Emerging Immunotherapeutic Approaches in Colorectal Cancer: From Checkpoint Inhibitors to CAR-T Cell and Viral Vector Vaccines

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Advanced CRC has a poor prognosis, with treatment primarily relying on chemotherapy combined with targeted therapies. Currently, immunotherapy based on immune checkpoint inhibitors is reserved exclusively for mismatch repair-deficient (dMMR) or microsatellite instability-high (MSI-H) tumors, which represent less than 10% of advanced CRC cases. Chimeric antigen receptor (CAR)-T cell therapy is a type of adoptive cell therapy involving modified T-lymphocytes engineered to express chimeric antigen receptors, enabling them to recognize surface antigens expressed by tumor cells. CAR-T cell therapy has demonstrated efficacy in treating hematological malignancies such as lymphoma, myeloma, and leukemia. However, its efficacy in solid tumors remains limited due to several limitations such as antigen heterogeneity, restricted CAR-T cell trafficking into the tumor area, and the presence of an immunosuppressive tumor microenvironment. Developing novel CAR-T cell therapies for solid tumors represents an unmet need, particularly for cases where immune checkpoint blockade is ineffective, such as CRC. Preclinical studies have shown the efficacy of various CAR-T cell models targeting a wide range of tumor-associated antigens in CRC, both in vitro and in vivo. Despite these promising results, the clinical efficacy of CAR-T cell therapy for CRC has been limited in early-phase clinical trials. Factors such as trial design or tumor characteristics, including antigen heterogeneity and the immunosuppressive microenvironment, should be considered. The development of innovative CAR-T cell models and the identification of novel antigens may improve the effectiveness of CAR-T cell therapy for CRC patients.

Immune checkpoint inhibitors, such as anti-programmed cell death-1, programmed cell death ligand-1, and cytotoxic T-lymphocyte antigen-4 monoclonal antibodies, have been notably effective in various types of cancers. Mismatch repair deficiency and microsatellite instability-high tumors have been established as striking biomarkers for response to immune checkpoint inhibitors. These biomarkers show a higher mutational burden, have cancer-associated neoantigens, and dense immune cell infiltration, which generates a robust immune response. For metastatic colorectal cancer, pembrolizumab and nivolumab, with or without ipilimumab, are recommended for chemotherapy-refractory patients, and pembrolizumab is recommended for chemotherapy-naive patients with mismatch repair deficiency and microsatellite instability-high tumors. Conversely, patients with mismatch repair-proficient and microsatellite-stable metastatic colorectal cancer showed no clinical benefit from immune checkpoint inhibitor monotherapy. Currently, combination therapy with anti-programmed cell death-1/programmed cell death ligand-1 and cytotoxic T-lymphocyte antigen-4 monoclonal antibodies or a combination of immune checkpoint inhibitors with molecular targeting agents or radiotherapy have been investigated to modulate immune cells and enhance therapeutic efficacy in mismatch repair-proficient and microsatellite-stable metastatic colorectal cancer. Furthermore, immune checkpoint inhibitors have been developed for neoadjuvant and adjuvant settings. In this review, we summarize the existing clinical data and discuss future perspectives with a focus on immune checkpoint inhibitor-based treatments for colorectal cancer.

Introduction: MSI-H and mismatch repair deficient (dMMR) tumors are associated with favorable immune checkpoint inhibitor (ICI) responses (PMID: 3326454). However, up to 95% of CRC tumors are MSS/proficient MMR (pMMR) leading to poorer prognosis and treatment outcomes than MSI-H/dMMR patients (PMID: 12867608, 26028255). In MSS/pMMR metastatic CRC, multiple combination therapies are being investigated despite a lack of biomarkers to guide therapy. Therefore, we developed an RNA-based MSS-PRS with a primary objective to identify MSS tumors that have MSI-H/dMMR molecular features indicative of ICI response. Experimental Procedures: The MSS-PRS was developed using the TCGA CRC cohort (COAD; n=268). Training labels were assigned using the MSI Mantis score and mutation status from 8 genes with reported MSI association. Samples called “activated” had both a gene mutation and an MSI Mantis score > 0.4 (i.e., MSI-H), and samples called “non-activated” were wild type for all genes and had MSI Mantis score < 0.4. All other samples were designated “ambiguous” and excluded from training. Two thirds of the non-ambiguous samples were assigned to the classifier training set and all remaining samples were assigned to the test set. Using ClaNC software (PMID: 16269418) and cross-validation in the training set, a nearest centroid classifier was developed from a set of high mean, high variance candidate genes to select an optimal gene set to separate activated and non-activated groups. The classifier performance was evaluated in the test set. Results: The MSS-PRS contained 112-genes enriched for mismatched DNA binding, DNA damage repair, PD-L1, innate and adaptive immune response, cellular immunity, and cytokines. In the test set, the classifier called 44 of 46 MSI-H samples activated; of the remaining 77 samples that were MSS tumors, 38 were called activated and 39 were called not activated. Visual inspection of heat maps of tumor by MSI/MSS-PRS status and their association with TMB and CRC-related mutations suggest greater differences between MSS tumors called activated and MSI-H tumors than between MSS tumors called activated and MSS tumors called not activated. In contrast, immune marker expression profiles in MSS tumors called activated were markedly more similar to MSI-H tumors compared to MSS tumors called non-activated. Summary and Conclusions: Herein we described the development of a novel MSS-PRS that captures an MSI-H/dMMR-like molecular phenotype in a subset of patients with MSS tumors despite their lack of high TMB or overt MSI defects. Based upon these initial findings, further development of the MSS-PRS and its clinical validation as a tool to select patients with MSS tumors who may benefit from ICI-containing treatment regimens is warranted. Citation Format: Kirk Pappan, Gregory Mayhew, Jonathan Shepherd, Yuelong Guo, Kirk Beebe, Joel Eisner, Michael Milburn. Development of a novel RNA-based microsatellite stable predictive response signature (MSS-PRS) to identify MSS colorectal cancer (CRC) patients with a microsatellite instability-high (MSI-H) molecular phenotype [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 1040.

PD-0005 - Prognostic Biomarkers in a Series of Stage II Colon Cancer

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells' glycocalyx is much thicker than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation, are observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Significant reduction in cytotoxicity is observed on going from early to longer-seeded CRC monolayers. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes. Similar enhanced interaction is observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. Enzymatic treatment significantly enhanced CAR T cell-mediated cytotoxicity and increased the percentage of TNF-α-secreting CAR T cells. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.

<h3>Background</h3> Solid tumors comprise &gt;90% of cancers. Non-small cell lung cancer (NSCLC), metastatic colorectal cancer (CRC), and pancreatic cancer are the leading causes of cancer-related mortality (5-year overall survival: 26%, 15%, and 11%, respectively).¹ Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies.^2,3 However, translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities.^4,5 To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust logic gate capable of killing tumor cells, while leaving healthy cells intact.^6,7 The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells (figure 1).^8,9 Human leukocyte antigen (HLA) loss of heterozygosity (LOH) offers a definitive tumor versus normal discriminator target for CAR T-cell therapy.¹⁰ The frequency of HLA LOH among advanced NSCLC, CRC, and pancreatic cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-23.1%.¹¹ LOH can be reliably detected using the Tempus xT-Onco next-generation sequencing (NGS) assay.^12,13 Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives: 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) Subsequent apheresis and manufacturing feasibility for the future EVEREST CEA or MSLN Tmod CAR T-cell studies. <h3>Methods</h3> BASECAMP-1 (NCT04981119) patient eligibility has 2 parts (figure 2): 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xT-Onco NGS testing; 2) If the tumor demonstrates HLA-A*02:01 LOH and the patient is eligible after screening, the patient will undergo apheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. <h3>Trial Registration</h3> ClinicalTrials. gov, NCT04981119 <h3>References</h3> American Cancer Society. <i>Cancer Facts &amp; Figures 2022</i>. Atlanta: American Cancer Society; 2022. Locke F, Miklos D, Jacobson C, <i>et al</i>. Axicabtagene ciloleucel as second-line therapy for large B-cell lymphoma. <i>N Engl J Med</i>. 2022;<b>386</b>(7):640-654. Maude S, Laetsch T, Buechner J, <i>et al</i>. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. <i>N Engl J Med</i>. 2018;<b>378</b>(5):439-448. Parkhurst M, Yang J, Langan R, <i>et al</i>. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther. 2011;<b>19</b>(3):620-626. Haas AR, Tanyi JL, O’Hara MH, <i>et al</i>. Phase I study of lentiviral-transduced chimeric antigen receptor-modified T cells recognizing mesothelin in advanced solid cancers.<i> Mol Ther</i>. 2019;<b>27</b>(11):1919-1929. Hamburger A, DiAndreth B, Cui J, <i>et al</i>. Engineered T cells directed at tumors with defined allelic loss. <i>Mol Immunol</i>. 2020;<b>128</b>:298-310. DiAndreth B, Hamburger AE, Xu H, Kamb A. The Tmod cellular logic gate as a solution for tumor-selective immunotherapy. <i>Clin Immunol</i>. 2022;<b>241</b>:109030. Sandberg ML, Wang X, Martin AD, <i>et al</i>. A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. <i>Sci Transl Med</i>. 2022;<b>14</b>(634):eabm0306. Tokatlian T, Asuelime GE, Mock JY, <i>et al</i>. Mesothelin-specific CAR-T cell therapy that incorporates an HLA-gated safety mechanism selectively kills tumor cells. <i>J Immunother Cancer</i>. 2022;<b>10</b>(1):e003826. Hwang MS, Mog BJ, Douglass J, <i>et al</i>. Targeting loss of heterozygosity for cancer-specific immunotherapy. <i>Proc Natl Acad Sci U S A</i>. 2021;<b>118</b>(12):e2022410118. Simeone DM, Hecht JR, Patel SP, <i>et al</i>. BASECAMP-1: Leveraging human leukocyte antigen (HLA) loss of heterozygosity (LOH) in solid tumors by next-generation sequencing (NGS) to identify patients with relapsed solid tumor for future logic-gated Tmod CAR T-cell therapy. Poster presented at: ASCO Annual Meeting; June 3-7, 2022; Chicago, IL. Abstract #TPS2676. Perera J, Mapes B, Lau D, <i>et al</i>. Detection of human leukocyte antigen class I loss of heterozygosity in solid tumor types by next-generation DNA sequencing. <i>J Immunother Cancer</i>. 2019, <b>7</b>(suppl 1):P103. Hecht JR, Kopetz S, Patel SP, <i>et al</i>. Next generation sequencing (NGS) to identify relapsed gastrointestinal (GI) solid tumor patients with human leukocyte antigen (HLA) loss of heterozygosity (LOH) for future logic-gated CAR T therapy to reduce on target off tumor toxicity. <i>J Clin Oncol</i>. 2022;<b>40</b>(4_suppl):190-190. <h3>Ethics Approval</h3> The study was approved by site IRBs

Chimeric antigen receptor (CAR) T-cell therapy is a method that extracts T cells from the patient's blood and virally introduces a genetically engineered T cell receptor targeting a specific cancer antigen and subsequently readministering these genetically engineered CAR T cells to the patient. These T cells are then better at identifying the tumors and attaching to these tumor cells resulting in a stronger cytotoxic immune response. The development of CAR T cells has been a huge success as an immunotherapy, especially for the targeting of non-solid tumors. Since their original inception in 1987, there are now six independent FDA approved CAR T cell therapies targeting a variety of blood cancers, with the first being approved in 2017. As a relatively new treatment, there is a continuous effort in improving the safety and efficacy of CAR T cell therapies. As mentioned previously, CAR T cells have undoubtedly been successful in the treatment of non-solid tumors, however their efficacy towards treatment of solid tumors has been limited. Additionally, the safety and long-term effects of CAR T cell treatments is still a concern. Combination therapy utilizing CAR-T cells and immune checkpoint inhibitors is being explored to potentially mitigate some of the limitations associated with CAR-T cells.

Exploring novel strategies of oncolytic viruses and gut microbiota to enhance CAR-T cell therapy for colorectal cancer.

TPS2676 Background: Solid tumors comprise &gt; 90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively) (ACS. 2021). Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies (Neelapu S. et al. N Engl J Med. 2017). Translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities (Parkhurst M, et al. Mol Ther. 2011; Tanyi J. Cellicon Valley '21). To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust NOT logic gate capable of killing tumor cells, while leaving healthy cells intact (Hamburger A, et al. Mol Immunol. 2020). The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells. The frequency of HLA LOH among advanced GI solid tumor cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-20.8% between colorectal, pancreatic, and gastroesophageal tumors (Hecht R. et al. ASCO-GI 2022. Abstract #190). As such, HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy. Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives of 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) To obtain leukapheresis and feasibility for the future EVEREST Tmod CAR T-cell trial. Methods: BASECAMP-1 (NCT04981119) patient eligibility has 2 parts: 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xT-Onco NGS testing. 2) If the tumor demonstrates HLA-A*02 LOH and the patient is eligible after screening, the patient will undergo leukapheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. Clinical trial information: NCT04981119.

ObjectiveColorectal cancer (CRC) presents significant treatment challenges, including immune evasion and tumor microenvironment (TME) suppression. Chimeric antigen receptor (CAR) T-cell therapy has shown promise in hematologic malignancies, but its effectiveness against solid tumors is hampered by the detrimental effects of the TME. This article aims to explore the potential of bispecific CAR T cells targeting programmed death-ligand 1 (PD-L1) and cancer-associated fibroblasts (CAFs) in CRC treatment.Material and MethodsDual-targeted CAR-T cells against PD-L1 and CAF were engineered using the GV400 lentiviral vector. Programmed death-1 (PD-1)/nanobody (Nb) and fibroblast activation protein (FAP)/Nb-encoding lentiviral vectors were generated, and CAR T cells were produced through a three-plasmid system in 293T cells. Human peripheral blood mononuclear cells (PBMCs) were separated, transduced with these vectors, and then expanded. Functional characterization of CAR-T cells was performed through enzyme-linked immunosorbent assay (ELISA), Western blot analysis, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and cell counting kit-8 (CCK-8) assay. Migration and invasion assays were conducted using Transwell chambers to assess the ability of FAP-PD-1/Nb CAR-T cells to migrate toward tumor cells and invade the extracellular matrix.ResultsWe developed dual-targeted CAR-T cells incorporating PD-L1 and CAF Nbs, which continuously secreted PD-1/Nb. Western blot confirmed PD-1/Nb expression in PD-1/Nb and FAP-PD-1/Nb CAR-T cells, with no expression in the untreated (UTD) group (P < 0.01). Flow cytometry showed a significantly higher cluster of differentiation (CD)25 and CD69 expression in FAP-PD-1/Nb CAR-T cells upon stimulation with FAP-positive target cells compared with the other groups (P < 0.01). TUNEL, flow cytometry, and CCK-8 assays revealed that FAP-PD-1/Nb CAR-T cells exhibited superior cytotoxicity and proliferation inhibition against FAP-positive HCT116 cells (P < 0.01). ELISA demonstrated increased interferon-gamma and tumor necrosis factor-alpha levels and reduced interleukin-10 (P < 0.01), suggesting enhanced cytokine modulation and antitumor immunity. Compared with single-target CAR-T cells and UTD, FAP-PD-1/Nb CAR-T cells showed notably enhanced Matrigel penetration and invasion (P < 0.01). Safety tests confirmed minimal cytotoxicity to normal PBMCs, indicating favorable safety.ConclusionThis study successfully developed dual-targeted CAR-T cells against PD-L1 and CAF and demonstrated their superior antitumor activity and immunomodulatory effects on CRC treatment. This novel therapeutic strategy was established using CAR T-cell technology for the treatment of CRC.

Background: The tremendous success of chimeric antigen receptor-T (CAR-T) cell therapy in haematological malignancies has not been recapitulated in solid tumours, owing to tumour-induced immunosuppression, tumour heterogeneity and inefficient tumour trafficking. One promising solution includes “armouring” CAR-T cells with therapeutic transgenes. Indeed, we demonstrated that CAR-T cells engineered to express dendritic cell growth factor Flt3L could effectively engage host anti-tumour immunity crucial for overcoming antigen-negative relapse1. However, synthetic promoters have demonstrated insufficiencies in driving tumour-restricted cytokine expression, which had caused systemic toxicities and trial termination2. The advent of CRISPR/Cas9 gene-editing tool has enabled the precise engineering of CAR-T cells for safety and efficacy enhancements. We previously showed that CRISPR/Cas9-mediated knock-out (KO) of immunosuppressive gene A2AR enhanced CAR-T cell function3. Now, we aim to exploit a CRISPR/Cas9-mediated knock-in (KI) strategy to leverage endogenous gene regulatory elements to restrict transgene expression to tumour for enhanced safety and efficacy. Methods: Genome-wide RNA sequencing was performed on CAR-T cells isolated from tumours and spleens of mice. 27 genes upregulated in intratumoural relative to splenic CAR-T cells were identified as potential KI sites. As KI disrupts target gene expression, the impact of each gene KO on CAR-T cell function/phenotype was first assessed. 7 genes without adverse impact following KO had GFP knocked in. Results: NR4A2 and RGS16 emerged as tumour-specific promoters upon KI. While NR4A2 was highly tumour-restricted and could deliver highly toxic cytokines (e.g., IL-12) without inducing toxicities in mice, RGS16 had high intratumoural expression and could mediate the efficacy of less potent cytokines (e.g., IL-2). Conclusions: Endogenous tumour-specific promoters enabled the generation of IL-12- and IL-2-expressing CAR-T cells with enhanced safety and efficacy in syngeneic and xenogeneic mouse models that was concomitant with improved CAR-T cell polyfunctionality and activation of host anti-tumour immunity. Notably, this CRISPR-KI strategy was applicable using patient-derived CAR-T cells, demonstrating its clinical translatability. Citation Format: Kah Min Yap, Amanda X. Y. Chen, Imran G. House, Phillip K. Darcy and Paul A. Beavis. Identifying Optimal Tumour-specific Promoters for CRISPR/Cas9 Engineering of Armoured CAR T Cells with Enhanced Safety and Efficacy [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr LT01.

Head and neck squamous cell carcinoma (HNSCC) remains among the most aggressive malignancies with limited treatment options, especially in recurrent and metastatic cases. Despite advances in surgery, radiotherapy, chemotherapy, and immune checkpoint inhibitors, survival rates remain suboptimal due to tumor heterogeneity, immune evasion, and treatment resistance. In recent years, Chimeric Antigen Receptor (CAR) T-cell therapy has revolutionized hematologic cancer treatment by genetically modifying T cells to target tumor-specific antigens like CD19, CD70, BCMA, EGFR, and HER2, leading to high remission rates. Its success is attributed to precise antigen recognition, sustained immune response, and long-term immunological memory, though challenges like cytokine release syndrome and antigen loss remain. Notably, its translation to solid tumors, including HNSCC, faces significant challenges, such as tumor microenvironment (TME)-induced immunosuppression, antigen heterogeneity, and limited CAR T-cell infiltration. To address these barriers, several tumor-associated antigens (TAAs), including EGFR, HER2 (ErbB2), B7-H3, CD44v6, CD70, CD98, and MUC1, have been identified as potential CAR T-cell targets in HNSCC. Moreover, innovative approaches, such as dual-targeted CAR T-cells, armored CARs, and CRISPR-engineered modifications, aim to enhance efficacy and overcome resistance. Notably, combination therapies integrating CAR T-cells with immune checkpoint inhibitors (e.g., PD-1/CTLA-4 blockade) and TGF-β-resistant CAR T designs are being explored to improve therapeutic outcomes. This review aimed to elucidate the current landscape of CAR T-cell therapy in HNSCC, by exploring its mechanisms, targeted antigens, challenges, emerging strategies, and future therapeutic potential.

e14069 Background: Immune checkpoint inhibitors and Chimeric Antigen Receptor (CAR) T-cell therapies have emerged as approaches to treat B-cell malignancies. Methods: PubMed/NCBI/MEDLINE databases were accessed with keywords "immune checkpoint inhibitors and B-cell malignancies" "CAR T-cell and B-cell malignancies", and various permutations including "clinical data" "toxicities", "reviews", "quality of life", and "adverse effects". Results: The first-in-class approved immune checkpoint inhibitor was ipilimumab, which is a fully humanized mAb that blocks the immunosuppressive signal by cytotoxic T-lymphocyte antigen. Thereafter, nivolumab was also approved for use in the treatment of Hodgkin's lymphoma in 2016. In phase I, open-label, dose-escalation, cohort-expansion study, patients with relapsed or refractory B-cell lymphoma received the anti-PD-1 monoclonal antibody nivolumab. Eighty-one patients were treated and drug-related adverse events occurred in 51 (63%) patients. Objective response rates were 40%, 36%, 15%, and 40% among patients with follicular lymphoma and other hematologic malignancies. Clinical trial results describing CD19-targeted CAR T-cell therapy of patients with relapsed B-ALL were published in 2015. In this study, all five patients treated with CAR T cells achieved minimal residual disease negative complete remission. Updated results describing the treatment of 16 patients with relapsed or refractory B-ALL treated with CAR T cells were published: the overall CR rate in this trial was 88% and 12 of 14 patients were classified as minimal residual disease negative. 44% of these patients went on to standard-of- care allogeneic hematopoietic stem cell transplant. Initial studies also reported potent anti-leukemic effects of CD19 CAR T cell therapy in three patients with refractory chronic lymphocytic leukemia where two of the three patients achieved MRD-CR. Infused CAR T cells proliferated up to 10,000-fold and persisted in recipients for at least 6 months and shown to retain antitumor activity after six months. Costs for CAR T-cell therapies remain exorbitant, reaching over $1M (USD) per patient. Conclusions: Clinical data reveal safety and efficacy, and also associated toxicities for both approaches.

&lt;div&gt;Abstract&lt;p&gt;Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment strategy for B-cell malignancies; however, its efficacy in solid tumors remains limited. VEGF-targeted drugs are used as antitumor agents to target abnormal tumor vasculature; however, toxicities associated with systemic VEGF blockade limit their maximal therapeutic benefit. Increasing evidence suggests a role for VEGF in the immunosuppressive tumor microenvironment, including through direct induction of T cell–effector dysfunction. In this study, we show that CAR T cells from patients treated with FDA-approved CAR T-cell products express members of the VEGF signaling pathway, and this expression is correlated with patient nonresponse. To overcome putative VEGF-induced CAR T-cell dysfunction and deliver local VEGF blockade, we generated CAR T cells that secrete a VEGF-targeting single-chain variable fragment to block T-cell and tumor-derived VEGF within the tumor microenvironment. These CAR T cells potently inhibited VEGF signaling and angiogenesis &lt;i&gt;in vitro&lt;/i&gt; and exhibited enhanced activation, cytotoxicity, proliferation, and effector function across different antigen and solid tumor contexts. VEGF single-chain variable fragment–secreting CAR T cells showed improved tumor control in immunocompromised murine metastatic and orthotopic models of ovarian and lung cancer. These findings suggest that CAR T cell–secreted VEGF blockade augments CAR T-cell performance, inhibits VEGF without systemic toxicity, and warrants further development.&lt;/p&gt;&lt;/div&gt;

Background: Success of Chimeric Antigen Receptor (CAR) T cells therapy for solid tumors are limited by the suppressive solid tumor microenvironment (TME), T cell exhaustion, lack of persistence, and poor trafficking to tumors. Strategies to improve this include therapeutic combinations, such as checkpoint inhibition (CPI) or cytokine support. However, CPI therapy has largely failed because of refractory and resistant tumors and cytokine administration can lead to lethal toxicities. To this end, we propose that enabling CAR T cells to secrete bi-functional fusion proteins consisting of cytokine modifiers (ie: IL12, IL15, or TGFβ-trap) combined with checkpoint inhibition (αPDL1 scFv) can provide tumor PDL1 sequestered cytokine activity and local tumor-immune modulation to boost solid tumor CAR T cell efficacy, enhance CPI impacts, and safely improve durable outcomes. Methods: Mouse T cells dual transduced to express surface CAR targeting prostate or ovarian solid tumors in addition to secretable cytokine fused to an αPDL1 scFv were generated. In vitro, the PDL1 blockade capacity and tumor-surface cytokine presentation of supernatants from dual transduced CAR T cells was assessed on PDL1 induced tumor cells. Further, secreted fusion protein transduced CAR T cells were assessed for function, phenotype, and cytokine release in a repetitive tumor rechallenge assay. In vivo, mouse CAR T cells dual transduced with fusion proteins and appropriate controls were assessed for anti-tumor efficacy, survival kinetics, and toxicity in immunocompetent solid tumor mouse models of prostate cancer and intraperitoneal disseminated ovarian cancer. Results: CAR T cells were shown to be successfully dual transduced, and secreted αPDL1-cytokine fusion proteins exhibiting functional PDL1 binding characteristics in vitro. CAR T cells engineered with αPDL1-IL12 fusion protein had greater anti-tumor activity and CAR specific expansion compared to other tested fusion proteins in in vitro co-culture assays. In a syngeneic prostate tumor model, mice receiving CAR T cells with αPDL1-IL12 fusion T cells out competed other cytokine fusion combinations. In a syngeneic ovarian tumor model we safely achieved 100% curative response rate with CAR secreting αPDL1-IL12 fusion in contrast to CAR with αPDL1mutIL12 control along with tumor regional PDL1 blockade on myeloid subsets within the TME. Conclusions: Our findings suggest that CAR T cells engineered to secrete αPD-L1-IL12 fusion protein show improved tumor control, less systemic toxicity, and enhanced expansion which promotes eradication of disease in two independent in vivo tumor models with two unique solid tumor CAR targets. We believe this strategy has the potential to improve solid tumor CAR T-cell efficacy and enhance durable innate immune responses. Citation Format: Lea Christian, John P. Murad, Lupita Lopez, Anthony Park, Jason Yang, Eric Lee, Candi Trac, Stephen Forman, Saul J. Priceman. Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3996.