T-cell Receptor Therapy Research Articles

Targeting epigenetic regulation and post-translational modification with 5-Aza-2’ deoxycytidine and SUMO E1 inhibition augments T-cell receptor therapy

BackgroundCellular immunotherapy using modified T cells offers new avenues for cancer treatment. T-cell receptor (TCR) engineering of CD8 T cells enables these cells to recognize tumor-associated antigens and tumor-specific neoantigens....

Sociodemographic Factors Influencing Access to Chimeric Antigen T-Cell Receptor Therapy for Patients With Non-Hodgkin Lymphoma

BackgroundChimeric antigen receptor T-cell (CAR-T) therapies are available for patients with Non-Hodgkin Lymphoma (NHL); however, their use has been limited in accessibility due to nondisease factors. Patients & MethodsWe conducted a retrospective study evaluating the influence of sociodemographic factors on access and outcomes after CAR-T therapy for adult patients with B-cell NHL in our institution treated between 2016 and 2023. ResultsAmong 154 patients treated with CAR-T, 43% were older than 65 years, 68% male, and 14% non-White (including Hispanic). Of those under 65, 66% had private insurance, while 82% over 65 had Medicare. Most patients (85%) were from in-state, 29% from areas below the national poverty level and 18% from nonmetropolitan areas. Distance to the treatment center was greater than 30, 60 or 120 miles for 52%, 40% and 29% of patients, respectively. No significant differences were found in the use of commercial versus investigational products among racial/ethnic minorities or those living >60 miles from the center. However, patients from nonmetropolitan areas and those below the national poverty level were less likely to receive commercial products. With a median follow-up of 11 months, the 1-year overall survival (OS) was 63.2% (95th CI 59.9%-66.8%). Poverty was associated with lower 1-year OS (HR 0.4, 95th CI 0.17-0.90, P = .031). ConclusionOur study shows that CAR-T therapy can be delivered across sociodemographic barriers and underscores the importance of considering social determinants of health to optimize access for all patients.

Novel Approaches to Managing Patients with Relapsed and Refractory Waldenström Macroglobulinemia.

Waldenström macroglobulinemia is a rare non-Hodgkin lymphoma (NHL) characterized by lymphoplasmacytic bone marrow infiltration associated with an immunoglobulin M (IgM) monoclonal gammopathy. Over the past two decades, a number of important novel therapies have emerged for the treatment of relapsed and refractory (R/R) WM. The purpose of this review is to discuss these novel agents. Chemoimmunotherapy which formed the basis treatment for R/R WM is slowly being replaced by novel targeted agents. These therapies, including Bruton's tyrosine kinase inhibitors, proteasome inhibitors, and B-cell lymphoma 2 inhibitors, have widened the landscape of management. Emerging therapies currently under investigation, such as bispecific T-cell engagers, chimeric antigen T-cell receptor therapy, and novel small molecule inhibitors, have additionally shown the potential to improve response and survival. The treatment of R/R WM has greatly evolved, in large part due to a greater understanding of the biology of WM, and the evaluation of novel targeted agents in the basket trials of NHL, showing early activity in the small WM cohorts. Combination regimens with these established and emerging novel therapies have the potential to further improve disease control and induce higher rates of deep responses. Strategies aimed at altering the disease trajectory would require randomized controlled trials to provide relevant data on optimal integration and sequencing of more effective and tolerable regimens earlier in the disease course.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Abstract 27: A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity

Abstract Background: Genetically engineered T cells with T cell receptor (TCR-T) cell therapy have shown great potential for cancer treatment by targeting tumor-associated antigens. Despite its success in treating various tumor types, such as synovial sarcoma, head and neck tumors, and cervical cancer, its application in liver cancer is limited. Alpha-fetoprotein (AFP), a highly expressed tumor-associated antigen in liver cancer, is an ideal target for TCR-T cell therapy. Although early human trials have confirmed the safety of AFP-targeting TCR-T products, their efficacy is still modest, highlighting the need for further research and development. Methods: We developed a robust strategy combining ex vivo stimulation and single-cell paired TCR sequencing to identify AFP-recognizing TCRs in liver cancer patients. To optimize TCR expression and minimize mispairing, we employed codon optimization and replaced the constant regions with murine counterparts. Furthermore, structural analysis guided the affinity optimization of the CDR3 region to enhance the antitumor function of the selected TCR. The anti-tumor efficacy of TCR-T cells was rigorously evaluated through in vitro cytotoxicity assays using the Real-Time Cell Analyzer (RTCA) system and in vivo antitumor assessment in immunodeficient mouse xenograft models. Antigen specificity, HLA restriction, and other potential toxicity were carefully evaluated in both in vitro and in vivo models. Results: We successfully cloned a highly effective TCR that recognizes the AFP158-166 epitope presented by HLA-A*0203. After sequence optimization, we further changed a single amino acid on the TCR vβ chain based on AI prediction. The expression and functionality of this TCR was significantly improved. When introduced into human T cells, the single amino acid mutated TCR demonstrated robust and specific anti-tumor activity in various models, including cultured tumor cells, patient-derived organoids, and mouse xenografts models. Notably, this TCR also redirected CD4 T cells to recognize tumor cells, induced the secretion of multiple cytokines, and maintained long-lasting anti-tumor effects. Our rigorous screening assays confirmed that the TCR showed no cross-reactivity with the human proteome or major HLA subtypes. Additionally, extensive preclinical safety assessments in tumor-bearing animals treated with ST08A01-AM14 TCR-T cells showed no significant safety concerns. Conclusion: In this study, we have developed an HLA-A*0203 restrict AFP158-166 specific TCR-T therapy, which exhibits exceptional anti-tumor effects while ensuring a favorable safety profile. The promising results support the further investigation against AFP-positive solid tumors in clinic. Citation Format: Chen-Song Huang, shudan Ou, Jun Li, Junjun Chu, Yanyan Han. A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity [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 27.

Precision Targeting and Genetically Modified T Cells for Targeting Cancer Cells

In this review cancer treatment, despite notable progress, challenges persist globally. Traditional methods like surgery, chemotherapy, and radiotherapy, while effective, often compromise patients' overall quality of life due to side effects. Immunotherapeutic strategies, especially Chimeric Antigen Receptor T cells, show promise by leveraging the immune system to target tumors independently of certain immune escape mechanisms. However, CAR-T cells' specificity to surface antigens limits their applicability. Precise cancer management demands ongoing research to refine and broaden these therapies. Employing CAR or T-cell receptor therapies, genetic engineering enhances T-cell antigenic specificity, optimizing cancer immunotherapy precision. CARs, synthetic receptors engineered for tumor antigen recognition, represent a groundbreaking approach, intertwining immunotherapy, gene therapy, and cancer therapy. The human immune system's ability to discern self from non-self-entities forms the basis of immunotherapy, fostering innovative modalities that selectively target cancer cells. CAR-T therapy, with FDA approval for leukemia and lymphoma, holds transformative potential but faces safety and efficacy challenges. Advances, including mitigating cytotoxicity and enhancing therapeutic efficacy, show promise. Utilizing genetic alteration, CARs have shown efficacy in the treatment of hematologic malignancies, particularly CD19 CARs in B cell blood cancers. Current study is investigating the potential uses of CAR-T cell treatment in patients with lymphoma and myeloma.

Management Patterns and Outcomes for Multiple Myeloma Patients with High-Risk Cytogenetics at an Urban Safety Net Hospital

Background: Over the past decade, there have been tremendous advancements and drug approvals for multiple myeloma (MM). This is especially true with the advent of therapies such as chimeric antigen T-cell receptor therapy (CART). As a result, patients with MM are living longer; with many patients living a decade or longer. However, there is still a small subset of patients with high-risk disease that unfortunately continue to do poorly. High-risk disease based on cytogenetics is defined by disease specific biologic features such as t(4;14), del17p, t(14;16), t(14;20), and gain of 1q. Patients with such features tend not to respond as well to induction therapies and autologous stem cell transplantation as those with standard risk cytogenetics. Here we report a retrospective cohort of patients with high-risk cytogenetics to evaluate patient characteristics, management patterns, and outcomes in an urban safety net hospital. Methods: We performed a retrospective chart review of all patients aged 18 years or older who presented to Harbor-UCLA Medical Center between 2008-2022 with the diagnosis of MM. Patients were included if they were 18 years or older, had MM based on IMWG criteria, and had high risk cytogenetics defined as having one or more of the following abnormalities: t(4;14), del17p, t(14;16), t(14;20), and gain of 1q. Data collected included demographics, induction regimens, transplant status, response, consolidation therapy, progression free survival (PFS) on first line therapy, and overall survival (OS). Results: We identified 28 MM patients with high-risk cytogenetics. The median age at diagnosis was 65, and the cohort was 79% Hispanic, 10% Asian, 7% African American, and 4% Caucasian. 54% of patients were female and 46% were male. Cytogenetic abnormalities were prevalent in the following order: deletion 17p (50%), gain of 1q (29%), t(4;14) (18%), t(14;16) (3%). The most used induction regimen was cyclophosphamide, bortezomib, and dexamethasone (CyBorD) (71%) followed by revlimid, velcade, and dexamethasone (RVD) (25%). Only 32% of patients received autologous stem cell transplantation. The most common drug used in the maintenance setting was velcade (50%). The median PFS for first line treatment in patients with high-risk cytogenetics was 14 months (18.1 months average). The median OS was 29 months (average 37.6 months). Conclusion: In this high-risk patient population, with few resources available, we found that the majority of patients were treated with CyBorD, primarily due to kidney disease or inability to access revlimid upfront. In addition, the majority of patients did not receive autologous stem cell transplantation, which is considered standard of care for upfront management of MM, especially in patients with high-risk cytogenetics. Our centers median PFS for first line treatment was 14 months, which was significantly lower compared to other published data with reported PFS of around 37 months. In addition, our median OS of 29 months was also lower compared to reported OS at other centers of around 60 months. This is most likely due to the low utilization of RVD upfront and transplantation, among other comorbidities that are unique to our population. Further research is needed to characterize real world outcomes and elucidate mechanisms of disparity.

The Landscape of Adoptive Cellular Therapies in Ovarian Cancer.

Ovarian cancers are typically poorly immunogenic and have demonstrated disappointing responses to immune checkpoint inhibitor (ICI) therapy. Adoptive cellular therapy (ACT) offers an alternative method of harnessing the immune system that has shown promise, especially with the success of chimeric antigen receptor T-cell (CAR-T) therapy in haematologic malignancies. So far, ACT has led to modest results in the treatment of solid organ malignancies. This review explores the possibility of ACT as an effective alternative or additional treatment to current standards of care in ovarian cancer. We will highlight the potential of ACTs, such as CAR-T, T-cell receptor therapy (TCR-T), tumour-infiltrating lymphocytes (TILs) and cell-based vaccines, whilst also discussing their challenges. We will present clinical studies for these approaches in the treatment of immunologically 'cold' ovarian cancer and consider the rationale for future research.

Comparative effectiveness of salvage chemotherapy regimens and chimeric antigen T-cell receptor therapies in relapsed and refractory diffuse large B cell lymphoma: a network meta-analysis of clinical trials

The optimal salvage chemotherapy regimen (SC) for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) prior to autologous stem cell transplant remains unclear. Moreover, although chimeric antigen receptor T cell (CAR-T) therapies were recently approved for primary refractory DLBCL, head-to-head comparisons are lacking. We searched MEDLINE, EMBASE and CENTRAL to July 2022, for randomized trials that enrolled adult patients with R/R DLBCL and performed network meta-analyses (NMA) to assess the efficacy of SC and CAR-T therapies. NMA of SC (6 trials, 7 regimens, n = 1831) indicated that rituximab with gemcitabine, dexamethasone, cisplatin (R-GDP) improved OS and PFS over compared regimens. NMA of 3 CAR-T trials (n = 865) indicated that both axi-cel and liso-cel improved PFS over standard of care, with no difference in OS. Our results indicate that R-GDP may be preferred for R/R DLBCL over other SC compared. Longer follow-up is required for ongoing comparative survival analysis as data from CAR-T trials matures.

Q&A: Philip Greenberg on T Cells, IO's Future.

Philip Greenberg, MD, assumed a 1-year term as president of the American Association for Cancer Research in April 2023. In this interview, he highlighted some tenure priorities, including improving scientists' ability to communicate with the public, and discussed his own research on T-cell receptor therapies, as well as the upcoming decade for immuno-oncology.

New therapeutics for soft tissue sarcomas: Overview of current immunotherapy and future directions of soft tissue sarcomas.

Soft tissue sarcoma is a rare and aggressive disease with a 40 to 50% metastasis rate. The limited efficacy of traditional approaches with surgery, radiation, and chemotherapy has prompted research in novel immunotherapy for soft tissue sarcoma. Immune checkpoint inhibitors such as anti-CTLA-4 and PD-1 therapies in STS have demonstrated histologic-specific responses. Some combinations of immunotherapy with chemotherapy, TKI, and radiation were effective. STS is considered a 'cold', non-inflamed tumor. Adoptive cell therapies are actively investigated in STS to enhance immune response. Genetically modified T-cell receptor therapy targeting cancer testis antigens such as NY-ESO-1 and MAGE-A4 demonstrated durable responses, especially in synovial sarcoma. Two early HER2-CAR T-cell trials have achieved stable disease in some patients. In the future, CAR-T cell therapies will find more specific targets in STS with a reliable response. Early recognition of T-cell induced cytokine release syndrome is crucial, which can be alleviated by immunosuppression such as steroids. Further understanding of the immune subtypes and biomarkers will promote the advancement of soft tissue sarcoma treatment.

Overcoming current challenges to T-cell receptor therapy via metabolic targeting to increase antitumor efficacy, durability, and tolerability.

The antitumor potential of personalized immunotherapy, including adoptive T-cell therapy, has been shown in both preclinical and clinical studies. Combining cell therapy with targeted metabolic interventions can further enhance therapeutic outcomes in terms of magnitude and durability. The ability of a T cell receptor to recognize peptides derived from tumor neoantigens allows for a robust yet specific response against cancer cells while sparing healthy tissue. However, there exist challenges to adoptive T cell therapy such as a suppressive tumor milieu, the fitness and survival of transferred cells, and tumor escape, all of which can be targeted to further enhance the antitumor potential of T cell receptor-engineered T cell (TCR-T) therapy. Here, we explore current strategies involving metabolic reprogramming of both the tumor microenvironment and the cell product, which can lead to increased T cell proliferation, survival, and anti-tumor cytotoxicity. In addition, we highlight potential metabolic pathways and targets which can be leveraged to improve engraftment of transferred cells and obviate the need for lymphodepletion, while minimizing off-target effects. Metabolic signaling is delicately balanced, and we demonstrate the need for thoughtful and precise interventions that are tailored for the unique characteristics of each tumor. Through improved understanding of the interplay between immunometabolism, tumor resistance, and T cell signaling, we can improve current treatment regimens and open the door to potential synergistic combinations.

Consensus Cachexia Criteria Are Independently Linked to Progression Free and Overall Survival in Multi-Site Analysis of Patients with Aggressive B-Cell Lymphomas Treated with CAR T-Cell Therapy

Background: Cachexia is a muscle wasting disorder present in 50% of cancer patients, 80% of advanced cancer patients, and is indirectly responsible for 20-30% of cancer-related mortality (von Haehling et al, JCSM. 2016). Prior studies have shown that cachexia is linked to poor treatment response and may be a proxy for an altered immune-state (Coss et al, CCR. 2018). While our pilot work suggested that serum-based cachexia markers are associated with poor survival after chimeric antigen T-cell receptor therapy (CART) (Roy et al., BrJH. 2022), the independent impact of cachexia, defined using consensus criteria, on short- and long-term outcomes after CART has not been explored. Our objective in this study was to evaluate whether consensus weight-based classifications of cachexia have an impact on short- and long-term outcomes after anti-CD19 CART in aggressive B-cell lymphomas. Methods: Adults with R/R aggressive B-cell NHL treated with anti-CD19 CART between 2015- 2020 from 13 academic centers in the US were identified. Demographic and clinical data were collected and analyzed as univariates via Chi-squared and Kaplan-Meier analysis. Cox multivariable regression analyses assessed impact of significant covariates on survival with α = 0.05. Cachexia (>5% body weight loss (BWL) or 2% BWL with BMI < 20), pre-cachexia (0-5% BWL), and non-cachexia (no BWL) statuses were defined at apheresis using consensus weight-based criteria (Fearon et al., Lancet. 2011). Results: 433 patients had available weight and BMI data to establish cachexia status. Incidence of cachexia was 37%, pre-cachexia was 18%, and non-cachexia was 43%. Median follow up after CART was 12.4 mo for the entire cohort and 18 mo for survivors. There was no significant univariate association between cachexia and age, sex, performance status, co-morbidity index, CART product, LDH elevation, or IPI score. Significant univariate associations included cachexia patients were less likely to be on a clinical trial (p<0.001), more likely to have auto-transplant pre-CART (p<0.01), and more likely to have ABC cell of origin (p<0.05), while increased lines of pre-CART therapy also trended towards association (p=0.08). Amongst short-term outcomes, there was no association between cachexia and post-treatment cytokine release syndrome (p=0.93), neurotoxicity (ICANS) (p=0.20), infection (p=0.25), overall response at day 180 (p=0.91), complete response rate at day 180 (p=0.52), or advancement to post-CART salvage therapy (p=0.15). In survival analyses (Figure 1), patients with cachexia had decreased median progression free survival (mPFS) of 6.7 mo, compared with 13 mo for non-cachexia (HR 1.3 (95%CI 1.0 -1.8), p=0.04) and 23.4 mo for pre-cachexia (HR 1.5 (95%CI 1.0-2.2), p=0.03). Additionally, median overall survival (mOS) was 16.5 mo for cachectic patients, significantly lower compared to mOS of 29 mo for non-cachexia (HR 1.7 (95%CI 1.2 - 2.2), p=0.005) and a mOS that was not reached for pre-cachexia. In a multivariate analysis (Table 1), independent predictors of decreased mPFS were cachexia (HR 1.6 (95%CI 1.1 - 2.3), p = 0.02), number of pre-CART therapies (HR 1.2 (95%CI 1.0 - 1.3), p = 0.01), and elevated LDH at time of CART (HR 1.6 (95%CI 1.1 - 2.4), p = 0.01). Independent predictors of decreased mOS were cachexia (HR 1.9 (95%CI 1.2 - 2.9), p = 0.003), and number of pre-CART therapies (HR 1.2 (95%CI 1.0 - 1.4), p = 0.01). Conclusion: As durable remission rates remain close to 40%, improved predictors of survival post-CART treatment for lymphoma are needed. In this multi-site study, our findings suggest that cachexia, defined by weight-based criteria, should be included as a prognostic marker for CART. Our data also indicate that, amongst CART patients, the weight/BMI threshold for cachexia criteria (>5% BWL or 2% BWL with BMI < 20) is more prognostically relevant than pre-cachexia (0-5% BWL). While prior mechanistic studies show that cachexia is driven by an altered inflammatory state, our study expands upon this notion. Our data show that although patients with cachexia have similar initial response rates to non-cachectic patients, there may be a long-term dampened immune response in this population that ultimately impacts survival. Further study into cachexia-focused therapies is needed and specific investigation of the role of cachexia in immune evasion mechanisms may yield new targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MiR-150 promotes progressive T cell differentiation via inhibiting FOXP1 and RC3H1

T cells used in immune cell therapy, represented by T cell receptor therapy (TCR-T), are usually activated and proliferated in vitro and are induced to a terminally differentiated phenotype, with limited viability after transfusion back into the body. T cells exhibited a robust proliferative potential and in vivo viability in the early stages of progressive differentiation. In this study, we identified microRNAs that regulate T cell differentiation. After microRNA sequencing of the four subsets: Naïve T cells (TN), stem cell-like memory T cells (TSCM), central memory T cells (TCM）, and effector memory T cells (TEM), miR-150 was identified as the most highly expressed miRNA among the four subsets and was lowly expressed in the TSCM cells. We predicted the target genes of miR-150 miRNA and performed Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses. We observed that the target genes of miR-150 were enriched in pathways associated with T-cell differentiation. FOXP1 and RC3H1 were identified as key target genes of miR-150 in the regulation of T-cell function. We examined the effects of miR-150 on the differentiation and function of healthy donor T-cells. We observed that miR-150 overexpression promoted T-cell differentiation to effector T-cells and effector memory T-cells, enhanced apoptosis, inhibited cell proliferation and increased secretion of pro-inflammatory cytokines such as IFN-γ and TNF-α. In addition, the expressions of early differentiation-related genes (ACTN1, CERS6, BCL2, and EOMES), advanced differentiation-related genes (KLRG1), and effector-function-related genes (PRF1 and GZMB) were significantly decreased after overexpression of miR-150. Collectively, our results suggested that miR-150 can promote progressive differentiation of T cells and the downmodulation of miR-150 expression while performing adoptive immunotherapy may inhibit T-cell differentiation and increase the proliferative potential of T cells.

Abstract 2526: Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients

Abstract Introduction: KK-LC-1 is a cancer testis antigen expressed notably in lung, breast, and gastric cancer making it an attractive target for T-cell receptor therapy. Here, we evaluate cell densities of NSCLC tissue specimens with known KK-LC-1 gene expression to better characterize the tumor immune microenvironment in such patients. Methods: A seven-plex mIF panel (FoxP3, PDL1, PanCK, PD1, CD8, KK-LC-1, and CD68) was developed based on a previously established mIF Lung-cancer panel and addition of KK-LC-1 following rigorous optimization of KK-LC-1 antibody titer and panel order with qualitative assessment of monoplex immunofluorescent assays. Serial sections from 23 NSCLC cases (14 biopsy samples and 9 excision specimens) with known KK-LC-1 gene expression values were collected. Tissue sections were stained and imaged on an Akoya Biosciences Vectra Polaris multispectral imaging platform. The regions of interest were identified and stamped with the Phenochart mIF image management software. Spectral unmixing, tissue segmentation and cell phenotyping were performed with Akoya Biosciences InForm image analysis software using the multispectral fields workflow. The 4-tier positive staining thresholds for PD-L1 and KK-LC-1 were determined by a pathologist (WDW). Cell densities in the tumor and adjacent stroma as well as the KK-LC-1 H-score were assessed by linear regression analysis. Student’s t-test was used to compare between the top quartile of tumors based on KK-LC-1 gene expression (Q4) and the remaining 3 quartiles (Q1-Q3). Results: The study consisted of 17/23 adenocarcinoma (73.9%), 4/23 squamous cell carcinoma (SCC) (17.3%), and 2/23 (8.6%) unclear or mixed histology tumors. Adenocarcinoma trended to have higher KK-LC-1 gene expression compared to SCC tumors (mean 17.34 vs. 0.65, p-value = 0.204). 13/23 (56.5%) were pan-wild type, 6/23 (26.0%) were EGFR mutated, 2/23 (8.7%) were KRAS mutated, and 1/23 (4.4%) were MET Exon 14 skip and multiple alterations. We saw significantly higher cell density of FoxP3 in tumor cells in Q4 versus Q1-Q3 (mean 7.17 vs. 0.8 cells/mm2, p-value = 0.004), and there was a trend towards a higher FoxP3/CD8 cell density ratio (mean 3.28 vs. 0.05, p = .110) and higher PD-1 cell density (mean 186.7 vs. 32.8 cells/mm2, p=0.1548) in tumor cells in Q4 versus Q1-Q3. There was significant correlation towards increased CD8+ T cell density ratio between tumor and stroma cells in cases with higher KK-LC-1 gene expression (r2= 0.307, p = 0.011). Conclusion: KK-LC-1 expression in lung cancer is seen in multiple molecular subsets and is associated with increase in both regulatory T cells in tumor and in CD8+ T-cells in tumor relative to stroma. Strategies to deplete regulatory T cells and to further amplify CD8+ T cells should be pursued in the development of T-cell receptor therapy targeting KK-LC-1. Citation Format: Ainaz Dory Barkhordarzadeh, Robert C. Hsu, Rongfu Wang, Sue E. Martin, Jorge J. Nieva, William Dean Wallace. Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2526.

First Drug Approved for Rare Eye Cancer.

Tebentafusp, the first drug shown to extend overall survival in people with uveal melanoma, was greenlighted by the FDA in late January to treat patients with inoperable or metastatic forms of the aggressive eye cancer. The drug is also the first T-cell receptor therapy to reach the market.

Molecular characterization of Kita-Kyushu lung cancer antigen (KK-LC-1) expressing carcinomas.

Cancer/testis antigens (CTAs) are strongly expressed in some solid tumors but minimally expressed in normal tissue, making them appealing therapeutic targets. KK-LC-1 (CXorf61) has cytoplasmic expression in gastric, breast, and lung cancer. We characterized the molecular subtypes of non-small cell lung cancer (NSCLC) expressing KK-LC-1 to inform rational clinical trials of T-cell receptor therapy (TCR-T) targeting KK-LC-1. 9790 NSCLC tumors that underwent whole transcriptome sequencing (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ) were analyzed. Tumors were split into quartiles based on KK-LC-1 expression and pathological and molecular differences were investigated. Adenocarcinoma had significantly higher KK-LC-1 expression than squamous cell carcinoma (median, 3.25 vs. 1.17 transcripts per million (TPM), p < 0.0001). Tumors with the highest quartile of KK-LC-1 expression had a greater proportion of tumors with high tumor mutation burden (TMB) (≥10 mutations per megabase; 44% vs. 28% in Q1, p < 0.001). Increased KK-LC-1 expression was associated with increased M1 macrophage abundance. Higher levels of KK-LC-1 expression were seen in pan-wild type and KRAS mutated tumors and associated with high TMB. TCR-T therapy directed against KK-LC-1 should be considered in patients whose clinical features reflect these characteristics.

T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas.

Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor-targeting guide RNAs. We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas. The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.

Patterns of Hematologic Function and Cytokine Release Syndrome Severity in ALL Patients Receiving CAR-T and TCR-T Therapies Using Pooled Clinical Trial Data

Background:Despite the potential for durable disease remission seen with Chimeric Antigen Receptor (CAR-T) and T-Cell Receptor (TCR-T) therapies, their use is limited by the potential for acute toxicity from Cytokine-Release Syndrome (CRS).Across all grades, CRS has seen rates as high as 100% in some trials of patients receiving CAR-T, and up to 15% receiving TCR-T. Prior research indicates an association of hematologic abnormalities with CRS. Due to smaller average trial size and limited adoption, to date, of CAR and TCR therapies, there have been no large-scale studies to date exploring these associations with CRS severity in a wide range of patients across treatment types. This study sought to address these evidence gaps using retrospective analysis of pooled clinical trial data in Acute Lymphocytic Leukemia (ALL), using, to our knowledge, the single largest data repository of of CAR-T and TCR-T clinical patient data, with high resolution measurements across a spectrum of clinical domains.Methods:Eligible Phase I, II and III completed clinical trials in Acute Lymphocytic Leukemia (ALL), with patients receiving either CAR-T or TCR-T, were identified from the Medidata Enterprise Data Store, which comprises over 22,000 historical clinical trials, for de-identified retrospective aggregate analyses. Baseline characteristics, including demographics, medical history, prior treatment regimens were assessed and stratified by treatment type. Pre-trial history of hematologic conditions, such as neutropenia and anemia, were also assessed. Using Common Terminology Criteria for Adverse Events (CTCAE) 4.03, patients were assigned to categories of any CRS, mild CRS (grade 1) and moderate-to-severe CRS (2+). Hematologic function was assessed at baseline through first exposure to treatment, including counts of erythrocytes, neutrophils, eosinophils and basophils. Baseline marrow blast cell percentage was assessed as a marker of tumor burden. Univariate analyses of associations between pre-treatment baseline variables and CRS were conducted using Wilcoxon signed rank tests.Results:The pooled CT data contained 1,410 ALL patients, of whom over 60% were 18 year of age or greater. Baseline blood chemistries indicated 21% with anemia, 12% with thrombocytopenia, 6% neutropenic and 5.3% with elevated LDH. Although CAR-T patients accounted for 14.9% of the cohort, 47% of CRS events observed were associated with CAR-T treatment. In line with expectations from prior literature, factors associated with moderate-to-severe CRS included prior history of anemia, reduced platelet levels, low neutrophil counts, and delayed neutrophil recovery. Nearly all cases of moderate-to-severe CRS occurred in subjects exhibiting both low neutrophil and low platelet counts (Figure 1). Similar associations were seen in patients with pretreatment history of anemia (Figure 2). Consistent with literature on tumor burden and CRS, patients without CRS tended to have lower marrow blast percentages. Lymphocyte levels at baseline were far lower in patients receiving CAR-T therapy, with slower recovery than in patients receiving TCR-T. While consistent with CAR-T pre-treatment lymphodepletion, this finding was noteworthy given the association of neutropenia with CRS.Conclusions:Overall findings suggest patterns of routine hematologic function at baseline can potentially be used to assess risk of moderate-to-severe CRS in patients receiving CAR-T and TCR-T agents. The association with these markers could also suggest a mechanism of CRS as a function of tumor cell concentration, modified by the strength and presence of innate immunity mechanisms such as granulocytes, and potentially mediated by intermediates such as macrophages, in line with emerging literature., Further analysis may facilitate development of predictive algorithms to identify patients at greater risk for severe CRS prior to as well as shortly after treatment. This has implications for enhancing supportive care for patients receiving CAR- and TCR therapies. Additionally, a data-driven stratification of patients by risk of CRS will allow improved utilization and management of care resources. [Display omitted] DisclosuresAgarwal: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Socolov: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Buderi: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Rusli: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Bouzit: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Talwai: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Itzkovich: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment. Galaznik: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment, Current equity holder in publicly-traded company. Aptekar: Medidata Acorn AI, a Dassault Systèmes Company: Current Employment.

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.