for patients with NSCLC. Citation Format: Yuta Tezuka, Kyoko Kawasaki, Yoshiki Sumitomo, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, demonstrates antitumor effects in combination with standard therapies for NSCLC [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 5310.

Abstract 709: Enhancing prostate cancer treatment: Synergistic effects of mechano-thermal focused ultrasound and radiation therapy

Abstract Current treatments for prostate cancer primarily rely on radiation and hormone deprivation therapy, yet they often lack effective immunotherapeutic effects. Furthermore, long-term hormone ablation has side effects, such as, impotency and osteoporosis. Addressing this gap, we introduce an innovative dual-frequency focused ultrasound (FUS) platform for non-ablative focal therapy, which induces protein unfolding, ER stress, and increases heat-shock protein expression along with translocation of endoplasmic reticular chaperone proteins (e.g., calreticulin) on the tumor cell surface, thereby sensitizing them for phagocytosis by dendritic cells for efficient antigen presentation and cross priming. We hypothesized that FUS-mediated acoustic immune priming can be combined with ablative therapies, such as, hormone ablation and/or radiation therapy (RT) for induction of tumor-specific adaptive immune response and better tumor control than monotherapy in murine prostate cancer models. Utilizing murine prostate cancer cell lines, MyC-CaP and RM1, we explored the cellular responses to androgen deprivation, observing distinct sensitivities. Twenty-four hours after FUS treatment tumor cells experienced mechano-thermal stress response with 15-fold increase in Hspa1a expression. Transmission Electron Microscopy studies showed formation of myelin figures, nuclear membrane deformation, and lipid droplet formation. In vivo, the combination of FUS (220w/cm2 intensity for 3 sec at focal point) and RT (8-10 Gy daily x two fractions) markedly delayed tumor growth and achieved complete cure in 14-17% of animals. Through multicolor flow cytometry, we detected significant alterations in the immune microenvironment, including increased CD8+ T cell infiltration and reduced tumor-associated macrophages in the tumor bed. However, we also observed a compensatory rise in pro-tumor immune cells such as PMN-MDSCs and Tregs with significant suppression of eosinophils (p<0.05). Interestingly, there was suppression of splenic PMN-MDSCs, Mo-MDSCs, and tumor draining lymph node macrophages, suggesting systemic immune modulation in peripheral lymphoid organs of FUS+RT-treated tumor-bearing animals. Our findings reveal that the combination therapy of FUS and RT improves tumor control in murine prostate cancer and significantly modulates the systemic and tumor immune microenvironment. This approach offers new avenues for immunotherapy, potentially transforming the treatment landscape for prostate cancer, typically resistant to conventional immunotherapeutic strategies. Citation Format: Soumya Chatterjee, Saurabh Singh, Stephen Barry, Chandan Guha. Enhancing prostate cancer treatment: Synergistic effects of mechano-thermal focused ultrasound and radiation therapy [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 709.

Abstract 4102: Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models

Abstract Background: Androgen receptor (AR) signaling inhibition is part of the mechanism behind many effective prostate cancer therapies, including androgen deprivation and next generation anti-androgens. However in castration resistant prostate cancer up to 50% of treatment resistance has been linked to AR amplification or expression of the constitutively active RNA splice variant, AR-V7. Although the use of immune checkpoint blockade has failed to demonstrate clinical success, immunotherapeutic interventions remain attractive as prostate cancer’s relatively slow progression provides time for anti-tumor immune responses to develop. Cancer specific vaccines have the potential to enable and enhance tumor specific immune responses by stimulating and directing T cell immunity to important oncologic targets. We hypothesized that a cancer vaccine designed to induce a T cell response against cells expressing high levels of AR would result in an anti-tumor effect and potentially prevent or delay the development of therapeutic resistance. Methods: Adenoviral vaccines against AR (Ad-AR) or AR splice variant 7 (Ad-AR-V7) were used to evaluate the potential of AR targeting vaccination in mouse models. We evaluated AR and AR variant specific adaptive responses produced by our vaccines with naïve and tumor-bearing mice by ex vivo peptide restimulation. We challenged vaccinated mice with multiple syngeneic subcutaneous tumor models that we engineered to express AR-V7, including a PTEN-/- p53-/- prostate tumor cell line. Finally we tested our vaccines in a therapeutic regimen, vaccinating mice after implanting our subcutaneous AR-V7 expressing models. Results: Across multiple strains of mice, AR or AR-V7 targeting adenoviral vaccination induced robust adaptive immune responses against the N-terminal domain of AR. Vaccination with either Ad-AR or Ad-AR-V7 resulted in significant anti-tumor responses, providing a significant overall survival benefit and complete tumor rejection by the majority of mice vaccinated against AR or AR-V7. Similarly tumor bearing mice treated with Ad-AR or Ad-AR-V7 vaccination displayed tumor growth inhibition or complete tumor regression resulting in an overall survival benefit. Additionally, we found that vaccine responses were unaffected by the use of the approved antiandrogen drug enzalutamide, suggesting that vaccinations could be deployed in tandem with standard-of-care therapies. Conclusions: Vaccination against AR and AR splice variants can induce AR specific adaptive immune responses capable of inhibiting the growth of AR-V7 expressing tumor models, illustrating the potential of AR and its splice variants as immunologic targets in prostate cancer. Citation Format: Robert D. Marek, Junping Wei, Tao Wang, Xiao-Yi Yang, Gangjun Lei, Zachary C. Hartman. Treatment with androgen receptor targeting vaccines provides an overall survival benefit in AR-V7 expressing tumor models [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 4102.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [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 5272.

Abstract 1609: Inferring the T cell repertoire dynamics in non-small cell lung cancer metastases

Abstract Non-small cell lung cancer (NSCLC) is a highly complex disease, characterized by extensive genomic heterogeneity and dynamic evolution under therapeutic pressure. This evolution can lead to alterations in the antigens expressed by cancer cells, thereby influencing the T cell repertoire (TCR) as T cells expand in response to foreign antigens. Despite the central role of TCR in cancer progression and treatment response, only few studies have investigated the temporal and spatial changes in the TCR repertoire. In this study, we delve into the role of the TCR repertoire in metastatic progression in NSCLC. We conducted TCR sequencing of multiregion primary tumors, normal-adjacent tissues, and post-mortem metastases sampled from a cohort of 22 patients enrolled in the TRACERx and PEACE studies. Our findings reveal that primary tumor regions and normal-adjacent tissues exhibit similar fractions of expanded clonotypes. However, a higher proportion of expanded intratumoral TCRs were privately expanded in the tumor samples, suggesting a unique TCR landscape within the tumor microenvironment. The TCR repertoire across multiregion primary tumor samples was largely homogenous, with most expanded clonotypes shared across at least two regions. However, only a small fraction of these clonotypes were tracked in metastases, indicating a significant shift in the TCR repertoire during metastatic progression. Notably, the expanded clonotypes that persisted over time from early to late stage disease were significantly enriched for clonotypes ubiquitously expanded in the primary tumor across all regions. This was not shown to be associated with an increased likelihood of generation via VDJ recombination, suggesting a tumor-specific immune response. The expanded TCR repertoire in the postmortem metastases clustered mainly by anatomical site, suggesting an immune response driven by tissue-resident T cells. Moreover, we found a significant positive correlation between repertoire dissimilarity and genomic distance, suggesting that T cell clones track with tumor clones across spatial sites within patients. Our study demonstrates the significant spatiotemporal dynamics of the TCR repertoire during the metastatic progression of NSCLC, orchestrated by both tissue-resident T cells and the genomic evolutionary trajectory of tumors. Such an exploration could provide valuable insights to inform future strategies for immunotherapy treatments and enhance clinical monitoring of patients. Citation Format: Corentin Richard, Sonya Hessey, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Gayathri Nageswaran, Crispin Hiley, Sergio Quezada, Benny Chain, TRACERx and PEACE Consortia, Charles Swanton, Mariam Jamal-Hanjani. Inferring the T cell repertoire dynamics in non-small cell lung cancer metastases [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 1609.

Abstract 6755: NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma

Abstract One sentence introduction: This study indicated the potential of combinational therapy of neoantigen vaccine and iNKT activator α-GalCer in Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a leading cause of death around the world. Current immune checkpoint blockade therapies only show effectiveness in some cancer patients, so there is an urgent need for more effective immunotherapy strategies. One alternative is the use of neoantigen-based cancer vaccines, which target tumor-specific antigens (neoantigens) on cancer cells and induce strong T-cell responses. Advances in technology now allow for precise identification of tumor neoantigens using bioinformatic analysis or mass spectrometry. While cancer vaccines have demonstrated strong anti-tumor immune responses and therapeutic effectiveness in some cancer types, their success in HCC remains limited. A contributing factor is the suppressive tumor microenvironment (TME) found in advanced HCC. Some studies suggest that combining neoantigen vaccines with anti-PD1/PD-L1 therapies can significantly enhance HCC treatment outcomes. Overcoming the suppressive immune microenvironment is crucial in cancer treatment, and reactivating intratumoral immunity is essential. In our study, we developed an HCC cancer vaccine using transcriptome sequencing and bioinformatics analysis. Our ELISpot assay results showed that this vaccine induced strong tumor-specific immune responses and significantly inhibited tumor growth in early-stage orthotopic HCC mouse models when administered five days after tumor inoculation. However, the effectiveness decreased in advanced tumors when administered ten days after tumor inoculation. We also discovered a significant relationship between the therapeutic effectiveness of the neoantigen vaccine and the activation of intratumoral NKT cells in HCC. Our study suggested that the type-I NKT cell activator, α-Galactosylceramide (α-GalCer), significantly improved the therapeutic outcomes of the cancer neoantigen vaccine in advanced orthotopic HCC mouse models. In some cases, tumors were almost completely eliminated. This indicates the potential of combining iNKT activators with neoantigen vaccines for HCC treatment. To further understand the role of NKT cells in HCC's TME, we plan to investigate intratumoral NKT cell subtypes following neoantigen vaccine therapy. Additionally, we will develop a new delivery system to deliver type I NKT cell activators and type II NKT inhibitors to tumor sites, aiming to reactivate the suppressive TME in HCC after neoantigen vaccine therapy. Citation Format: Renhao Li, Jing-Chu Hu, Li Rong, Yangfan Wu, Yige He, Jiandong Huang. NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma [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 6755.

Abstract 6666: Leveraging an obligate anaerobic salmonella strain for targeted cargo delivery in the tumor microenvironment to restore immunotherapy sensitivity

Abstract Tumor immunotherapy is often hindered by unresponsive cold tumor microenvironments (TME). Although Salmonella-based immunotherapy has shown potential in overcoming these cold immune environments, safety concerns remain. Our study introduces a genetically reprogrammed Salmonella typhimurium strain (OASL) with a dual safety lock to enhance safety and modify immunosuppressive TME, sensitizing tumors to immunotherapies. We propose using OASL to alter the cold TME and locally release therapeutic cargos such as PDL1 nanobodies and tumor-specific neoantigen peptides identified through bioinformatic algorithms, activating immune cells within the tumor while minimizing side effects. We selected YB1, an obligate anaerobic Salmonella strain that thrives in hypoxic environments, as our base strain and incorporated a self-lysis genetic circuit for controlled bacterial growth and cargo release upon reaching a specific population density within the tumor core. We assembled and evaluated the self-lysis gene circuit in vitro, documenting the event using an optical density (OD) reader and confirming OASL's ability to express genetic cargos through western blot analysis. We then investigated OASL's therapeutic effect in vivo and characterized TME changes using flow cytometry and immunohistochemistry staining. Our results indicate that OASL undergoes lysis and releases cargos upon reaching a specific population density in vitro. Additionally, OASL-delivered neoantigen peptides elicited a tumor-specific immune response in vivo. OASL also demonstrated the capacity to modify the TME and restore sensitivity to PDL1 in previously unresponsive tumors, enhancing therapeutic effects. Citation Format: Yige He, Jiandong Huang. Leveraging an obligate anaerobic salmonella strain for targeted cargo delivery in the tumor microenvironment to restore immunotherapy sensitivity [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 6666.

Abstract 6643: Hyperthermic induction of immunotherapeutic response in in vitro melanoma model

Abstract Immunotherapy has revolutionized the treatment of advanced melanoma, yet challenges persist with non-responders, development of resistance, and dosage concerns. Our strategy to enhance checkpoint inhibitor efficacy is to combine it with hyperthermia. Moderate hyperthermia, 39-45°C, is an interesting anti-neoplastic therapy which induces production of tumor associated antigens rendering tumor sites more amenable to immunotherapy. To investigate temperature-induced alterations on carcinogenic phenotypes in vitro, B16-F10 melanoma cells were grown at 37°C under normal culture conditions, or incubated at 41°C for 2 hours for moderate, febrile hyperthermic induction. Hyperthermia-induced changes were analyzed by trypan blue exclusion assay, scratch wound assay, collection of conditioned media for cytokine array, and protein isolation for Western Blot. Conditioned media from the hyperthermia-treated group showed an inflammatory shift in cytokine and chemokine expression, with increased TNFa and decreased IL-4 expression. Prior studies found the hyperthermia-induced apoptotic effect is directly correlated to endogenous TNFa levels, elucidating a potential molecular mechanism. Treatment for two hours at 41°C decreased proliferation of B16-F10 cells by 62% after 48 hours and 94% after 72 hours. Additionally, hyperthermia decreased cellular migration by 70% after 24 hours. Further evaluation of cellular interactions in important pathways responsible for these hyperthermia-induced changes were investigated via Western blot. Expression of constitutively active MAPK signaling cascade effectors pERK and ERK was decreased by 86% and 50%, respectively, helping to regulate pro-tumorigenic proliferative signaling. Some cancer cells have been demonstrated to resist hyperthermia via mechanisms to inhibit induction of caspase-3, however, in our model caspase-3 expression increased by 31% following 41°C treatment, thus allowing for induction of apoptosis via caspase-3 in this model. Cellular stress may induce these cell death pathways, as well as heat shock responses. Following febrile hyperthermic induction, hsp70 expression increased by 188% compared to cells cultured at 37°C. Hsp70 has a well-established role in supporting tumor-specific immune responses. Induction of hyperthermia in combination with anti-PDL1, anti-PD-1, or IL-15 immunotherapy is currently being evaluated in a C57BL/6 mouse model by our group. These studies aim to assess if hyperthermia augments response to immunotherapies in vivo. Our current works suggests that moderate, non-ablative hyperthermia helps initiate anti-tumor immune responses, assisting in the induction of anti-tumorigenic cell programs and, potentially, the production of additional tumor associated antigens, thereby making it an interesting candidate for combination with immune checkpoint inhibition and other immunotherapeutic approaches. Citation Format: Tara Jarboe, Danielle Quaranto, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Raj K. Tiwari, Mark D. Hurwitz. Hyperthermic induction of immunotherapeutic response in in vitro melanoma model [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 6643.

Abstract 7297: Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA

Abstract The V-domain Ig Suppressor of T cell Activation (VISTA) is expressed on cells of the myeloid and lymphoid lineages and is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Furthermore, a tripeptide small molecule antagonist of VISTA, CA170, exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. We have previously demonstrated that KRAS or EGFR vaccines are effective in preventing lung tumorigenesis driven by either KRAS or EGFR in a pure prevention setting but are less effective in mice carrying existing lesions. Here, we determined if combining an MHC class II-restricted multi-antigen vaccine targeting KRAS or EGFR with CA170 will provide enhanced efficacy in preventing lung cancer progression. Using tetracycline-inducible KRAS and EGFR transgenic mice, we found that lung tumor development was significantly suppressed when CA170, delivered by aerosol inhalation to minimize systemic toxicity, was combined with either KRAS or EGFR MHCII-directed peptide vaccines using a post-initiation model. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 significantly increases tumor infiltrating CD8+ T cells and enhances effector-memory T cell frequencies and functions of both CD4+ and CD8+ T cells. These changes coincide with significant reductions in G-MDSCs (granulocytic myeloid-derived suppressor cells) and Foxp3+ Treg populations within lung tumors from mice treated with CA170. We found that the KRAS and EGFR preventive vaccines primarily induced the expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory, and regulatory T cells, and drive an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS and EGFR preventive vaccines in transgenic mouse models of lung cancer. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Ming You. Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA [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 7297.

Oncolytic mineralized bacteria as potent locally administered immunotherapeutics.

Oncolytic bacteria can trigger innate immune activity. However, the antitumour efficacy of inactivated bacteria is poor, and attenuated live bacteria pose substantial safety risks. Here we show that intratumourally injected paraformaldehyde-fixed bacteria coated with manganese dioxide potently activate innate immune activity, modulate the immunosuppressive tumour microenvironment and trigger tumour-specific immune responses and abscopal antitumour responses. A single intratumoural administration of mineralized Salmonella typhimurium suppressed the growth of multiple types of subcutaneous and orthotopic tumours in mice, rabbits and tree shrews and protected the cured animals against tumour rechallenge. We also show that mineralized bacteria can be administered via arterial embolization to treat orthotopic liver cancer in rabbits. Our findings support the further translational testing of oncolytic mineralized bacteria as potent and safe antitumour immunotherapeutics.

Programmed Nanocloak of Commensal Bacteria-Derived Nanovesicles Amplify Strong Immunoreactivity against Tumor Growth and Metastatic Progression.

Recent discoveries in commensal microbiota demonstrate the great promise of intratumoral bacteria as attractive molecular targets of tumors in improving cancer treatment. However, direct leveraging of in vivo antibacterial strategies such as antibiotics to potentiate cancer therapy often leads to uncertain effectiveness, mainly due to poor selectivity and potential adverse effects. Here, building from the clinical discovery that patients with breast cancer featured rich commensal bacteria, we developed an activatable biointerface by encapsulating commensal bacteria-derived extracellular vesicles (BEV) with a responsive nanocloak to potentiate immunoreactivity against intratumoral bacteria and breast cancer. We show that the interfacially cloaked BEV (cBEV) not only overcame serious systemic side responses but also demonstrated heightened immunogenicity by intercellular responsive immunogenicity, facilitating dendritic cell maturation through activating the cGAS-STING pathway. As a preventive measure, vaccination with nanocloaked cBEVs achieved strong protection against bacterial infection, largely providing prophylactic efficiency against tumor challenges. When treated in conjunction with immune checkpoint inhibitor anti-PD-L1 antibodies, the combined approach elicited a potent tumor-specific immune response, synergistically inhibiting tumor progression and mitigating lung metastases.