Costimulatory Domain Research Articles

IMMU-08. PRECLINICAL EVALUATION OF GAL-1-SPECIFIC CAR-T CELL FUNCTION IN AN IN VITRO AND EX VIVO MODEL OF GLIOBLASTOMA

Abstract BACKGROUND Human glioblastoma (GBM), especially those exhibiting anti-VEGF resistance, overexpress the carbohydrate-binding protein galectin-1 (Gal-1). To develop novel anti-GBM approaches, we developed Gal-1 targeted CAR-T cells to investigate potential antitumor effect with in vitro and ex vivo slice culture models. METHODS Uniform GBM brain slice cultures were collected using NICO’s Myriad resection tool and their Automated Preservation System (APS), designed to obtain fresh brain tumor samples maintaining tumor cytoarchitecture and microenvironment. To prepare Gal-1 CAR-T cells, we used creative laboratory to utilize a third-generation Gal-1-CAR construct with high binding and codon optimized scFv-Gal-1 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB (Gal-1 scFv-CD28-4-1BB-CD3ζ). We then transfected into the T cells, from peripheral blood, mononuclear cells using a lentiviral system. The anti-tumor capabilities of CAR-T cells were evaluated by co-culturing with GBM cells and ex vivo APS-GBM slices. RESULTS The collected APS- GBM slices samples were clear of debris and blood product, not requiring post-surgical mechanical slices, and exhibited viability up to 12 days over traditionally prepared ex vivo cultures. With this, we found Gal-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotypic and biological attributes such as cell viability, potency, migration, and T cell activation. Gal-1 scFv-CD28-4-1BB-CD3ζ -CAR-T cells killed more than 80% of GBM tumor cells within 24 hours in Gal-1+ve, but not Gal-1-ve GBM cells in vitro. Additionally, these CAR-T cells selectively caused significant release of IL-2, IFN-γ and TNF-α only from Gal-1+ve co-cultures and induced significant Gal-1+ve GBM cell death in an ex vivo model without any general toxicity. CONCLUSION Based on in vitro cell culture and ex vivo APS-brain slice culture results, we conclude that the novel Gal-1 scFv-CD28-4-1BB-CD3ζ CAR-T cell therapy may offer an effective therapeutic option. Further pre-clinical and clinical studies need to be performed to validate this novel approach

Application of CAR-T cell therapy in B-cell lymphoma: a meta-analysis of randomized controlled trials.

This study aims to compare the efficacy and safety of chimeric antigen receptor T-cell (CAR-T) immunotherapy with standard treatment for B-cell lymphoma, providing evidence-based support for the more efficient use of CAR-T cell immunotherapy. We conducted a comprehensive literature search of high-quality randomized controlled trials (RCTs) on CAR-T therapy for B-cell lymphoma in the following databases: Wanfang, Web of Science, CNKI, VIP database, and PubMed, up to February 2024. The outcome measures included objective remission rate (ORR), complete remission rate (CRR), and incidence of adverse reactions. Subgroup analysis was performed based on the differences in co-stimulatory domains. Meta-analysis was conducted using Review Manager 5.4 and Stata software. A total of five RCTs involving 1670 patients were included in this meta-analysis. The results showed that the CAR-T treatment group had significantly higher ORR (RR: 1.47, 95% CI 1.23-1.76, I2 = 80%, p < 0.0001), CRR (RR: 2.19, 95% CI 2.16-3.79, I2 = 93%, p = 0.005), cytokine release syndrome (CRS) incidence (RR: 34.51, 95% CI 2.27-523.78, I2 = 98%, p = 0.01), neurotoxicity (NT) incidence (RR: 6.00, 95% CI 1.82-19.75, I2 = 80%, p = 0.003), neutropenia incidence (RR: 1.39, 95% CI 1.02-1.88, I2 = 93%, p = 0.03), leukopenia incidence (RR: 1.39, 95% CI 1.04-1.87, I2 = 61%, p = 0.03), and headache incidence (RR: 1.56, 95% CI 1.25-1.95, I2 = 34%, p < 0.0001) compared to the standard treatment group. Subgroup analysis based on co-stimulatory domains revealed that the 4-1BB subgroup had higher incidences of CRR, CRS, NT and leukopenia than the CD28 subgroup; however, the CD28 subgroup exhibited higher ORR and neutropenia than the 4-1BB subgroup. CAR-T cell immunotherapy demonstrates superior efficacy compared to standard therapy in treating B-cell lymphoma. However, CAR-T treatment can lead to adverse reactions such as CRS and NT. Infusion of an appropriate dose of CAR-T cells (e.g., 100 × 106) may be a strategy to mitigate the risk of CRS and NT.

Preclinical assessment of the efficacy of B7-H3 CAR-T in renal cell carcinoma

B7-H3 is a type I transmembrane protein that belongs to the B7 immune checkpoint protein family, is aberrantly expressed in cancer cells, but rarely expressed in normal tissues, making it an attractive target for cancer therapy. Here, we found B7-H3 is highly expressed in the renal cell carcinoma (RCC) tumor tissues and RCC cell lines, but is undetectable in normal renal tissues. Therefore, we engineered second-generation CAR-T cells targeting B7-H3, incorporating either CD28 or 4–1BB co-stimulatory domains. Both CAR-T cell variants demonstrated potent antitumor activity against RCC tumors in vitro and in metastatic and orthotopic RCC mouse models. Furthermore, the B7-H3 CAR-T cells exhibited remarkable proliferation and robust cytokine release when co-cultured with RCC cancer cells. These findings demonstrated that targeting B7-H3 by CAR-T cells potentially offering a new treatment option for RCC patients.

MEK inhibition prevents CAR-T cell exhaustion and differentiation via downregulation of c-Fos and JunB

Clinical evidence supports the notion that T cell exhaustion and terminal differentiation pose challenges to the persistence and effectiveness of chimeric antigen receptor-T (CAR-T) cells. MEK1/2 inhibitors (MEKIs), widely used in cancer treatment due to their ability to inhibit aberrant MAPK signaling, have shown potential synergistic effects when combined with immunotherapy. However, the impact and mechanisms of MEKIs on CAR-T cells remain uncertain and controversial. To address this, we conducted a comprehensive investigation to determine whether MEKIs enhance or impair the efficacy of CAR-T cells. Our findings revealed that MEKIs attenuated CAR-T cell exhaustion and terminal differentiation induced by tonic signaling and antigen stimulation, thereby improving CAR-T cell efficacy against hematological and solid tumors. Remarkably, these effects were independent of the specific scFvs and costimulatory domains utilized in CARs. Mechanistically, analysis of bulk and single-cell transcriptional profiles demonstrates that the effect of MEK inhibition was related to diminish anabolic metabolism and downregulation of c-Fos and JunB. Additionally, the overexpression of c-Fos or JunB in CAR-T cells counteracted the effects of MEK inhibition. Furthermore, our Cut-and-Tag assay revealed that MEK inhibition downregulated the JunB-driven gene profiles associated with exhaustion, differentiation, anergy, glycolysis, and apoptosis. In summary, our research unveil the critical role of the MAPK-c-Fos-JunB axis in driving CAR-T cell exhaustion and terminal differentiation. These mechanistic insights significantly broaden the potential application of MEKIs to enhance the effectiveness of CAR-T therapy.

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

Antigen-independent activation is critical for the durable antitumor effect of GUCY2C-targeted CAR-T cells

BackgroundChimeric antigen receptor (CAR)-T cells face many obstacles in solid tumor therapy, including heterogeneous antigen expression and inefficient T cell persistence. Guanylyl cyclase C (GUCY2C) has been identified as a...

FGFR4-specific CAR-T cells with inducible caspase-9 suicide gene as an approach to treat rhabdomyosarcoma.

Metastatic rhabdomyosarcoma is associated with poor survival and unsatisfactory treatment outcomes. Therefore, new immunotherapeutic methods are urgently required. Fibroblast growth factor receptor 4 (FGFR4), a new therapeutic target for rhabdomyosarcoma, plays a crucial role in its onset and development. This study aimed to generate FGFR4 single-chain variable fragment-based chimeric antigen receptor (CAR) T cells without causing evident toxicity and incorporating an inducible caspase-9 (iCasp9) suicide gene system to enhance their safety. FGFR4 antigen expression was evaluated in normal murine tissues, normal human tissues, and specimens from patients with rhabdomyosarcoma. Combined with a 4-1BB co-stimulatory domain, a CD3ζ signaling domain, and an iCasp9 suicide gene, CAR-T cells with an FGFR4-specific single-chain variable fragment were developed. The specific cytotoxic effects, T-cell proliferation, cytokine secretion, apoptosis induction by chemical dimerization (AP20187), and toxicity of FGFR4 CAR-T cells were investigated in vitro and in vivo. FGFR4 CAR-T cells generated a variety of immune-promoting cytokines, including tumor necrosis factor α, interleukin 2, and interferon γ, and displayed effective cytotoxic activity against FGFR4-overexpressing rhabdomyosarcoma cells in vitro. FGFR4 CAR-T cells were relatively effective against FGFR4-overexpressing rhabdomyosarcoma, with tumor regression and poor survival in a subcutaneous xenograft model. The iCasp9 gene was incorporated into FGFR4 CAR-T cells and it was demonstrated that effective and reliable suicide gene activity depends on the administration of AP20187. By making use of the cross-reaction of FGFR4 CAR-T cells with murine FGFR4 in a syngeneic tumor model, this study found that FGFR4 CAR-T cells could regulate the growth of tumors without evident toxicity. Our study demonstrates that FGFR4 is a prospective target for CAR-T cell therapy in rhabdomyosarcoma without serious on-target off-tumor toxicity. FGFR4 CAR-T cells with the iCasp9 suicide gene system as a safety switch to limit toxicity may broaden the clinical applications of cellular therapy.

Preclinical Evaluation of AZD6422, an Armored Chimeric Antigen Receptor T Cell Targeting CLDN18.2 in Gastric, Pancreatic, and Esophageal Cancer.

CLDN18.2 is a surface membrane protein crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, often characterized by the presence of immune and stromal cells secreting high levels of transforming growth factor beta (TGF-β). Addition of TGF-β armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T showing effectiveness in CLDN18.2-positive gastric, esophageal, and pancreatic tumor models. The lead lentivirus product contains a unique single-chain variable fragment, CD28 and CD3z costimulatory and signaling domains, and dominant negative TGF-β receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product, AZD6422. AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-β levels, as determined by immunohistochemistry. Efficacy of armored CAR-Ts in tumor models with elevated TGF-β was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T. AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.

High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy.

Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity. Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain. We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells. First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity.

CD19 CAR T cells for B cell malignancies: a systematic review and meta-analysis focused on clinical impacts of CAR structural domains, manufacturing conditions, cellular product, doses, patient’s age, and tumor types

CD19-targeted chimeric antigen receptors (CAR) T cells are one of the most remarkable cellular therapies for managing B cell malignancies. However, long-term disease-free survival is still a challenge to overcome. Here, we evaluated the influence of different hinge, transmembrane (TM), and costimulatory CAR domains, as well as manufacturing conditions, cellular product type, doses, patient’s age, and tumor types on the clinical outcomes of patients with B cell cancers treated with CD19 CAR T cells. The primary outcome was defined as the best complete response (BCR), and the secondary outcomes were the best objective response (BOR) and 12-month overall survival (OS). The covariates considered were the type of hinge, TM, and costimulatory domains in the CAR, CAR T cell manufacturing conditions, cell population transduced with the CAR, the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age. Fifty-six studies (3493 patients) were included in the systematic review and 46 (3421 patients) in the meta-analysis. The overall BCR rate was 56%, with 60% OS and 75% BOR. Younger patients displayed remarkably higher BCR prevalence without differences in OS. The presence of CD28 in the CAR’s hinge, TM, and costimulatory domains improved all outcomes evaluated. Doses from one to 4.9 million cells/kg resulted in better clinical outcomes. Our data also suggest that regardless of whether patients have had high objective responses, they might have survival benefits from CD19 CAR T therapy. This meta-analysis is a critical hypothesis-generating instrument, capturing effects in the CD19 CAR T cells literature lacking randomized clinical trials and large observational studies.

Bayesian network analysis of risk classification strategies in the regulation of cellular products

Cell therapy, a burgeoning therapeutic strategy, necessitates a scientific regulatory framework but faces challenges in risk-based regulation due to the lack of a global consensus on risk classification. This study applies Bayesian network analysis to compare and evaluate the risk classification strategies for cellular products proposed by the Food and Drug Administration (FDA), Ministry of Health, Labour and Welfare (MHLW), and World Health Organization (WHO), using real-world data to validate the models. The appropriateness of key risk factors is assessed within the three regulatory frameworks, along with their implications for clinical safety. The results indicate several directions for refining risk classification approaches. Additionally, a substudy focuses on a specific type of cell and gene therapy (CGT), chimeric antigen receptor (CAR) T cell therapy. It underscores the importance of considering CAR targets, tumor types, and costimulatory domains when assessing the safety risks of CAR T cell products. Overall, there is currently a lack of a regulatory framework based on real-world data for cellular products and a lack of risk-based classification review methods. This study aims to improve the regulatory system for cellular products, emphasizing risk-based classification. Furthermore, the study advocates for leveraging machine learning in regulatory science to enhance the assessment of cellular product safety, illustrating the role of Bayesian networks in aiding regulatory decision-making for the risk classification of cellular products.

Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-Stimulatory Signal Domain Exhibits Exhaustion-Resistant Properties.

Improving chimeric antigen receptor (CAR)-T cell therapeutic outcomes and expanding its applicability to solid tumors requires further refinement of CAR-T cells. We previously reported that CAR-T cells bearing a herpes virus entry mediator (HVEM)-derived co-stimulatory signal domain (CSSD) (HVEM-CAR-T cells) exhibit superior functions and characteristics. Here, we conducted comparative analyses to evaluate the impact of different CSSDs on CAR-T cell exhaustion. The results indicated that HVEM-CAR-T cells had significantly lower frequencies of exhausted cells and exhibited the highest proliferation rates upon antigenic stimulation. Furthermore, proliferation inhibition by programmed cell death ligand 1 was stronger in CAR-T cells bearing CD28-derived CSSD (CD28-CAR-T cells) whereas it was weaker in HVEM-CAR-T. Additionally, HVEM-CAR-T cells maintained a low exhaustion level even after antigen-dependent proliferation and exhibited potent killing activities, suggesting that HVEM-CAR-T cells might be less prone to early exhaustion. Analysis of CAR localization on the cell surface revealed that CAR formed clusters in CD28-CAR-T cells whereas uniformly distributed in HVEM-CAR-T cells. Analysis of CD3ζ phosphorylation indicated that CAR-dependent tonic signals were strongly sustained in CD28-CAR-T cells whereas they were significantly weaker in HVEM-CAR-T cells. Collectively, these results suggest that the HVEM-derived CSSD is useful for generating CAR-T cells with exhaustion-resistant properties, which could be effective against solid tumors.

CAR T-cells targeting FGFR4 and CD276 simultaneously show potent antitumor effect against childhood rhabdomyosarcoma

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.

Targeting GPC2 on Intraocular and CNS Metastatic Retinoblastomas with Local and Systemic Delivery of CAR T Cells.

Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy. GPC2 expression and its regulation by the E2F1 transcription factor were studied in retinoblastoma patient samples and cellular models. In vitro, we performed functional studies comparing GPC2 CAR T cells with different costimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T cells was evaluated in intraocular and leptomeningeal human retinoblastoma xenograft models. Retinoblastoma tumors, but not healthy retinal tissues, expressed cell surface GPC2, and this tumor-specific expression was driven by E2F1. GPC2-directed CARs with 4-1BB costimulation (GPC2.BBz) were superior to CARs with CD28 stimulatory domains (GPC2.28z), efficiently inducing retinoblastoma cell cytotoxicity and enhancing T-cell proliferation and polyfunctionality. In vivo, GPC2.BBz CARs had enhanced persistence, which led to significant tumor regression compared with either control CD19 or GPC2.28z CARs. In intraocular models, GPC2.BBz CAR T cells efficiently trafficked to tumor-bearing eyes after intravitreal or systemic infusions, significantly prolonging ocular survival. In central nervous system (CNS) retinoblastoma models, intraventricular or systemically administered GPC2.BBz CAR T cells were activated in retinoblastoma-involved CNS tissues, resulting in robust tumor regression with substantially extended overall mouse survival. GPC2-directed CAR T cells are effective against intraocular and CNS metastatic retinoblastomas.

The Spectrum of CAR Cellular Effectors: Modes of Action in Anti-Tumor Immunity.

Chimeric antigen receptor-T cells have spearheaded the field of adoptive cell therapy and have shown remarkable results in treating hematological neoplasia. Because of the different biology of solid tumors compared to hematological tumors, response rates of CAR-T cells could not be transferred to solid entities yet. CAR engineering has added co-stimulatory domains, transgenic cytokines and switch receptors to improve performance and persistence in a hostile tumor microenvironment, but because of the inherent cell type limitations of CAR-T cells, including HLA incompatibility, toxicities (cytokine release syndrome, neurotoxicity) and high costs due to the logistically challenging preparation process for autologous cells, the use of alternative immune cells is gaining traction. NK cells and γδ T cells that do not need HLA compatibility or macrophages and dendritic cells with additional properties such as phagocytosis or antigen presentation are increasingly seen as cellular vehicles with potential for application. As these cells possess distinct properties, clinicians and researchers need a thorough understanding of their peculiarities and commonalities. This review will compare these different cell types and their specific modes of action seen upon CAR activation.

Preclinical studies show that Co-STARs combine the advantages of chimeric antigen and T cell receptors for the treatment of tumors with low antigen densities.

Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.

Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo

Background aimsHu8F4 is a T-cell receptor–like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor–expressing cells. MethodsWe generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor–expressing cells in vivo. ResultsThe primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. ConclusionsHerein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding–induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Outpatient administration of CAR T-cell therapies using a strategy of no remote monitoring and early CRS intervention

AbstractRecent studies demonstrating the feasibility of outpatient chimeric antigen receptor (CAR)–modified T-cell therapy administration are either restricted to CARs with 41BB costimulatory domains or use intensive at-home monitoring. We report outcomes of outpatient administration of all commercially available CD19- and B-cell maturation antigen (BCMA)–directed CAR T-cell therapy using a strategy of no remote at-home monitoring and an early cytokine release syndrome (CRS) intervention strategy. Patients with hematologic malignancies who received CAR T-cell therapy in the outpatient setting during 2022 to 2023 were included. Patients were seen daily in the cancer center day hospital for the first 7 to 10 days and then twice weekly through day 30. The primary end point was to determine 3-, 7-, and 30-day post–CAR T-cell infusion hospitalizations. Early CRS intervention involved administering tocilizumab as an outpatient for grade ≥1 CRS. Fifty-eight patients received outpatient CAR T-cell infusion (33 myeloma, 24 lymphoma, and 1 acute lymphoblastic leukemia). Of these, 17 (41%), 16 (38%), and 9 patients (21%) were admitted between days 0 to 3, 4 to 7, and 8 to 30 after CAR T-cell infusion, respectively. The most common reason for admission was CAR T-cell–related toxicities (33/42). Hospitalization was prevented in 15 of 35 patients who received tocilizumab for CRS as an outpatient. The nonrelapse mortality rates were 1.7% at 1 month and 3.4% at 6 months. In conclusion, we demonstrate that the administration of commercial CAR T-cell therapies in an outpatient setting is safe and feasible without intensive remote monitoring using an early CRS intervention strategy.

IMMU-06. CAR DESIGN SHAPES THE BRAIN TUMOR IMMUNE MICROENVIRONMENT: IMPACT ON ACTIVITY AND PERSISTENCE OF ANTI-GLIOMA ADOPTIVE T CELL IMMUNOTHERAPY

Abstract BACKGROUND B7-H3 CAR T-cells effectively eradicate brain tumors in xenograft models and have proven safe in clinical trials, yet no objective responses were observed in patients. The limited efficacy is partly attributed to the immunosuppressive tumor microenvironment (TME), a characteristic not fully recapitulated in investigations using xenograft models. In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on the brain TME using an immunocompetent glioma model. METHODS We generated a panel of fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry and single-cell RNA sequencing were then used to investigate changes in the brain TME following CAR T-cell therapy. RESULTS While five out of six B7-H3 CARs with single co-stimulatory domains exhibited potent functionality in vitro, optimizing co-stimulation and signaling failed to enhance their anti-glioma efficacy in vivo. To further enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 co-stimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. Although this design significantly improved anti-glioma efficacy in vitro, it conferred no additional benefits in vivo. Analysis of the TME revealed that CAR T-cell therapy influenced the immune composition of the brain TME. Successful anti-tumor responses were dictated by the recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ containing specific subsets of macrophages and endogenous T-cells. Interestingly, depletion of brain macrophages using CSF1R inhibitor markedly inhibited CAR T-cell anti-tumor activity. CONCLUSION Our study highlights the critical role of CAR structural design and its modulation of the TME in mediating the efficacy of CAR T-cell therapy in brain tumors. Yet, more specific targeting and/or reprogramming of the TME is warranted for the successful design of effective adoptive immunotherapies for high-grade glioma.

Zevorcabtagene Autoleucel: First Approval.

Zevorcabtagene autoleucel () is a fully humanised B cell maturation antigen (BCMA)-targeting specific chimeric antigen receptor (CAR) T-cell therapy being developed by CARsgen for the treatment of multiple myeloma. Zevorcabtagene autoleucel is an autologous CAR T cell comprising a fully human BCMA-specific scFv (25C2), a CD8α hinge region and transmembrane domain, a 4-1BB costimulatory domain and a CD3-ζ T cell activation domain. Zevorcabtagene autoleucel recognizes and induces selective toxicity against BCMA-expressing tumour cells leading to their elimination. In February 2024, zevorcabtagene autoleucel received its first approval in China for the treatment of adults with relapsed or refractory multiple myeloma who have progressed after ≥ 3 prior lines of therapy (including ≥ 1 proteasome inhibitor and an immunomodulatory agent). Clinical studies of zevorcabtagene autoleucel are underway in Canada and the US. This article summarizes the milestones in the development of zevorcabtagene autoleucel leading to this first approval for relapsed or refractory multiple myeloma.