Specific Cancer Immunotherapy Research Articles

CAR-engineered NK cells versus CAR T cells in treatment of glioblastoma; strength and flaws.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that carries a grim prognosis. Because of the dearth of treatment options available for treatment of GBM, Chimeric Antigen Receptor (CAR)-engineered T cell and Natural Killer (NK) therapy could provide alternative strategies to address the challenges in GBM treatment. In these approaches, CAR T and NK cells are engineered for cancer-specific immunotherapy by recognizing surface antigens independently of major histocompatibility complex (MHC) molecules. However, the efficacy of CAR T cells is hindered by GBM's downregulation of its targeted antigens. CAR NK cells face similar challenges, but, in contrast, they offer advantages as off-the-shelf allogeneic products, devoid of graft-versus-host disease (GVHD) risk as well as anti-cancer activity beyond CAR specificity, potentially reducing the risk of relapse or resistance. Despite CAR T cell therapies being extensively studied in clinical settings, the use of CAR-modified NK cells in GBM treatment remains largely in the preclinical stage. This review aims to discuss recent advancements in NK cell and CAR T cell therapies for GBM, including methods for introducing CARs into both NK cells and T cells, addressing manufacturing challenges, and providing evidence supporting the efficacy of these approaches from preclinical and early-phase clinical studies. The comprehensive evaluation of CAR-engineered NK cells and CAR T cells seeks to identify the optimal therapeutic approach for GBM, contributing to the development of effective immunotherapies for this devastating disease.

The Significance of Artificial Intelligence and Machine Learning in the Identification of Immunotherapy Targets for Cancer: Advances, Challenges, and Future Directions

Cancer immunotherapy has revolutionized cancer treatment by leveraging the immune system to target malignant cells, yet resistance in many cancers highlights the need for novel therapeutic targets. Artificial intelligence (AI) and machine learning (ML) have emerged as transformative tools for identifying new immunotherapy targets by analyzing vast datasets from genomics, proteomics, and clinical studies. This review explores the role of AI and ML in advancing the discovery of cancer-specific immunotherapy targets, such as tumor antigens and immune pathways. Key advances include the integration of big data, neoantigen prediction, biomarker discovery, and single-cell analysis. Despite these advancements, challenges remain, including data quality and standardization, interpretability of AI models, computational costs, and the need for biological validation of AI-driven discoveries. As AI and ML technologies continue to evolve, they hold the potential to overcome these barriers, leading to personalized immunotherapy solutions. This review also discusses future directions for AI-driven immunotherapy, emphasizing the need for improved models, ethical considerations, and clinical integration. Keywords: Artificial Intelligence, Machine Learning, Immunotherapy, Cancer, Advances, Challenges, Future Directions

CRISPR-based molecule-regulatory expression platform for specific immunotherapy of cancer.

Cancer is still a major challenge of human health. The abnormality of intracellular cancer-related signal pathways is an important mechanism for the occurrence of cancer. We used a molecular-senor to act on the endogenous signal molecules within the cell to redirect the abnormal signal flows in the cell to treat cancer. Based on CRISPR-dCas12f procedures, we combined aptamers and ribozymes to construct riboswitches, which served as molecular switches to reprogram sgRNAs, so that CRISPR-dCas12f redirected the intracellular anti-cancer signal flows after sensing specific input signal molecules. In addition, the activated molecular sensors and the inhibitory molecular sensors were constructed by combining transcription factors (VP64) and transcription inhibitors (KRAB) to specifically activate and inhibit target genes of interest. Our experimental results showed that the molecular sensors that we designed and constructed specifically sensed the endogenous signal molecules and then redirect the cancer related signal networks of cancer cells. In addition, corresponding logic gates were constructed to distinguish cancer cells from normal cells and redirect anticancer signal flows to trigger specific cancer immunotherapy. The constructed molecular sensors constructed specifically recognized the signal molecules within the cell and redirected the endogenous signal pathway to reprogram the fate of cancer cells.

Abstract 4111: Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy

Abstract Background and objective: Kirsten rat sarcoma viral oncogene homologue (KRAS) is frequently mutated in human cancers. KRAS mutants offer the potential for the development of cancer-specific immunotherapy, as epitopes harbouring mutations are strong neoantigens to which the immune system generates cancer-specific anti-tumor effects. However, previous clinical trials of KRAS-targeted vaccines failed to generate satisfactory anti-cancer efficacy. Evidence suggested that CD4 T cell activity plays a critical role in augmenting the cytotoxic effect of CD8 T cells. Here, we design long peptides based on the MHC II hotspots on the entire KRAS protein that activate both CD4 and CD8 T cells to kill KRAS-driven cancer cells. Long peptides also cover a wider range of HLA alleles to increase their utility in a broader spectrum of patients. Our objective is to create a multi-peptide vaccine simultaneously targeting the mutant and wild-type (WT) regions of human KRAS protein. Methods: By selecting MHC II hotspots via multiple epitope prediction algorithms, we designed and synthesized 8 long peptides with lengths between 25 to 30mers. One of them harboured a G12D mutation, while the others covered the WT regions of KRAS. In addition, the corresponding keyhole limpet hemocyanin (KLH) conjugated peptides were also generated. We first demonstrated their immunogenicity in mouse in vivo models. Different combinations of peptides adjuvanted with either CpG oligodeoxynucleotide + aluminum (CpG+alum) or complete/incomplete Freud’s adjuvant (CFA/IFA) were immunized into Balb/c mice before determining the immune response by mouse interferon γ (mIFNγ) ELISPOT assay. Subsequently, an in vivo prophylactic anti-tumor study was conducted by immunizing this multi-peptide vaccine in Balb/c mice and observing the growth inhibition of the CT26-derived tumors. Results: T cell responses were observed in two peptide mixes with or without G12D mutation, suggesting that both mutant-harbouring peptide and WT peptides were immunogenic. We showed that KLH-conjugated peptides and CpG+alum elicited stronger immune responses than the naked peptides and CFA/IFA respectively. Furthermore, peptide mapping showed that the G12D mutant-harbouring peptide and two other WT peptides may contribute to the overall T cell response in the Balb/c mouse. In the prophylactic anti-tumor study (n = 10), immunization of KRAS naked peptide and KLH-conjugated peptide vaccines significantly inhibited tumor growth by 51.2% (****p < 0.0001) and 44.7% (***p < 0.001) respectively, compared to control. Subsequent mIFNγ ELISPOT assay showed that the tumor inhibitory effect was attributed to the activation of antigen-specific T cell response of the vaccines. Conclusion: Our multi-peptide vaccines targeting both the mutant and WT regions of KRAS could generate KRAS-specific T cell responses and elicit significant anti-tumor effect in mouse models. Citation Format: Chi Han Samson Li, Melvin Toh. Multi-peptide cancer vaccines targeting KRAS induce significant anti-tumor efficacy [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 4111.

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TC2N.

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor related antigens is a promising approach for malignant tumor immunotherapy. TC2N, a recently identified tumor associated antigen from human glioblastoma, is regarded as a promising target of tumor-specific immunotherapy. As one of the most widely used histocompatibility molecules in Chinese is HLA-A*0201, we were able to identify the TC2N peptides that are provided by this molecular type. A panel of antigenic peptides produced from TC2N were predicted by using a computer tool. The binding affinities of three peptides with the highest predicted score to the HLA-A*0201 molecule were evaluated after synthesis. In vitro and in vivo stimulation of the main T-cell response against the predicted peptides. The results demonstrated that TC2N (152-160) was able to release IFN-γ and lyse U251 cells in vitro as well as in vivo by eliciting peptide-specific CTLs. Our results indicated that peptide TC2N (152-160) (RLYGSVCDL) was a novel HLA-A2.1-restricted CTL epitope capable of inducing TC2N specific CTLs in vitro. As TC2N might qualify as a viable target for immunotherapeutic approaches for patients with GBM, we speculated that the newly identified epitope RLYGSVCDL would be of potential use in peptide-based, cancer-specific immunotherapy against GBM.

Immunogenicity and disease control induced by a multineoantigen vaccine (ADXS-503) in patients with metastatic non–small cell lung cancer who have progressed on pembrolizumab.

9042 Background: The administration of a lung cancer-specific immunotherapy with 22 tumor-associated antigens (ADXS-503, A503), has been evaluated as an add-on therapy for patients (pts) with metastatic non-small-cell lung cancer (NSCLC) who have progressed on pembrolizumab (pembro) as last therapy [Haigentz M et al. ASCO 2021]. The present study explores the immunogenicity and potential reversal of immune resistance with A503 when added-on to pembro at the time of progressive disease (PD). Methods: A phase 2 study of A503 + pembro is being conducted in pts with metastatic squamous or non-squamous NSCLC. In Part B of the study, A503 was added-on to pembro within 12 weeks after the first scan showing disease progression following pembro therapy (per RECIST criteria v1.1). Both A503 (1x108 CFU) and pembro (200 mg) were infused by IV every 3 weeks until disease progression or dose-limiting toxicity. Immunogenicity assays included serum cytokine and chemokine levels; flow cytometry; and in-vitro stimulation FluoroSpot assay with 4 different antigen-pools represented in A503 [i.e., hot spot mutations, heteroclitic/wild-type tumor-associated antigens and other antigens not included in the A503 construct (antigen spreading)]. Results: A total of 14 pts have been treated in Part B, of which 13 are clinically evaluable and up to 11 have immune assessments. Combination therapy was well tolerated with transient increased secretion of cytokines for several hours after infusion of A503 consistent with the expected immune activation and transient ‘flu-like’ syndrome. The objective response rate (16%) and disease control rate (46%) were encouraging with 2 partial responses (PR), 4 stable diseases (SD) and 7 pts with PD. Pts with disease control, in particular, generated CD8+ T cells reactive to neoantigens in 1 or more of the 4 antigen pools tested in FluoroSpot. Also, activation of NK cells and of cytotoxic- and memory-CD8+ T cells was mainly observed in pts with PR or SD, but not in those with PD, as shown in the table. Conclusions: Adding A503 to pembro after PD appears to induce innate and adaptive immune responses that may restore or enhance sensitivity to checkpoint inhibitors in pts with clinical benefit. Clinical trial information: NCT03847519. [Table: see text]

Equipping Cancer Cell Membrane Vesicles with Functional DNA as a Targeted Vaccine for Cancer Immunotherapy.

By inducing tumor-specific immune responses, tumor vaccines have recently aroused great research interest. Herein, we design a targeted nanovaccine by equipping cell membrane vesicles (CMVs) harvested from tumor cells with functional DNA including CpG oligonucleotide, an agonist for toll-like receptor 9, as well as an aptamer targeting the dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin (DC-SIGN) receptor overexpressed on DCs. Such DNA-modified CMVs could target DCs and further stimulate their maturation. Notably, our nanovaccines could trigger robust antitumor immune responses to effective delay the tumor growth. Moreover, the combination of CMV-based nanovaccines with an immune checkpoint blockade could result in improved therapeutic responses by eliminating the majority of the tumors as well as long-term immune memory to prevent tumor recurrence. Therefore, by simply assembling functional DNA on CMVs harvested from tumor cells, we propose a general platform of DC-targeted personalized cancer vaccines for effective and specific cancer immunotherapy.

027 2Gy low-dose total body irradiation facilitates antitumoral Th1 immune responses in tumor antigen specific Th1 cell and immune checkpoint inhibitor-based cancer immunotherapy

CD4+ T helper cells are capable of mediating long-term antitumoral immune responses. We developed a combined immunotherapy (COMBO) using tumor antigen-specific T helper 1 cells (Tag-Th1), dual PD-L1/LAG-3 immune checkpoint blockade, and a low-dose total body irradiation (TBI) of 2 Gy, that was highly efficient in controlling the tumor burden of non-immunogenic RIP1- Tag2 mice with late-stage endogenous pancreatic islet carcinomas. In this study, we aimed to explore the impact of 2 Gy TBI on the treatment efficacy and the underlying mechanisms to boost CD4+ T cell-based immunotherapies by positron emission tomography, optical imaging and flow cytometric analyses.

MICA-G129R: A bifunctional fusion protein increases PRLR-positive breast cancer cell death in co-culture with natural killer cells.

Breast cancer cells were reported to up-regulate human prolactin receptor (PRLR) to assist their growth through the utilization of prolactin (PRL) as the growth factor, which makes PRLR a potential therapeutic target for breast cancer. On the other hand, advanced cancer cells tend to down-regulate or shed off stress signal proteins to evade immune surveillance and elimination. In this report, we created a fusion protein consisting of the extracellular domain of MHC class I chain-related protein (MICA), a stress signal protein and ligand of the activating receptor NKG2D of natural killer (NK) cells, and G129R, an antagonistic variant of PRL. We hypothesize that the MICA portion of the fusion protein binds to NKG2D to activate NK cells and the G129R portion binds to PRLR on breast cancer cells, so that the activated NK cells will kill the PRLR-positive breast cancer cells. We demonstrated that the MICA-G129R fusion protein not only binds to human natural killer NK-92 cells and PRLR-positive human breast cancer T-47D cells, but also promotes NK cells to release granzyme B and IFN-γ and enhances the cytotoxicity of NK cells specifically on PRLR-positive cells. The fusion protein, therefore, represents a new approach for the development of breast cancer specific immunotherapy.

Targeting loss of heterozygosity for cancer-specific immunotherapy

Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Tumor-Associated Macrophages in Human Breast, Colorectal, Lung, Ovarian and Prostate Cancers.

Tumor-associated macrophages (TAMs) are major innate immune cells that constitute up to 50% of the cell mass of human tumors. TAMs are highly heterogeneous cells that originate from resident tissue-specific macrophages and from newly recruited monocytes. TAMs’ variability strongly depends on cancer type, stage, and intratumor heterogeneity. Majority of TAMs are programmed by tumor microenvironment to support primary tumor growth and metastatic spread. However, TAMs can also restrict tumor growth and metastasis. In this review, we summarized the knowledge about the role of TAMs in tumor growth, metastasis and in the response to cancer therapy in patients with five aggressive types of cancer: breast, colorectal, lung, ovarian, and prostate cancers that are frequently metastasize into distant organs resulting in high mortality of the patients. Two major TAM parameters are applied for the evaluation of TAM correlation with the cancer progression: total amount of TAMs and specific phenotype of TAMs identified by functional biomarkers. We summarized the data generated in the wide range of international patient cohorts on the correlation of TAMs with clinical and pathological parameters of tumor progression including lymphatic and hematogenous metastasis, recurrence, survival, therapy efficiency. We described currently available biomarkers for TAMs that can be measured in patients’ samples (tumor tissue and blood). CD68 is the major biomarker for the quantification of total TAM amounts, while transmembrane receptors (stabilin-1, CD163, CD206, CD204, MARCO) and secreted chitinase-like proteins (YKL-39, YKL-40) are used as biomarkers for the functional TAM polarization. We also considered that specific role of TAMs in tumor progression can depend on the localization in the intratumoral compartments. We have made the conclusion for the role of TAMs in primary tumor growth, metastasis, and therapy sensitivity for breast, colorectal, lung, ovarian, and prostate cancers. In contrast to other cancer types, majority of clinical studies indicate that TAMs in colorectal cancer have protective role for the patient and interfere with primary tumor growth and metastasis. The accumulated data are essential for using TAMs as biomarkers and therapeutic targets to develop cancer-specific immunotherapy and to design efficient combinations of traditional therapy and new immunomodulatory approaches.

Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced γδT cells against lung cancer cells.

The present study analyzed the antitumor effect of γδT cells transduced with the TCR of cancer‐specific CTLs to establish forceful cancer‐specific adoptive immunotherapy. We cloned the TCRαβ genes from CTLs showing HLA‐B15 restricted recognition of Kita‐Kyushu lung cancer antigen‐1 (KK‐LC‐1), a cancer/germline gene antigen, identified in a lung adenocarcinoma case (F1121). The TCRαβ and CD8 genes were transduced into γδT cells induced from PBLs of healthy volunteers stimulated with zoledronate and IL‐2. The KK‐LC‐1‐specific TCRαβ‐CD8 γδT cells showed cytotoxic activity against the KK‐LC‐1 positive lung cancer cell line F1121L and produced IFN‐γ against F1121L and KK‐LC‐1 peptide‐pulsed F1121 EBV‐B cells. These responses were blocked by HLA class I and HLA‐B/C antibodies. An in vivo assay using NOD/SCID mice with xenotransplantation of human lung cancer cells was performed, and the TCRαβ‐CD8 transduced γδT cells (TCRαβ‐CD8 γδT cells) were intravenously injected. Growth inhibition of KK‐LC‐1+, HLA‐B15+ lung cancer cells was confirmed in mice with injection of the TCRαβ‐CD8 γδT cells from 1 wk after xenotransplantation of cancer cells but not in those treated 2 wk after xenotransplantation. The resected specimens of the tumor, 2 wk after xenotransplantation, highly expressed FasL but not programmed death ligand‐1 (PD‐L1) by immunohistochemical staining. FasL highly expressed cancer cells xenotransplanted 2 wk ago were resistant to TCRαβ‐CD8 γδT cells injection. These results suggested that apoptosis of Fas‐positive TCRαβ‐CD8 γδT cells may be induced by a Fas‐mediated signal after interacting with FasL‐positive cancer cells.

Abstract B67: Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia

Abstract Lymphoproliferative malignancies form a major group of cancers and are responsible for nearly 10% of all cancer deaths in developed countries. Adoptive transfer of engineered T cells has been shown to be a promising treatment approach; hence, screening and evaluation of cellular antigens for T-cell therapy with higher specificity and efficacy is a rapidly progressing area of research. Cancer-specific antigens derived by somatic mutations acquired during tumor development, so called “neoantigens,” represent a very advantageous target repertoire. Here we aimed to develop T-cell receptors (TCRs) targeting recurrent neoantigens in lymphoma and leukemia, for a truly cancer-specific immunotherapy. With this purpose, we have generated T-cell lines from multiple healthy donors targeting one of the most common driver mutations found in B-cell lymphomas: a missense mutation on adaptor protein MyD88 changing leucine at position 265 to proline (L265P). T cell lines generated by autologous in vitro priming were reactive selectively against the predicted mutant epitope restricted to HLA-B7, but not against the corresponding wild-type peptide. TCR alpha and beta nucleotide sequences from these T-cell lines were successfully cloned into a retroviral vector and expressed on T cells of healthy donors for further characterization. All cloned TCRs have shown mutation-specific and HLA-restricted reactivity with varying functional avidity. TCR-transduced T cells were able to selectively recognize and kill engineered target cells expressing MYD88 mutation, proving that the mutant epitope can be naturally processed by human proteasome and presented on HLA-B7. In order to further evaluate the therapeutic potency, we tested TCR-transduced T cells with non-Hodgkin lymphoma cell lines naturally harboring MyD88 L265P, and again observed HLA-restricted and mutation-specific reactivity and cytotoxicity, accompanied by antigen-induced proliferation. Taken together, our data suggest that mutation-specific TCRs can be used to target cancer cells with MyD88 L265P, and hold promise for immunotherapy of B-cell malignancies. Therefore, an application to the European Patent Office for variable region sequences of the TCRs has been made (EP19152801.7) in collaboration with Charité Technology Transfer Office. Preclinical safety screening of the TCRs and in vivo efficacy assessment in mice are in progress. Citation Format: Özcan Çinar, Antonia Busse, Antonio Pezzutto. Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B67.

Tumor antigens heterogeneity and immune response-targeting neoantigens in breast cancer.

Breast cancer is both the most common type of cancer and the most frequent cause of cancer mortality in women, mainly because of its heterogeneity and limited immunogenicity. The aim of specific active cancer immunotherapy is to stimulate the host's immune response against cancer cells directly using a vaccine platform carrying one or more tumor antigens. In particular, the ideal tumor antigen should be able to elicit T cell and B cell responses, be specific for the tumor and be expressed at high levels on cancer cells. Neoantigens are ideal targets for immunotherapy because they are exclusive to individual patient's tumors, are absent in healthy tissues and are not subject to immune tolerance mechanisms. Thus, neoantigens should generate a specific reaction towards tumors since they constitute the largest fraction of targets of tumor-infiltrating T cells. In this review, we describe the technologies used for neoantigen discovery, the heterogeneity of neoantigens in breast cancer and recent studies of breast cancer immunotherapy targeting neoantigens.

Harnessing T-cell activity against prostate cancer: A therapeutic microparticulate oral cancer vaccine

Prostate Cancer specific immunotherapy in combination with immune stimulating adjuvants may serve as a viable strategy for facilitating tumor regression and preventing recurrence. In this study, an oral microparticulate vaccine encapsulating tumor associated antigens (TAA) extracted from a murine prostate cancer cell line, TRAMP-C2, was formulated with the help of a spray dryer. Microparticles were characterized in vitro to determine their physicochemical properties and antigenicity. Formulated microparticles had an average size of 4.92 ± 0.5 μm with a zeta potential of 7.92 ± 1.2 mV. In order to test our formulation for its ability to demonstrate adequate antigen presentation and co-stimulation, microparticles were tested in vitro on murine dendritic cells. In vitro biological characterization demonstrated the activation of specific immune system markers such as CD80/86, CD40, MHC-I and MHC-II. Following in vitro characterization, in vivo anti-tumor efficacy of the oral microparticulate vaccine was evaluated in C57BL/6 male mice. Combination therapy of vaccine microparticles with cyclophosphamide and granulocyte macrophage-colony stimulating factor (GM-CSF) demonstrated a five-fold reduction in tumor volume as compared to non-vaccinated mice. At the cellular level, cyclophosphamide and GM-CSF augmented the vaccine response as indicated by the reduced tumor volume and significant elevation of cytotoxic T-cell (CTL) CD8+ and (T-helper) CD4+ T-cells compared to mice receiving vaccine microparticles alone. Furthermore, our studies indicate a significant reduction in T-regulatory cells (T-regs) in mice receiving vaccine along with GM-CSF and cyclophosphamide, one of the immune escape mechanisms linked to tumor growth and progression. Thus, oral microparticulate vaccines have the potential to trigger a robust anti-tumor cellular response, and in combination with clinically relevant agents, significantly resist tumor growth and progression.

Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma

BackgroundAlthough mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs.MethodsIn-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data.ResultsThe approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations.ConclusionsThis study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy. MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells. ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity. DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.

Abstract 1709: Targeting immune checkpoint therapy to the lung tumor microenvironment

Abstract Lung cancer is the leading cause of cancer deaths worldwide. Many lung cancer patients are diagnosed with advanced disease. These patients have a low 5-year survival rate and limited treatment options. Novel therapies, which target biomarkers that are overexpressed in lung cancer but have basal expression in benign tissue, are desperately needed. The goal of this work was to develop a targeted agent for immune checkpoint therapy of lung cancer. Recently, immune checkpoint inhibitors have been approved for use in lung cancer and many more are being tested in clinical trials. The current immune checkpoint inhibitor agents are not tumor-targeted. Targeting the immune checkpoint inhibitor to tumor cell-surface markers should concentrate the conjugate in the tumor microenvironment and enhance the immune response in the tumor while reducing the systemic dosages needed, resulting in lower systemic toxicity. The delta opioid receptor (DOR) is expressed in some lung cancers, but is not expressed or is expressed only at basal levels in normal tissues outside the brain. We have previously synthesized fluorescently-labeled DOR-targeted imaging agents based on a synthetic peptide antagonist (DORL). These targeted fluorescent agents have high affinity and selectivity for DOR, and exhibit good pharmacokinetic (PK) and biodistribution (BD) profiles, i.e. specific tumor uptake with rapid systemic clearance and no uptake in tissues of concern, e.g. brain. We are now developing lung cancer-specific immunotherapy agents that target the DOR by conjugating DORL to immunomodulatory molecules. In the current work, we synthesized a fluorescently-labeled DOR targeting ligand and conjugated it to an anti-PD1 antibody (DORL-PD1). We synthesized immunoconjugates with several targeting ligand-to-antibody ratios (TARs). We engineered murine lung cancer cells to constitutively express the DOR. By lanthanide time-resolved fluorescence (LTRF) competitive binding assays, we have shown that the agents have high avidity for the DOR in vitro with higher TARs resulting in higher binding avidity. We characterized the uptake of DORL-PD1 in vitro using live-cell fluorescence microscopy. Using syngeneic engraftment tumor models in immunocompetent mice, we performed longitudinal fluorescence imaging studies to determine the agent circulation time (PK), tumor selectivity and tissue distribution (BD). Immune checkpoint efficacy studies were performed using the DOR negative mouse tumor models. In conclusion, we have synthesized fluorescent DOR-targeted immune checkpoint therapy agents, DORL-PD1; demonstrated avidity and selectivity for the DOR in vitro and in vivo; and immune checkpoint therapy efficacy in vivo. Future studies will evaluate the efficacy of DORL-PD1 in immune competent mice bearing DOR positive tumors. These agents could be useful for increasing the efficacy and reducing systemic toxicity of immune-checkpoint therapy of lung cancer. Citation Format: Allison S. Cohen, Michael L. Doligalski, Hong Zheng, Narges K. Tafreshi, Veronica Estrella, Nella Delva, Jonathan Nguyen, Amer Beg, Mark L. McLaughlin, David L. Morse. Targeting immune checkpoint therapy to the lung tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2017-1709

Abstract 4624: Natural killer cell licensing contributes to clearance of lung cancer

Abstract a) Natural Killer (NK) cell Inhibitory receptors function in a dichotomous role. Their engagement by MHC Class I upon encountering a target results in the inhibition of cytotoxicity. During NK development, however, their interaction with MHC Class I mediates the process of licensing or education, leading to an increase in responsiveness and cytotoxicity. For this reason the role of inhibitory receptors in controlling the clearance of MHC Class I+ solid tumors such as lung cancer is poorly defined. Since NK cells comprise the primary cytotoxic lymphocyte responsible for clearance of lung cancer, we set out to determine whether the licensing status of NK cells contributes to lung cancer susceptibility. b) Growth of MHC Class I+ murine lung cancer was evaluated in MHC Class I- mice, with unlicensed NK cells in direct comparison to MHC Class I+ mice with licensed NK cells. IFN-γ production was evaluated after plate bound antibody stimulation through the NKp46 and NKG2D pathways both flow cytometrically and by quantitative RT-PCR. Bone marrow transplantation was performed by injection of 10x106 unfractionated bone marrow cells after 500cGy irradiation and NK and CD4+ T cell depletion of recipient host. c) Injection of Lewis Lung Carcinoma (LLC) revealed a significant decrease in pulmonary tumor burden in mice with licensed NK cells (230.4±21.6 vs. 442.7±53.8 mg) that disappeared after NK depletion (421.4±36.8 vs. 421.9±66.3 mg). Freshly isolated licensed NK cells lysed LLC more robustly than unlicensed NK cells (28.7±.16% vs. 4.9±.2% specific lysis at 100:1 ratio respectively). Lung cancer expresses stress ligands recognized by NKG2D and NKp46 activating receptors. Licensed NK cells produced significantly more IFN-γ than their unlicensed counterparts after plate-bound stimulation with anti-NKp46 and anti-NKG2D (17.9±2.8% vs. 5.4±.8% respectively for dual NKp46 and NKG2D stimulation). No differences were evident between licensed and unlicensed NK cells in ribosomal mass, making it unlikely that translational defects were responsible for altered cytokine production. Licensing status also did not affect stability of IFN-γ but did affect mRNA levels. This suggests transcriptional control of licensing-mediated NK activity. IFN-γ production could be significantly increased in unlicensed NK cells by reintroduction of MHC Class I through non-myeloablative bone marrow transplantation from MHC Class I-sufficient congenic donors. Blockade of H2Kb-Ly49c (MHC Class I-inhibitory receptor) interaction during NK development in C57BL/6 mice using a non-depleting anti-Ly49c antibody (clone 4LO) decreased IFN-γ production in NK cells. d) The role of NK cell licensing and tumor-specific cytotoxicity is highly controversial. We now demonstrate that licensed NK cells mediate a superior lung cancer-specific immune response in a murine model. Our data suggests that altering the licensing status of NK cells may be an avenue of lung cancer-specific immunotherapy. Citation Format: Shi Lei, Kang Li, Saeed Arefanian, Alexander S. Krupnick. Natural killer cell licensing contributes to clearance of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2017-4624

MTE08.01 Immunotherapy in Early and Locally Advanced NSCLC: Challenges and Perspectives

MTE08.01 Immunotherapy in Early and Locally Advanced NSCLC: Challenges and Perspectives