Tumor-specific Immune Responses Research Articles

Antigen-Capturing Dendritic-Cell-Targeting Nanoparticles for Enhanced Tumor Immunotherapy Based on Photothermal-Therapy-Induced in-situ Vaccination.

In-situ vaccines have revolutionized immunotherapy as they can stimulate tumor-specific immune responses, with the cancer being the antigen source. However, the heterogeneity of tumor antigens and insufficient dendritic cell (DC) activation result in low cancer immunogenicity and hence poor vaccine response. Here, we designed a new in-situ vaccine composed of acid-responsive liposome-coated polydopamine (PDA) nanoparticles modified with mannose and loaded with resiquimod (R848) to promote the efficacy of immunotherapy. The in-situ vaccine can actively target the tumor site based on the decomposition of the liposome, while the PDA nanoparticles promote photothermal therapy and capture the immunogenic cell-death-induced tumor-associated antigens based on the adsorption effect of dopamine-mimetic mussels. The PDA nanoparticles, which were modified with a mannose ligand, target the DCs and release R848 for activated antigen presentation. As a result, the in-situ vaccine not only effectively activates the maturation of the DCs but also significantly enhances their effect on cytotoxic T lymphocyte cells. Furthermore, the vaccine effectively inhibits the distant recurrence and metastasis of tumors via long-term immune memory effects. Therefore, the in-situ vaccine provides a potential strategy for improving the efficacy of cancer immunotherapy. This article is protected by copyright. All rights reserved.

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.

TPS635 Background: Local tumor destruction with cryoablation (cryo) induces inflammation and releases antigens that can activate tumor-specific immune responses. Pre-clinically, cryo with immune checkpoint inhibition (ICI) augmented tumor-specific immune responses and prevented recurrence. Clinically, we established that peri-operative (peri-op) cryo with ICI [ipilimumab (ipi) +/- nivolumab (nivo)] was safe in patients (pts) with operable, early stage breast cancer (ESBC) and generated robust intra-tumoral and systemic immune responses. In this phase 2 study, we evaluate the disease specific impact of peri-op ICI in women with residual triple negative breast cancer (TNBC) after neoadjuvant chemotherapy (NAC), a subset at high risk of early relapse. 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. As of 2/14/21, 16/80 pts have been enrolled and treated with ipi/nivo/cryo followed by breast surgery and adjuvant nivo. Pts undergo percutaneous, image-guided cryo with concurrent research core biopsy 7-10 days prior to surgery and received ipi (1mg/kg IV) with nivo (240mg IV) 1 to 5 days prior to cryo. After surgery, pts received 3 additional doses of nivo at 240mg IV Q2 weeks. To reflect the US FDA approval of curative-intent pembrolizumab (pembro) in 2021, the protocol was recently amended to allow standard-of-care pembro as an alternative ICI option. Adjuvant capecitabine or olaparib is recommended for all patients per local standard-of-care. Patients will be 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. Clinical trial information: NCT03546686 .

Standardizing the strategy in glioblastoma trials to promote success in cell and gene therapy clinical development.

e13604 Background: Glioblastoma is the most common and aggressive brain tumor, with a high recurrence rate and low survival. We are increasing our understanding of its biology, and more targeted therapeutical agents are available. However, the GBM prognosis has not improved over the last decades. Multiple clinical trials on Cell and Gene Therapies are ongoing but, there is a huge need to define standardized and innovative strategies to promote better patient selection, implement adaptative designs and specific laboratory assays, and tailor the clinical endpoint assessments. Methods: Medical experts panel agreed on the following insights and gave advice to improve the design and conduct of GBM trials. Results: The criteria for selecting patients are not uniform among studies. The 2021 WHO Classification of central nervous system tumors has yet to be universally adopted, and different tumor entities are being treated as GBM according to the prior 2016 classification. Furthermore, the required genetic testing panel is only available in some countries as part of routine testing. A wide range of CGT therapies are being studied with different mechanisms of action and are well suited to be administered as combinatory regimens and multiple-dosing schedules to boost the antitumor effect. The implementation of master protocols offers the possibility to test different schedules to identify the most promising for further investigation during early phase development. There is a lack of biomarkers for the patient- or cancer-specific sensitivity and the efficacy of CGT. This is important not only to target clinical treatments to the appropriate patients and identify responding/non-responding patients early during treatment but also to improve the preclinical models and identify targets for further therapeutic development. Investigators should use standard endpoint criteria when reporting clinical outcomes. It has been common to refrain from introducing any preliminary-effect objective in the early stages. This tendency changes after guidance from experts and regulatory agencies about maximizing early-phase data. Another vital endpoint in the early phases is assessing patient immunogenicity against CG therapy based on humoral and cell virus-specific and tumor-specific immune responses. This is especially important as there are significant concerns in translating preclinical safety results to the clinic due to the species-specificity of different viruses and critical differences between immune systems. Conclusions: Today, the purpose of treatment for GBM is to extend the survival period and to manage the disease while preserving the patient’s quality of life. The goal of CGT is a durable complete response, but to achieve this target, we will need a deep analysis of how we perform clinical trials and move to an expert and tailored research environment.

Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

Post-resection tumors with microscopic foci and immunosuppressive microenvironments have high risk of recurrence and metastasis but respond poorly to various therapies. Herein, we propose a biomimetic hydrogel as a biocompatible, biodegradable and bioadhesive postoperative dressing that could be formed in situ by NaIO4-initiated thiourea-catechol crosslinking after syringe-injection into the resection cavity. The thiourea or catechol-bearing hyaluronic acid (HA) precursors are also separately engineered with phenylboronic acid and β-cyclodextrin (β-CD) groups, potentiating the reversible immobilization of (1S, 3R) RAS-selective lethal 3 (RSL3) and glycosylated granulocyte macrophage-colony stimulating factor (GM-CSF) without invasive chemical reactions. Meanwhile, the interconnected porous superstructure of the hydrogels allows the incorporation and self-regulated delivery of PD-L1 antibody (aPD-L1). RSL3-induced immunogenic ferroptosis and GM-CSF could cooperatively trigger robust adaptive tumor-specific immune responses, while aPD-L1 further alleviates the accumulated immunoresistance of tumor cells due to interferon γ-mediated PD-L1 upregulation, thus stimulating potent local and whole-body antitumor immunity to prevent postoperative tumor recurrence and metastasis. The biomimetic hydrogel may serve as a promising solution for the postoperative treatment of solid tumors.

Integrated proteogenomic characterization reveals an imbalanced hepatocellular carcinoma microenvironment after incomplete radiofrequency ablation

BackgroundEfforts to precisely assess tumor-specific T-cell immune responses still face major challenges, and the potential molecular mechanisms mediating hepatocellular carcinoma (HCC) microenvironment imbalance after incomplete radiofrequency ablation (iRFA) are unclear. This study aimed to provide further insight into the integrated transcriptomic and proteogenomic landscape and identify a new target involved in HCC progression following iRFA.MethodsPeripheral blood and matched tissue samples were collected from 10 RFA-treated HCC patients. Multiplex immunostaining and flow cytometry were used to assess local and systemic immune responses. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were explored via transcriptomic and proteogenomic analyses. Proteinase-3 (PRTN3) was identified in these analyses. And then, the ability of PRTN3 to predict overall survival (OS) was assessed in 70 HCC patients with early recurrence after RFA. In vitro CCK-8, wound healing and transwell assays were conducted to observe interactions between Kupffer cells (KCs) and HCC cells induced by PRTN3. The protein levels of multiple oncogenic factors and signaling pathway components were detected by western blotting. A xenograft mouse model was built to observe the tumorigenic effect of PRTN3 overexpression on HCC.ResultsMultiplex immunostaining revealed no immediate significant change in local immune cell counts in periablational tumor tissues after 30 min of iRFA. Flow cytometry showed significantly increased levels of CD4+ T cells, CD4+CD8+ T cells, and CD4+CD25+CD127− Tregs and significantly decreased the levels of CD16+CD56+ natural killer cells on day 5 after cRFA (p < 0.05). Transcriptomics and proteomics revealed 389 DEGs and 20 DEPs. Pathway analysis showed that the DEP-DEGs were mainly enriched in the immunoinflammatory response, cancer progression and metabolic processes. Among the DEP-DEGs, PRTN3 was persistently upregulated and closely associated with the OS of patients with early recurrent HCC following RFA. PRTN3 expressed in KCs may affect the migration and invasion of heat stress-treated HCC cells. PRTN3 promotes tumor growth via multiple oncogenic factors and the PI3K/AKT and P38/ERK signaling pathways.ConclusionsThis study provides a comprehensive overview of the immune response and transcriptomic and proteogenomic landscapes of the HCC milieu induced by iRFA, revealing that PRTN3 promotes HCC progression after iRFA.Trial registrationChiCTR2200055606, http://www.chictr.org.cn/showproj.aspx?proj=32588.

Mature tertiary lymphoid structures are key niches of tumour-specific immune responses in pancreatic ductal adenocarcinomas

ObjectiveTo better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation...

A General Biomineralization Strategy to Synthesize Autologous Cancer Vaccines with cGAS-STING Activating Capacity for Postsurgical Immunotherapy.

Autologous cancer vaccines constructed by nonproliferative whole tumor cells or tumor lysates together with appropriate adjuvants represent a promising strategy to suppress postsurgical tumor recurrence. Inspired by the potency of cytosolic double-stranded DNA (dsDNA) in initiating anticancer immunity by activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, we herein report the concise synthesis of a cGAS-STING agonist through dsDNA-templated biomineralization growth of calcium carbonate (CaCO3) microparticles. The yielded DNA@CaCO3 can activate the intracellular cGAS-STING pathway of dendritic cells (DCs) by promoting endosomal escape of dsDNA, triggering their maturation and activation as a potent immune stimulator. Upon intratumoral injection, DNA@CaCO3 can reverse the immunosuppressive tumor microenvironment by simultaneously provoking innate and adaptive antitumor immunity, thereby effectively suppressing the growth of murine CT26 and B16-F10 tumors in mice. Furthermore, via CaCO3-based biomineralization of complete tumor lysates, we constructed a personalized autologous cancer vaccine with intrinsic cGAS-STING activation capacity that could provoke tumor-specific immune responses to not only delay the growth of challenged tumors but also synergize with anti-PD-1 immunotherapy to suppress postsurgical tumor recurrence. This study highlights a CaCO3-based biomineralization method to prepare autologous cancer vaccines in a concise manner, which is promising for personalized immunotherapy and clinical translation.

ITCep: a deep learning framework for identification of T cell epitopes by harnessing fusion features.

Neoantigens recognized by cytotoxic T cells are effective targets for tumor-specific immune responses for personalized cancer immunotherapy. Quite a few neoantigen identification pipelines and computational strategies have been developed to improve the accuracy of the peptide selection process. However, these methods mainly consider the neoantigen end and ignore the interaction between peptide-TCR and the preference of each residue in TCRs, resulting in the filtered peptides often fail to truly elicit an immune response. Here, we propose a novel encoding approach for peptide-TCR representation. Subsequently, a deep learning framework, namely iTCep, was developed to predict the interactions between peptides and TCRs using fusion features derived from a feature-level fusion strategy. The iTCep achieved high predictive performance with AUC up to 0.96 on the testing dataset and above 0.86 on independent datasets, presenting better prediction performance compared with other predictors. Our results provided strong evidence that model iTCep can be a reliable and robust method for predicting TCR binding specificities of given antigen peptides. One can access the iTCep through a user-friendly web server at http://biostatistics.online/iTCep/, which supports prediction modes of peptide-TCR pairs and peptide-only. A stand-alone software program for T cell epitope prediction is also available for convenient installing at https://github.com/kbvstmd/iTCep/.

"To be or not to Be": Regulatory T cells in melanoma.

In spite of progresses in the therapy of different malignancies, melanoma still remains as one of lethal types of skin tumor. Melanoma is almost easily treatable by surgery alone with higher overall survival rates when it is diagnosed at early stages. However, survival rates are decreased remarkably upon survival if the tumor is progressed to advanced metastatic stages. Immunotherapeutics have been prosperous in the development of anti-tumor responses in patients with melanoma through promotion of the tumor-specific effector T cells in vivo; nonetheless, suitable clinical outcomes have not been satisfactory. One of the underlying causes of the unfavorable clinical outcomes might stem from adverse effects of regulatory T (Treg) cell, which is a prominent mechanism of tumor cells to escape from tumor-specific immune responses. Evidence shows that a poor prognosis and low survival rate in patients with melanoma can be attributed to a higher Treg cell number and function in these subjects. As a result, to promote melanoma-specific anti-tumor responses, depletion of Treg cells appears to be a promising approach; even though the clinical efficacy of different approaches to attain appropriate Treg cell depletion has been inconsistent. Here in this review, the main purpose is to assess the role of Treg cells in the initiation and perpetuation of melanoma and to discuss effective strategies for Treg cell modulation with the aim of melanoma therapy.

Targeting complement C5a to improve radiotherapy sensitivity in non-small cell lung cancer.

Tumor local and distant relapse recurrence after radiotherapy (RT) is one of the critical factors leading to poor prognosis. The effective antitumor effects of RT are dependent upon the participation of innate and adaptive components of the immune system. C5a/C5aR1 signaling can regulate antitumor immune effect in the tumor microenvironment (TME). Thus, exploring the changes and mechanism in the TME induced by RT-mediated complement activation may provide a novel perspective for reversing radioresistance. First, fractionated radiation of 8 Gy ×3 fractions were targeted at Lewis lung carcinoma (LLC) tumor-bearing female mice to measure the infiltration of CD8+ T cell and analyze the RNA sequencing (RNA-seq) in RT-recruited CD8+ T cells. Second, tumor growth was measured in LLC tumor-bearing mice treated with RT either with or without C5aR1 inhibitor to clarify the antitumor effect of RT combined with C5aR1 inhibitor. Third, we detected the expression of C5a/C5aR1 and their signaling pathways on radiated tumor tissues. Furthermore, we investigated the expression of C5a in tumor cells at different time points after different doses of RT. In our system, RT induced the increased infiltration of CD8+ T cells and local activation of complement C5a/C5aR. Concurrent administration of RT and blocking of C5aR improved radiosensitivity and tumor-specific immune response, which was reflected by high C5aR expression in CD8+ T cells. The AKT/NF-κB pathway was found to be an important signaling pathway in C5a/C5aR axis mediation by RT. RT promotes the release of C5a from tumor cells and leads to up-regulation of C5aR1 expression via the AKT/NF-κB pathway. Inhibition of the combination of complement C5a and C5aR could improve RT sensitivity. Our work provides evidence that the combination of RT and C5aR blockade opens a new window of opportunity to promote anti-tumor therapeutic effects in lung cancer.

Simultaneous targeting of PD-1 and IL-2Rβγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rβ and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific Tcell activation while reducing regulatory Tcell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ Tcell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ Tcells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ Tcells, Tcell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Cryoablation inhibits the recurrence and progression of bladder cancer by enhancing tumour‐specific immunity

Recurrence and metastasis of bladder cancer are major factors affecting patient prognosis. Endoscopic cryoablation achieved a better clinical outcome among clinical patients and could be synergistic with ICIs. Thus, this study aimed to evaluate the immunological mechanism of cryoablation for bladder cancer to reveal the therapeutic mechanism. We systematically reviewed the clinical prognosis of patients underwent cryoablation at Huashan Hospital in these first-in-human studies (ChiCTR-INR-17013060). Murine models were constructed to explore cryoablation-induced tumour-specific immunity, which was further confirmed by primary bladder tumour organoids and autologous lymphocytes cocultured system. Cryoablation improved progression-free survival and recurrence-free survival respectively. Assessment of murine models after cryoablation confirmed microenvironment remodelling and tumour-specific T cells expansion. Enhanced antitumour effects were found after coculture of organoids with autologous lymphocytes collected from post-cryoablation. We also demonstrated cryoablation-induced tumour elimination required IFNGR expression on tumour cells. In addition, a long-lasting antitumour memory response is achieved by cryoablation and could be enhanced after combination with ICIs. This study revealed endoscopic cryoablation is an efficient and safe therapy for bladder tumour treatment. The tumour-specific immune responses induced by cryoablation could reduce tumour recurrence and metastasis.

NeoAdjuvant pembrolizumab and STEreotactic radiotherapy prior to nephrectomy for renal cell carcinoma (NAPSTER): A phase II randomised clinical trial

BackgroundSurgery remains the standard of care for localised renal cell carcinoma (RCC). Nevertheless, nearly 50% of patients with high-risk disease experience relapse after surgery, with distant sites being common. Considering improved outcomes in terms of disease-free survival with adjuvant immunotherapy with pembrolizumab, we hypothesise that neoadjuvant SABR with or without the addition of pembrolizumab before nephrectomy will lead to improved disease outcomes by evoking better immune response in the presence of an extensive reserve of tumor-associated antigens. Methods and analysisThis prospective, open-label, phase II, randomised, non-comparative, clinical trial will investigate the use of neoadjuvant stereotactic ablative body radiotherapy (SABR) with or without pembrolizumab prior to nephrectomy. The trial will be conducted at two centres in Australia that are well established for delivering SABR to primary RCC patients. Twenty-six patients with biopsy-proven clear cell RCC will be recruited over two years. Patients will be randomised to either SABR or SABR/pembrolizumab. Patients in both arms will undergo surgery at 9 weeks after completion of experimental treatment. The primary objectives are to describe major pathological response and changes in tumour-responsive T-cells from baseline pre-treatment biopsy in each arm. Patients will be followed for sixty days post-surgery. Outcomes and significanceWe hypothesize that SABR alone or SABR plus pembrolizumab will induce significant tumor-specific immune response and major pathological response. In that case, either one or both arms could justifiably be used as a neoadjuvant treatment approach in future randomized trials in the high-risk patient population.

Chemotherapeutic and targeted drugs-induced immunogenic cell death in cancer models and antitumor therapy: An update review.

As traditional strategies for cancer treatment, some chemotherapy agents, such as doxorubicin, oxaliplatin, cyclophosphamide, bortezomib, and paclitaxel exert their anti-tumor effects by inducing immunogenic cell death (ICD) of tumor cells. ICD induces anti-tumor immunity through release of, or exposure to, damage-related molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), calreticulin, adenosine triphosphate, and heat shock proteins. This leads to activation of tumor-specific immune responses, which can act in combination with the direct killing functions of chemotherapy drugs on cancer cells to further improve their curative effects. In this review, we highlight the molecular mechanisms underlying ICD, including those of several chemotherapeutic drugs in inducing DAMPs exposed during ICD to activate the immune system, as well as discussing the prospects for application and potential role of ICD in cancer immunotherapy, with the aim of providing valuable inspiration for future development of chemoimmunotherapy.

In Situ Tumor Vaccine for Lymph Nodes Delivery and Cancer Therapy Based on Small Size Nanoadjuvant.

Tumor vaccine is a promising cancer treatment modality, however, the convenient antigens loading in vivo and efficient delivery of vaccines to lymph nodes (LNs) still remain a formidable challenge. Herein, an in situ nanovaccine strategy targeting LNs to induce powerful antitumor immune responses by converting the primary tumor into whole-cell antigens and then delivering these antigens and nanoadjuvants simultaneously to LNs is proposed. The in situ nanovaccine is based on a hydrogel system, which loaded with doxorubicin (DOX) and nanoadjuvant CpG-P-ss-M. The gel system exhibits ROS-responsive release of DOX and CpG-P-ss-M, generating abundant in situ storage of whole-cell tumor antigens. CpG-P-ss-M adsorbs tumor antigens through the positive surface charge and achieves charge reversal, forming small-sized and negatively charged tumor vaccines in situ, which are then primed to LNs. Eventually, the tumor vaccine promotes antigens uptake by dendritic cells (DCs), maturation of DCs, and proliferation of T cells. Moreover, the vaccine combined with anti-CTLA4 antibody and losartan inhibits tumor growth by 50%, significantly increasing the percentage of splenic cytotoxic T cells (CTLs), and generating tumor-specific immune responses. Overall, the treatment effectively inhibits primary tumor growth and induces tumor-specific immune response. This study provides a scalable strategy for in situ tumor vaccination.

Heterologous prime-boost vaccine using antigen-loaded microparticles and adenovirus (encoding antigen) enhances cellular immune responses and antitumor activity

Heterologous prime-boost vaccines have the potential to promote higher immune responses than homologous prime-boost vaccines and were used in this murine study to investigate the effect on the magnitude of the cellular (and humoral) antigen-specific immune responses and antitumor efficacy when a microparticle formulation (prime) is combined with an adenoviral vaccine (boost). Specifically, the prime comprised chick egg ovalbumin (OVA; 25 µg/dose), used here as a model tumor antigen (TA), encapsulated in microparticles (∼700 nm diameter) made from the biodegradable polymer, 50:50 poly(lactic-co-glycolic acid) (PLGA); while attenuated adenovirus (type 5) encoding OVA (Ad5OVA; 108 PFU/dose) was employed as the boost. The ability of OVA-loaded microparticles to enhance OVA-specific antibody responses, OVA-specific CD3 + CD8 + T cell responses and antitumor activity (i.e., protection against OVA-expressing tumor-challenge) to the heterologous prime-boost vaccine was investigated; and it was found that this prime-boost combination could significantly enhance OVA-specific cellular responses compared to all other vaccination groups and was the only group to confer a significant survival advantage over the unvaccinated group (naïve) in a prophylactic animal tumor model. This finding illustrates the potential for combining TA-loaded PLGA-based microparticles with other vaccine formats to improve tumor-specific cellular immune responses.

Abstract 6543: Comparative analysis of immune landscape according to EGFR mutation and tumor PD-L1 proportion scores in non-small cell lung cancer at single cell resolution

Abstract Introduction: In EGFR-mutant non-small cell lung cancer (NSCLC), the efficacy of immune checkpoint blockade (ICB) is limited with higher incidence of toxicity profiles. EGFR-mutant NSCLC is associated with immune suppressive microenvironment, including lower programmed death-ligand 1 (PD-L1), tumor mutational burden (TMB), and tumor-infiltrating CD8+ T lymphocytes. However, tumors expressing high levels of PD-L1 show improved and durable clinical benefit in immunotherapy. In addition, Understanding the dynamics of tumor microenvironment may elucidate immune response in terms of immune composition, and help overcome resistance to immune checkpoint inhibitors. Here, we demonstrate that conventional type 1 dendritic cells (cDC1s) is one of the key tumor microenvironment, resulting in priming of CD8+ T cells and production of chemokines, such as CXCL9/CXCL10 and cytokines that augment anti-tumor immunity. Methods: A total of 31 surgical samples of NSCLC, including EGFR wild and mutant NSCLC of stages I-III, and 16 normal lung tissue samples were collected. PD-L1 expression in EGFR wild type was categorized into tumor proportion score (TPS) of &amp;lt; 50% and ≥ 50% using immunohistochemistry (IHC). Single cell RNA sequencing was performed with the Scanpy toolkit on the retrospective cohorts of normal (n=16), EGFR mutant (n=14, L858R=6, E19Del=8), EGFR wild TPS ≥ 50% (n=10), and TPS &amp;lt; 50% (n=7). Results: We observed that an overall tumor specific immune response occurred in EGFR wild-type TPS ≥ 50% compared with others. XCL1+ innate-like T cells, which act as an orchestra of tumor specific immune response, were found. These cells had high expression of FCER1G and IL2RB, a receptor for IL-15, However, expression of CD8A/B, CD4 and FCGR3A was low. Interestingly, in these innate-like T cells, the expression of ITGAE, which interacts with E-cadherin in cancer cells, and CD81, which interacts with CD3 and enhances immunological synapse, was observed in EGFR wild TPS ≥ 50%. According to these results, XCR1+ cDC1s were enriched in EGFR wild-type TPS ≥ 50%. CXCL9/CXCL10, which recruits CXCR3+ effector T cells which are primed at the lymph node, was highly expressed in activated DCs at EGFR wild TPS ≥ 50%. CD8+ T cells were significantly enriched in EGFR wild-type TPS ≥ 50%. These cells also expressed IFNG and FLT3LG, which maintain the function of intra-tumoral dendritic cells (DCs), and were also up-regulated in EGFR wild-type TPS ≥ 50%. Among them, exhausted CD8+ T cells expressing PDCD1 were observed in EGFR wild TPS ≥ 50%, and these cells also expressed ITGAE and CD81. In addition, these cells also showed high expression of CXCR3, a receptor for CXCL9/CXCL10 which were expressed in the activated DCs. Conclusion: cDC1s-mediated tumor specific immune response occurred in patients with EGFR wild TPS ≥ 50%, which could be an important mechanism to predict anti-PD-1 response. Citation Format: YoungJoon Park, Su-Jin Choi, Jii Bum Lee, Dong Kwon Kim, Sun Min Lim, Seong Gu Heo, Ke Jia Qi, Byoung Chul Cho, Kyoung-Ho Pyo. Comparative analysis of immune landscape according to EGFR mutation and tumor PD-L1 proportion scores in non-small cell lung cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6543.

Abstract 6399: Hyperthermic intrathoracic chemotherapy (HITOC) improves malignant pleural mesothelioma control through a tumor specific cytotoxic immune response

Abstract Introduction: The management of malignant pleural mesothelioma (MPM) remains challenging with poor patient survival. Local therapies such as hyperthermic intrathoracic cisplatin (HITOC) have shown good tumor control in selected patients. HITOC was shown to increase MPM drug exposure while limiting systemic side effects but alternative mechanisms for HITOC are still lacking. Here, we hypothesized that HITOC induces an immune response directed against MPM which decreases cancer related mortality. Methods: We implanted AB12-luc MPM cells in the pleural cavity of BALB/c mice. A chemotherapy perfusion circuit was downsized to administer cisplatin (80mg/m2 equivalent dose) in the thoracic cavity at normo (37°C, ITOC) or hyperthermic (39°C, HITOC) conditions for 30 minutes. Tumor growth (visualized by bioluminescence) and mouse survival were then assessed. We determined tumor platinum content and distribution by inductively coupled plasma mass spectrometry (ICP-MS) and by laser ablation ICP-MS (LA-ICP-MS) respectively. We also questioned the impact of (H)ITOC on the MPM immune microenvironment (innate and adaptive immune cells, activity and checkpoint expression) by 16-colour flow cytometry and immunohistochemistry. Finally, tumor response to (H)ITOC was assessed in BALB/c athymic mice implanted with AB12-luc cells. Results: MPM tumor control and mouse survival were significantly improved by HITOC compared to controls (ITOC, saline 37 and 39°C). Tumor platinum content was significantly higher in HITOC compared to ITOC but was majorly located at the surface of tumors. HITOC enhanced MPM infiltration by CD8+Granzyme B+ T-cells and decreased the levels of MCHII−/CD80− (M2-like) macrophages compared to controls at day 7. Interestingly, immune checkpoint expression of PD1 and CTLA4 was significantly enhanced in CD8+ lymphocytes in HITOC treated MPM compared to controls at day 7. Finally, the lack of T lymphocytes (BALB/c athymic mice) abrogated the impact of HITOC on MPM control and mouse survival. Conclusion: HITOC improves MPM control through a T lymphocyte immune mediated response. The enhanced immune checkpoint (PD1/CTLA4) expression in CD8+ lymphocytes opens perspectives for the combination of HITOC with immune checkpoint inhibitors. Citation Format: Yameng Hao, Aspasia Gkasti, Louis-Emmanuel Chriqui, Damien Marie, Michel Gonzalez, Thorsten Krueger, Solange Peters, Paul J. Dyson, Etienne Meylan, Johanna Joyce, Sabrina Cavin, Jean Y. Perentes. Hyperthermic intrathoracic chemotherapy (HITOC) improves malignant pleural mesothelioma control through a tumor specific cytotoxic immune response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6399.

Abstract 6638: Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model

Abstract Background: Although anti-PD1 therapy is standard of care in multiple tumor types, unmet need remains for those non-responsive to immunotherapy. Pulsed Electric Field (PEF) treatment may alter the tumor micro-environment, converting an immune “cold” tumor into an immune “hot” tumor, triggering an adaptive immune response and abscopal effect. We evaluated PEF in combination with anti-PD1 in a highly aggressive orthotopic triple negative breast tumor mouse model. Methods: Murine mammary 4T1 cells (2 × 105) non-responsive to anti-PD1 therapy were orthotopically implanted in mammary fat of 48 female Balb/C mice. Once tumors were established (5 mm diameter), mice were randomized into six groups: Sham+IgG controls, aPD-1-only, PEF (1X), PEF (2X), aPD-1 + PEF (1X), and aPD-1 + PEF (2X). Groups 4 and 6 received two PEF treatments, five days apart. Anti-PD1 and isotype matched control IgG was administered (200 ug, intraperitoneal) weekly, starting 3 days after PEF treatment. Tumor volumes were recorded 3x/week and standard survival analyses were conducted per IACUC protocol. In the cohort of tumors collected 30 days after PEF, flow cytometry (FC) evaluated T-cell immune infiltrates and associated markers. Blood was collected at different time points to evaluate systemic immunological response. Tumor-specific cytotoxic lymphocytes (CTL) were isolated from tumor-infiltrating and circulating lymphocytes by gp70 MHC tetramer formation assay. Results: Single PEF reduced tumor growth compared to IgG and aPD-1-only groups by 65% and 62%, respectively. Double PEF treatment reduced tumor growth by 74% and prolonged survival. FC demonstrated circulating helper T-cells and CTL frequency was increased two weeks post-PEF compared to the control group. The tetramer detected substantial increases in antigen-specific T-cell populations in both single and double PEF treatment groups. PEF groups had significant tumor and spleen infiltration of CD8 and CD4 T-cells, including increased tetramer levels. Anti-PD1-only animals showed no meaningful reductions in tumor growth, prolonged survival, or significant changes in CD8, CD4 T cells or in the tetramer assay. Conclusions: PEF treatments prior to initiation of anti-PD1 in orthotopic 4T1 mouse model triggers a tumor-specific adaptive immune response in an immune “cold” tumor model, irrespective of anti-PD-1 therapy. PEF increases circulating and tumor-infiltrating T-cells and antigen-specific T-cells, with these effects observed after a single or two treatments with PEF. Collectively, our results demonstrate that PEF treatment prior to initiation of anti-PD1 treatment can reduce tumor growth, prolong survival, and activate an adaptive immune response in an immune “cold” tumor. These findings suggest that PEF treatment merits further evaluation in IO-non-responsive cancer patients. Citation Format: Ebtesam Nafie, Mukta Wagh, Chiara Pastori, Mo Trikha, Robert Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6638.

Abstract 691: Irradiated whole tumor cells pulsed with mannan-BAM, TLR ligands and anti-CD40 antibody serve as a potent tumor cell vaccine against glioblastoma

Abstract Despite numerous therapeutic advances in cancers, the treatment of glioblastoma multiforme (GBM) remains a challenge. Boosting T-lymphocyte mediated responses against GBM offers a promising approach towards solving this problem. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response with subsequent tumor eradication. This strategy consists of subcutaneous injection of irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands (LTA, Poly (I:C), and R-848), and anti-CD40 agonistic antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in mouse syngeneic GBM tumor models with GL261 and SB28 cells. In GL261 GBM model, complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Of note, the therapeutic efficacy of rWTC-MBTA was abolished in CD4-T and/or CD8-T lymphocyte depleted mice. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated increased antigen presenting cells (dendritic cells and MHC II+ monocytes) and increased cytotoxic IFNγ, TNFα, and granzyme B-secreting CD4-T and CD8-T cells. All three CR mice that were rechallenged with GL261 cells intracranially 14 months after their last rWTC-MBTA treatment resisted tumor development, confirming the establishment of long-term immunological memory. In SB28 GBM model, 80% (8/10) rWTC-MBTA treated mice survived past 95 days after tumor cell implantation without any GBM-related symptoms, with median survival being only 35 days in control groups. In summary, our study demonstrated that rWTC-MBTA strategy can induce potent adaptive immune response against GBM in pre-clinical models. Citation Format: Herui Wang, Rogelio Medina, Juan Ye, Samik Chakraborty, Ondrej Uher, Mitchell Sun, Jan Zenka, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang. Irradiated whole tumor cells pulsed with mannan-BAM, TLR ligands and anti-CD40 antibody serve as a potent tumor cell vaccine against glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 691.