Target In Solid Tumors Research Articles

Innovative pan-tumor target strategy for CAR-T therapy: cancer-specific exons as novel targets for pediatric solid and brain tumors.

Chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved remarkable success in treating chemotherapy-refractory hematological malignancies. However, its efficacy in solid and brain tumors remains limited due to challenges such as insufficient target antigens, poor T-cell adaptability, inefficient tumor site trafficking, and the immunosuppressive tumor microenvironment. To address these challenges, Shaw and colleagues proposed an innovative strategy targeting cancer-specific exons (CSEs) in pediatric solid and brain tumors. Using RNA sequencing data from 16 tumor types, the study identified 157 highly tumor-specific targets, including both known and novel proteins. The researchers validated several targets, including FN1 and COL11A1, demonstrating their therapeutic potential in in vitro and in vivo models. The study's approach of integrating exon-level analysis with a broad search for extracellular matrix proteins offers a new frontier for CAR-T therapy, providing valuable insights for improving immunotherapy in pediatric solid tumors. Although promising, the study also highlights the need for further evaluation of tumor recurrence and CAR-T cell exhaustion. The identification of novel pan-tumor targets may revolutionize CAR-T therapy design and expand its application in pediatric cancer treatment.

5-Fluorouracil/curcumin loaded silk fibroin hydrogel for the adjuvant therapy in colorectal cancer

This study employed silk fibroin (SF) as a carrier material to encapsulate curcumin (CUR) and 5-fluorouracil (5-FU), forming a highly effective drug-loaded hydrogel. The process involved mixing SF solution containing 5-FU with curcumin solution dissolved in acetone (AC), leading to the formation of composite drug-loaded nanospheres with particle sizes ranging from 77.87 nm to 299.22 nm, demonstrated enhanced permeability and retention (EPR) effects, enabling passive targeting of solid tumors. After the formation of the nanospheres, they were dispersed into a solution containing SF and polyethylene glycol (PEG). Following gelation and PEG removal, a SF hydrogel loaded with 5-FU and CUR (5-FU/CUR@SF hydrogel) was obtained. Results indicated that the 5-FU/CUR@SF hydrogel exhibited excellent drug release properties, with 5-FU and CUR achieving sustained release of 59.66 ± 3.76 % and 47.94 ± 5.03 %, respectively, over a 400-h of sustainable releasing period. Human colorectal cancer cell line (HT-29) and normal human colon epithelial cell line (NCM-460) were cultured with the 5-FU/CUR@SF hydrogel, resulting an apoptosis rate of only 17.38 ± 1.98 % for NCM-460 cells, whereas the apoptosis rate for HT-29 cells significantly increased to 72.31 ± 2.18 %, and its cell viability dropped to 59.77 ± 0.55 %. These findings suggest that the 5-FU/CUR@SF hydrogel exhibits low cytotoxicity toward normal NCM-460 cells, while exerting significant and sustained inhibitory effects on HT-29 cancer cells. In conclusion, the SF-based drug-loaded composite hydrogel holds great potential as a novel adjuvant therapeutic strategy for the treatment of CRC.

Solid cancer-directed CAR T cell therapy that attacks both tumor and immunosuppressive cells via targeting PD-L1

Chimeric antigen receptor (CAR) T-cell therapy has encountered limited success in solid tumors. The lack of dependable antigens and the immunosuppressive tumor microenvironment (TME) are major challenges. Within the TME, tumor cells along with immunosuppressive cells employ an immune-evasion mechanism that upregulates programmed death ligand 1 (PD-L1) to deactivate effector T cells; this makes PD-L1 a reliable, universal target for solid tumors. We developed a novel PD-L1 CAR (MC9999) using our humanized anti-PD-L1 monoclonal antibody, designed to simultaneously target tumor and immunosuppressive cells. The antigen-specific antitumor effects of MC9999 CAR T-cells were observed consistently across four solid tumor models: breast cancer, lung cancer, melanoma, and glioblastoma multiforme (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting penetration of the blood-brain barrier. The proof-of-concept data demonstrate the cytolytic effect of MC9999 CAR T-cells against immunosuppressive cells, including microglia HMC3 cells and M2 macrophages. Furthermore, MC9999 CAR T-cells elicited cytotoxicity against primary tumor-associated macrophages within GBM tumors. The concept of targeting both tumor and immunosuppressive cells with MC9999 was further validated using CAR T-cells derived from cancer patients. These findings establish MC9999 as a foundation for the development of effective CAR T-cell therapies against solid tumors.

Molecular imaging supports the development of multispecific cancer antibodies.

Multispecific antibodies are engineered antibody derivatives that can bind to two or more distinct epitopes or antigens. Unlike mixtures of monospecific antibodies, the binding properties of multispecific antibodies enable two specific molecules to be physically linked, a characteristic with important applications in cancer therapy. The field of multispecific antibodies is highly dynamic and expanding rapidly; to date, 15 multispecific antibodies have been approved for clinical use, of which 11 were approved for oncological indications, and more than 100 new antibodies are currently in clinical development. Nevertheless, substantial challenges limit the applications of multispecific antibodies in cancer therapy, particularly inefficient targeting of solid tumours and substantial adverse effects. Both PET and single photon emission CT imaging can reveal the biodistribution and complex pharmacology of radiolabelled multispecific antibodies. This Review summarizes the insights obtained from preclinical and clinical molecular imaging studies of multispecific antibodies, focusing on their structural properties, such as molecular weight, shape, target specificity, affinity and avidity. The opportunities associated with use of molecular imaging studies to support the clinical development of multispecific antibody therapies are also highlighted.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity

Adoptive cell therapy using chimeric antigen receptor (CAR) T-cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this are cancer-associated fibroblasts (CAFs), that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce “T cell dysfunction”. Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in “on-target off-tumor” cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR-T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T-cells” are designed to express a constitutive CAR, targeting FAP+ CAFs in solid tumors. Additionally, a second CAR targeting a Tumor Associated Antigen (TAA) such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin-CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated Dual CAR T-cells which efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Characterizing clinical trials for CAR T targeting solid tumors from 2018 to 2023: A systematic review.

e15080 Background: In the last decade, there have been many advances in the treatment of solid tumors, including new chemotherapy agents, immunotherapy, and most recently, cellular therapies. Chimeric antigen receptor T-cell (CAR T) therapy, first approved for acute lymphoblastic leukemia in 2017, represented a paradigm shift in hematologic malignancies, producing response rates that dramatically outpaced alternative regimens in a relapsed/refractory patient setting. Approvals in large B-cell lymphomas and multiple myeloma followed. Excited by the potential of CAR T, investigators worldwide have initiated trials to investigate the use of CAR T in solid tumors. This review explores the trials conducted globally, evaluating the potential molecular targets and the status of current clinical trials. Methods: This systematic review identified CAR T clinical trials specifically targeting solid tumors starting in 2018 to 2023 registered at ClinicalTrials.gov as of January 8, 2024. Trial-related variables including trial phase, primary trial site location, primary endpoints, start and end dates, and molecular target were captured. Results: We screened 110 clinical trials, ultimately including 103 studies for analysis after excluding trials without a CAR T construct in the methods. The number of new clinical trials increased over time, with a significantly larger increase (440%) seen with non-US sponsors compared to US sponsors (100%), p< 0.0001. Only 19 trials (18%) are/were being conducted in the United States in the studied time period. There were 33 unique molecular targets used, with mesothelin (17%), and the claudin protein family (13%) as the most frequented targets. Conclusions: The success that CAR T has demonstrated in hematologic malignancies has created great hope in solid tumor malignancies, although tempered by differences between the two categories. Unlike hematologic cancers, solid tumors exhibit significant biomarker variation, with interpatient biomarker expression varying greatly, even within the same tissue diagnosis. To deal with this issue, the majority of clinical trials that were reviewed here were not restricted to a single disease location such as breast cancer or esophageal cancer but were rather restricted by the expression of the targeted epitope, to allow for the heterogeneity inherent in these tumors. As presented here, substantial efforts are being made worldwide to improve these technologies, but the United States appears to be lagging in clinical trial efforts, compared to other nations. Further investigation is warranted into this topic, as the usage of CAR T quickly spreads from a therapy only used in hematology to one with promise in solid tumors, infectious diseases, and beyond.

Outstanding safety and efficacy data of IMC002, an autologous CLDN18.2-targeting CAR-T, in CLDN18.2+ advanced solid tumors.

e16012 Background: Claudin 18.2 (CLDN18.2) is a promising therapeutic target for advanced solid tumors with CLDN18.2 expression. IMC002 is an autologous CLDN18.2-targeting CAR-T cell directed by a CLDN18.2-specific VHH, the specificity of which was confirmed by a membrane proteome array assay. Preclinical studies showed that IMC002 exhibited high tumor specificity, anti-tumor efficacy, and excellent safety profile in both cell and animal models. Methods: This is an open label first in human Phase 1 dose escalation study to evaluate the safety and efficacy of IMC002 in advanced solid tumors with CLDN18.2 expression which is defined as CLDN18.2 expression of ≥1+ membrane staining intensity in ≥10% tumor cells. Accelerated titration followed by Bayesian optimal interval design was adopted with a total of 4 dose levels planned at 1, 3, 5 and 7 million (mil) CAR-T cells/kg respectively. All patients (pts) were given a single dose of IMC002 infusion after lymphodepletion chemotherapy. Dose limiting toxicity (DLT) was evaluated during the 28-day period after IMC002 infusion. Safety, anti-tumor activity, pharmacokinetics and pharmacodynamics were explored in this study. Here, we present preliminary safety and efficacy data for the pts dosed with up to 3 mil CAR-T cells/kg. Results: As of January 17, 2024, 3 advanced unresectable gastric cancer pts received IMC002 infusion, 1 patient at 1 mil CAR-T cells/kg and 2 pts at 3 mil CAR-T cells/kg. No DLT or SAE was reported. Manageable CRS (grade 1) was reported in all 3 pts. Other adverse events related to cell therapy included grade 1-2 lipase increase (3/3), diarrhea (1/3), pruritis (1/3) and γ-GGT increase (1/3). All adverse events recovered quickly. Preliminary efficacy data showed that best overall response of the 3 pts was all stable disease as evaluated by RECIST 1.1. The two pts at the dose level of 3 mil CAR-T cells/kg underwent successful radical surgery at week (W) 11 and 44 after IMC002 infusion respectively. Pathological complete response (pCR) was achieved in the patient with surgery at W44. Distinct CAR-T cell expansion in peripheral blood was observed in all 3 pts, with the peak number of CAR+ cells reaching on day 7-11 in the peripheral blood after the infusion. Moreover, CAR-T cell infiltration was detected in the W11 and W44 surgical samples. Significant elevations of serum levels of IFN-γ, IL-10, IP-10, IL-2 and IL-6 were observed in the 3 pts. The trial is ongoing. Conclusions: IMC002 showed a favorable safety profile, encouraging anti-tumor activities with observed pCR and persistent expansion in the tumor tissue at the low dosage levels in pts with advanced CLDN18.2+ gastric cancer. In addition, our trial has successfully provided a surgical treatment opportunity after CAR-T therapy downstaging of unresectable gastric cancers. Clinical trial information: NCT05472857 .

Phase 1 study of ASP1002, a bispecific antibody targeting claudin 4 (CLDN4) and CD137, in patients with locally advanced (LA) or metastatic solid tumors that express CLDN4.

TPS2670 Background: The lack of benefit for many cancers from newer-generation combination chemotherapies has stimulated research into targeted agents. CLDN4, a claudin protein family member that regulates tight junction formation, is highly expressed in multiple tumor types including non-small cell lung cancer (NSCLC), urothelial carcinoma (UC), colorectal cancer (CRC), prostate adenocarcinoma (PA), ovarian cancer (OC), and triple-negative breast cancer (TNBC). Following primary T cell activation, CD137 provides a costimulatory signal that enhances T cell proliferation and cytokine production. Together CLDN4 and CD137 represent promising therapeutic targets for solid tumors. ASP1002 is a bispecific antibody designed to target CLDN4 and CD137, thereby enhancing the antitumor response of T cells against CLDN4-expressing tumor cells. Methods: This phase 1 first-in-human, open-label, multicenter study (NCT05719558) is evaluating the safety and tolerability, maximum tolerated dose (MTD), and/or recommended phase 2 dose (RP2D) of ASP1002 in adult patients with LA or metastatic solid tumors that express CLDN4. The primary endpoint is safety and tolerability, as assessed by dose-limiting toxicities (DLTs), adverse events (AEs), serious AEs, laboratory tests, vital signs, ECGs, PEs, and ECOG performance status. Secondary endpoints are pharmacokinetic characteristics; confirmed objective response rate (ORR), duration of response, and disease control rate per iRECIST and RECIST 1.1; and incidence rate and expression levels of CLDN4 by immunohistochemistry. Dose escalation will include patients with LA or metastatic NSCLC, UC, CRC, PA, OC, or TNBC. ASP1002 will be administered at escalating doses Q1W IV on days 1, 8, 15 in a 21-day cycle until discontinuation to determine MTD and/or candidate RP2D regimens. At doses ≥10 mg, multi-participant cohorts will be included. Safety follow-up visits will occur within 7 days posttreatment, and on 30 and 90 days posttreatment. Next, dose expansion will enroll patients with LA or metastatic NSCLC, UC, CRC, and other tumor types with confirmed complete response (CR) or partial response (PR) in dose escalation. Candidate RP2D regimens based on dose escalation data will be evaluated in ≤40 patients per dose level, per tumor type in dose expansion. ORR (confirmed CR or PR) per iRECIST will be evaluated in tumor-specific expansion cohorts. The minimum number of responders needed to establish activity is 4 (NSCLC), 3 (CRC), and 7 (UC). Study enrollment is currently ongoing in the United States. Clinical trial information: NCT05719558 .

A phase 1 dose escalation study of a novel coupled CAR T cell therapy, GCC19CART, for patients with metastatic colorectal cancer.

e15572 Background: GCC19CART is a multitargeted CAR-T cell therapy designed to overcome the limitations of conventional CAR-T cells in solid malignancies by pairing a solid tumor targeting CAR with additional CD19-targeting CARs. This addition is meant to amplify proliferation, activation, and persistence of the CAR-T cells with a goal to overcome the inherent resistance of solid tumors to CAR-T cells. Guanylate cyclase-C (GCC) has been reported to be expressed in over 80% of colorectal cancers with normal expression largely restricted to the luminal side of the GI tract, making it an appealing target for CAR-T cell therapy. A Phase 1 investigator-initiated clinical trial in China reported preliminary results with an ORR of 15.4% (2/13), mOS of 13.3 months in dose level 1 (1x106 CAR-T cells/kg) and 50% (4/8), mOS of 26.1 months in dose level 2 (2x106 CAR-T cells/kg). Common adverse events included cytokine release syndrome (CRS), diarrhea/colitis, and immune effector cell-associated neurotoxicity syndrome (ICANS). Methods: A phase 1 study in the United States was initiated in August, 2022, for patients with refractory metastatic colorectal cancer. As of a data cutoff of December 31, 2023, with a prespecified analysis after the DLT window for the first patient treated at dose level 2, 5 subjects have been reinfused: 4 in dose level 1 and 1 in dose level 2. Subjects were screened for GCC expression. Over 95% of participants screened positive, so this requirement was removed. Eligible subjects underwent leukapheresis, lymphodepleting chemotherapy with a single dose of fludarabine 30mg/m2 and cyclophosphamide 300mg/m2, and infusion of GCC19CART. Responses were assessed by RECIST v1.1 by site investigator and an independent third-party imaging review. Results: Five subjects have been treated, 4 in dose level 1 (1x106 cells/kg) and 1 in dose level 2 (2x106 cells/kg), and have completed the 30 day DLT period. The most common CAR-T related adverse events were CRS in 5/5 subjects (grade 1: 2/5 (40%) and grade 2: 3/5 (60%)), diarrhea in 4/5 subjects (grade 1: 1/5 (20%), grade 2: 2/5 (40%), and grade 3: 1/5 (20%)), and ICANS (grade 2: 1/5 (20%)). All adverse events resolved with therapy. The ORR was 40% (2/5) by independent review: 2 subjects demonstrated a partial response however 1 additional subject had partial metabolic response on PET/CT with stable disease. Progressive disease was seen in 2 participants by RECIST 1.1, but 1 has a falling tumor marker suggesting tumor flare. Conclusions: Preliminary results demonstrate that GCC19CART has an acceptable safety profile and meaningful clinical activity in refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented, including from patients treated at dose level 2 (2x106 CAR-T cells/kg). Clinical trial information: NCT05319314 .

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Establishing an immunocompetent mouse model of CAR T anti-tumor efficacy for studies of therapeutic resistance

Abstract Chimeric antigen receptor (CAR) T cell therapies redirect a patient's T-cells to recognize tumor antigens, and are an effective clinical strategy in the treatment of several hematological malignancies. However, targeting of solid tumors remains challenging, in part due to features of the solid tumor microenvironment that impair the expansion, persistence, and function of CAR T cells. To date, most studies employ xenograft models, thus limiting a mechanistic understanding of CAR T biology in the context of an intact immune system. To model the resistance of solid tumors to CAR T therapy and dissect how endogenous immune populations impact this process, we have generated an immunocompetent mouse model of CAR T therapy. Murine CARs recognizing human CD20 have been engineered with an extracellular scFv domain and intracellular CD3z signaling and 4-1BB costimulatory domains. Using flow cytometry and live-cell imaging assays, we demonstrate potent, antigen-dependent in vitro cytotoxicity and cytokine release by huCD20-targeting CAR T cells against solid (MC38) or heme (EL4) tumor cells engineered to express cell-surface huCD20. Moreover, huCD20 CAR T cells exhibit in vivo efficacy against both EL4-huCD20 and MC38-huCD20 tumor models, while CAR T that recognize an irrelevant antigen do not affect tumor growth. These results highlight a platform to explore resistance mechanisms and potential immune cell interactions that may impair CAR T therapy directed against solid tumors.

Theranostic Bottle-Brush Polymers Tailored for Universal Solid-Tumor Targeting.

Designing an efficient nanocarrier to target multiple types of cancer remains a major challenge in the development of cancer nanomedicines. The majority of systemically administered nanoparticles (NPs) are rapidly cleared by the liver, resulting in poor tumor-targeting efficiency and severe side effects. Here, we present a delicately tailored design and synthesis of fluorescent bottle-brush polymers and screen nine derived NPs, each varying in size and surface coatings, for tumor imaging and targeted delivery. Our optimized polymer bearing (oligo(ethylene glycol) methyl ether methacrylate) in the side chains shows reduced macrophage uptake, prolonged blood-circulation time (up to 27 h), and exceptionally high accumulation in the tumor compared to the liver, elucidating an immune-evasion-induced tumor-targeting mechanism. High tumor accumulation significantly improved the antitumor efficacy. The outstanding tumor-targeting ability has been further validated across five distinct tumor models, including orthotopic glioblastoma and pancreatic cancer, which demonstrate the universality of our polymeric nanocarrier for tumor-targeting delivery.

Abstract LB340: Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice

Abstract FDA approval for chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers has unleashed the potential of utilizing live T cells as therapeutics. However, CAR-mediated targeting of solid tumors is currently limited by a paucity of suitable tumor antigens that are both specific and highly expressed on the tumor cell surface. In this report we explore the possibility of harnessing tumor-specific expression of endogenous retroviruses (ERVs) as a universal target for CAR T cell therapy. We found that gp70, the envelope protein from a murine ERV, is highly expressed on the surface of tumor cell lines derived from a diverse array of murine tumor types. We utilized the B16 melanoma model as a representative poorly inflamed tumor that does not respond to existing immunotherapies to examine efficacy of gp70 CAR T cells. T cells engineered to express a gp70 CAR suppress tumor growth in vivo and recognize B16 tumor cells with improved sensitivity compared to T cells expressing two previously described dominant tumor antigens (gp100 or P15e) due to low expression of MHC-I on B16 tumors. Co-incubation of B16 tumor cells with gp70 CAR T cells results in upregulation of MHC-I on the tumor cell surface suggesting that in addition to killing tumor cells directly, gp70 CAR T cells could facilitate recognition of the tumor by endogenous T cells through local release T cell derived IFNγ. We postulate that utilization of CAR T cells specific for cell surface ERV envelope proteins offers a promising strategy that could be harnessed to achieve local release of T cell derived payloads specifically within the tumor microenvironment. Citation Format: Sophie M. Lehar, Srirupa Roy, Lisa Liao, Haiyin Chen, Yongmei Chen, Mayra Cruz Tleugabulova, Eric Janezic, Azadeh Madjidi, Tamaki Jones, Vincent Javinal, James Ziai, Ira Mellman, Jill Schartner, Andrey Shaw. Harnessing the gp70 endogenous retrovirus envelope protein as a universal tumor antigen for T cell therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB340.

Abstract 1329: Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors

Abstract T cell receptor (TCR)-T based therapies, such as MAGE-A4 TCR-T cells, have shown compelling data demonstrating effective infiltration into and targeting of solid tumors with clinical responses across various solid cancers. However, heterogeneity and down-regulation of target antigen and HLA expression limit the durability and curative potential of these types of treatments. On the other hand, owing to their clinical potency, favorable safety profile and applicability as “off the shelf” therapy, natural killer (NK) cells have emerged as promising modalities in recent years. Unguided NK cells have shown limited potential against solid cancers, due to lacking infiltration into tumors. With our proprietary TCR-NK platform, we combine the solid tumor targeting capabilities of TCRs with the pan-cancer sensing and cytotoxic potential of highly potent killer cells, redirecting and arming NK cells with a fully functional TCR-CD3 complex. Here we present our lead product, an optimized TCR-NK cell product, ZI-MA4-1a, expressing an affinity enhanced TCR (KVL-a) directed to the HLA class I-restricted clinically validated cancer-testis antigen MAGE-A4, that is broadly expressed across solid tumors. The KVL-a TCR was successfully engineered from a naturally occurring wild-type TCR, increasing the affinity to MAGE-A4-peptide-HLA complex and resulting in enhanced recognition of tumor cells expressing the target antigen. ZI-MA4-1a is produced by transducing healthy peripheral blood derived NK cells with the full CD3 complex, the KVL-a TCR and a CD8 coreceptor. The process has been optimized to enrich TCR-NK cells, increasing process robustness, diminishing the impact of donor variability, and producing a higher performing TCR-NK cell product. We show the dual functionality of ZI-MA4-1a: TCR driven cytotoxicity as well as innate NK sensing of antigen knockout or HLA negative cancers. We demonstrate killing of heterogenous tumor cell models in vitro, as well as the multifunctionality of ZI-MA4-1a with killing, degranulation and multicytokine secretion. Interestingly, we observe enhanced proliferation of ZI-MA4-1a cells after activation by MAGE-A4 expressing tumor cells, indicating that TCR-NK cells acquire further proliferative capacity when the TCR is triggered. Finally, we demonstrate higher activity of ZI-MA4-1a compared to KVL-a and benchmark TCR transduced T cells against antigen positive targets, and activity against antigen negative tumor cells escaping TCR-T cell recognition. Citation Format: Sarah Vollmers, Artur Cieslar-Pobuda, Justyna Kmiecik, Giulia Malachin, Michelle L. Sætersmoen, Frida Høsøien Haugen, Margherita Boieri, Maja Sandve, Dennis Clement, Ines Cardoso, Pimthanya Wanichawan, Martha E. Haugstøyl, Anja Oldenburg, Liliane Christ, Pranav Oberoi, Luz Mora-Velandia, Anders Holm, Namir J. Hassan, Emilie Gauthy, Julia Ino, Sylvie Pollmann, Luise U. Weigand. Development of the first optimised “off the shelf” MAGE-A4 targeting TCR-NK cells for advancement into the clinic for the treatment of solid tumors [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 1329.

Abstract 1868: Target-dependent considerations for the design of bispecific T-cell engagers

Abstract In this study, we present data from four T-cell engager (TCE) programs in which we leveraged our highly diverse CD3-binding antibodies to generate functional bispecifics for each tumor target. By aggregating tumor-cell killing and cytokine data across four tumor targets with different properties and target expression levels, we have gained novel insights into target-specific considerations for the development of CD3 TCEs. TCEs recapitulate a synapse between T cells and target cells. T cell function downstream of this synapse is determined by the complex interplay between multiple independent factors. These include properties of the CD3- and tumor-binding arms, such as binding kinetics and epitope, as well as target-dependent factors such as cell type and target density. We used our diverse CD3 antibodies to engineer bispecific TCEs against four solid tumor targets: PSMA, B7-H4, 5T4, and the peptide-MHC target MAGE-A4. For each target, we engineered and characterized hundreds of bispecific molecules using high-throughput assays, including T-cell dependent tumor-cell killing, cytokine release, developability assessments, and structural studies. Here, we compare data from across these programs to elucidate the impact of target-dependent parameters on TCE function. These data provide important insights for the selection of appropriate TCE targets and efficient design of bispecific antibodies with high therapeutic potential. Citation Format: Matt Mai, Raffi Tonikian, Peter Bergqvist, Alaa Amash, Nathalie Blamey, Gabrielle Conaghan, Valentine de Puyraimond, Patrick Farber, Allison Goodman, Ahn Lee, Jessica Fernandes Scortecci, Cindy-Lee Crichlow, Akram Khodabandehloo, Tova Pinsky, Kate Caldwell, Jessica Patterson, Philippe Pouliot, Davide Tortora, Oscar Urtatiz, Ping Xiang, Irene Yu, Kirstin Brown, Kelly Bullock, Andrea Chee, Stephanie K. Masterman, Neil Aubuchon, Lindsay Devorkin, Bryan C. Barnhart, Tim Jacobs. Target-dependent considerations for the design of bispecific T-cell engagers [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 1868.

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [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 5006.

Abstract 3125: TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors

Abstract Tumor-associated carbohydrate antigens (TACAs) are short oligosaccharides expressed on the surface of cancer cells and play key roles in cellular communication, immune evasion, apoptosis and metastasis. TACAs are therefore interesting targets for antibody-based therapies in cancer. We have generated an antibody (TCX-101) against a TACA structure with improved binding affinity compared to other monoclonal antibodies (mAbs) against the same target as shown by surface plasmon resonance (~ 10x superior KD), as well as high level of specificity against glycan species related to the target, as accessed by glycan array, flow cytometry, and immunocytochemistry or immunofluorescence. Immunohistochemistry studies on fresh-frozen tissue microarrays showed high expression of the target in various solid tumors (e.g. gastrointestinal, lung, and breast) and only low expression in normal tissues. In vitro, TCX-101 has ~ 4x higher maximum binding, ~ 2x faster binding kinetics, ~ 4x faster cellular internalization and induces ~ 2x higher antibody-dependent cellular cytotoxicity than a reference antibody. Monomethyl auristatin E (MMAE) was used in a drug-antibody ratio (DAR) of 4 to generate an antibody-drug conjugate (ADC). This ADC was effective in in vitro toxicity assays against murine B16 melanoma cells induced to express the target (measurable effect on WT cells), as well as human breast cancer MCF7 WT cells (~ 25x less activity on target-knockout MCF7 cells, Table 1). When tested in vivo, TCX-101 was highly effective in two different xenograft mouse models (B16, MCF7) with more than 95% tumor growth inhibition compared to vehicle, and no signs of toxicity (no weight loss, no histophatological findings). A fully humanized version of TCX-101 with improved binding properties (EC50 6.27 nM vs. 8.13 nM) was generated and will be evaluated in further in vivo experiments. In conclusion, TCX-101 is a promising ADC for the treatment of various solid tumors. IC50 [nM] TCX-101-MMAE 4 DAR B16 KI 7.64 B16 WT no killing observed MCF7 WT 1.97 MCF7 KO 45.97 Citation Format: Fausto G. Gomes, Francesco Muraca, Gustavo M. Schmidt Garcia Moreira, Kathrin Rösch, Maria Bräutigam, Peter Sondermann, Matthias Ocker. TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors [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 3125.

Abstract 6312: Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against blood malignancies. However, prominent barriers, including limited proliferation, CAR T cell persistence, and poor T cell effector function, hinder its potential, especially in solid tumors. Various engineering approaches attempt to address these issues by armoring CAR T cells with factors that enhance the function and maintenance of T cells in the anti-tumor response. We previously described a human orthogonal IL-2/IL-2R system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (oRβ) that does not significantly respond to its native ligand, wild type IL-2. This system provides a “private IL-2 signal” in engineered oRβ-expressing cells while avoiding signaling in bystander immune cells, reducing toxicities. We have demonstrated STK-009/oRβ’s ability to selectively expand oRβ-expressing CAR T cells (SYNCAR T cells) targeting heme (SYNCAR-001, CD19) and solid tumor (SYNCAR-002, GPC3) targets at will. Importantly, the system enhanced anti-tumor control driving deep and durable responses in both bulky lymphoma and hepatocellular carcinoma (HCC) tumor models. Additionally, in a non-human primate model, STK-009 did not potentiate wild type T or NK cells and exhibited no evidence of toxicity. Currently, the combination of STK-009 + SYNCAR-001 is undergoing Phase I clinical trials (NCT05665062). Here, we conducted a direct comparison of several armoring approaches including the SYNCAR platform, the overexpression of c-Jun, an IL-15/IL-15Rα fusion, and IL-18 in human CAR T cells targeting GPC3. To assess their effects in overcoming CAR T cell dysfunction, cells were subjected to continuous antigen exposure in vitro. STK-009 treated SYNCAR-002 T cells displayed an enhanced proliferative capacity, sustained effector cytokine secretion, and an increased activation state when compared to other armoring technologies and PBS treatment. In a HCC tumor model, treatment with STK-009/SYNCAR-002 demonstrated superior tumor control with minimal toxicities. Interestingly, IL-18 overexpression also controlled tumors, but was accompanied by uncontrolled CAR T cell expansion and toxicity resembling severe GVHD. scRNAseq analyses revealed STK-009-treated SYNCAR-002 T cells simultaneously activated effector genes and repressed an exhaustion gene signature. This analysis also identified a novel mechanism by which STK-009/oRβ signaling induces a superior synthetic effector state via transcriptional repression of the stress response/proteosome. These findings support the advantages of an orthogonal platform that selectively drives and sustains potent T cell effector function without safety concerns associated with constitutively expressed armored approaches. Citation Format: Ethan Jung, Helena Silva, Marie Semana, Ivan Cheng, Somya Singh, Michele Bauer, Mohammed Ali, Henry Rosas, Ryan Burgess, George Zeng, Woei Chang, Navneet Ratti, Deepti Rokkam, Patrick J. Lupardus, Martin Oft, Paul-Joseph Aspuria. Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches [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 6312.