CAR-T Construct Research Articles

The Role of CAR T-Cell Therapy in Relapsed/Refractory Adult B-ALL.

CAR T-cell therapy is a recent therapeutic advancement that has transformed the management of relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). To date, there are 2 FDA-approved CAR-T products for R/R B-ALL: tisagenlecleucel in patients aged <26 years and brexucabtagene autoleucel in those aged ≥18 years. This review summarizes the pivotal clinical trials that led to FDA approval of these 2 products and highlight emerging data addressing key questions pertinent to CAR-T utilization in the rapidly evolving landscape of R/R ALL management. These include optimal sequencing of CAR-T among other novel immunotherapeutic agents, the role of consolidation and maintenance following CAR-T, novel CAR-T constructs currently under clinical development, and strategies to optimize use of commercially available CAR-T products to improve patient outcomes.

AMPK agonism optimizes the in vivo persistence and anti-leukemia efficacy of chimeric antigen receptor T cells.

Chimeric antigen receptor T cell (CART) therapy has seen great clinical success. However, up to 50% of leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism ex vivo can improve in vivo T cell function and we hypothesized that exposure to an agonist targeting the metabolic regulator AMP-activated protein kinase (AMPK), would create CARTs capable of both efficient leukemia clearance and increased in vivo persistence. CART cells were generated from healthy human via lentiviral transduction. Following activation, cells were exposed to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, followed one week later by either 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. Human T cells expanded in Compound 991 activated AMPK without limiting cellular expansion and gained both mitochondrial density and improved handling of reactive oxygen species (ROS). Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance, prolonged recipient survival, and increased CD4+ T cell yields at early times post-injection. Ex vivo, 991 agonist treatment mimicked nutrient starvation, increased autophagic flux, and promoted generation of mitochondrially-protective metabolites. Ex vivo expansion processes are necessary to generate sufficient cell numbers, but often promote sustained activation and differentiation, negatively impacting in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during CART expansion metabolically reprograms cells without limiting T cell yield, enhances in vivo anti-leukemia efficacy, and improves CD4+ in vivo persistence. Importantly, AMPK agonism achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and overall efficacy of cancer-targeting T cells.

Potential and pitfalls of repurposing the CAR-T cell regimen for the treatment of autoimmune disease

Chimeric antigen receptors (CARs) are synthetic proteins designed to direct an immune response toward a specific target and have been used in immunotherapeutic applications through the adoptive transfer of T...

Analyzing shopping cart abandonment enablers: an ISM and MICMAC approach

PurposeThe primary purpose of this study is to examine the factors leading to shopping cart abandonment and construct a model depicting interrelationship among them using interpretive structural modeling (ISM) and Matriced Impact Croises Multiplication Appliquee an un Classement (MICMAC).Design/methodology/approachInitially, 20 factors leading to shopping cart abandonment were extracted through a systematic literature review and expert opinions. Fifteen factors were finalized using the importance index and CIMTC method, for which consistency has been checked in SPSS software through a statistical reliability test. Finally, ISM and MICMAC approach is used to develop a model depicting the contextual relationship among finalized factors of shopping cart abandonment.FindingsThe ISM model depicts a technical glitch (SC8), cash on delivery not available (SC4), bad checkout interface (SC9), just browsing (SC11), and lack of physical examination (SC12) are drivers or independent factors. Additionally, four quadrants have been formulated in MICMAC analysis based on their dependency and driving power. This facilitates technical managers of e-commerce companies to focus more on factors leading to shopping cart abandonment according to their dependency and driving power.Research limitations/implicationsTaking an expert’s opinion as a base may affect the results of the study due to biases based on subjectivity.Practical implicationsThis study’s outcomes would accommodate practitioners, researchers, and multinational or national companies to indulge in e-commerce to anticipate factors restricting the general public from online shopping.Originality/valueFor the successful running of an e-commerce business and to retain the confidence of e-shoppers, every e-commerce company must make a strategy for controlling factors leading to shopping cart abandonment at the initial stage. So, this paper attempts to highlight the main factors leading to shopping cart abandonment and interrelate them using ISM and MICMAC approaches. It provides a clear path to technical heads, researchers, and consultants for handling these shopping cart abandonment factors.

Abstract 3609: Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies

Abstract Cytokine activation of T cells can promote T cell proliferation, memory and improve T cell function in driving tumor cell killing. However, systemic administration of cytokines at therapeutic doses is also associated with unacceptable toxicities. To overcome this limitation, we have engineered a system of private cytokine signaling to affect T cell homeostasis, identity, and function. Specifically, we have engineered chimeric cytokine receptors (CCRs) composed of single-chain variable fragments (scFvs) that bind specifically to small molecules such as fluorescein or DOTA. These scFvs are fused to transmembrane and intracellular domains of all members of the common gamma-chain (γ-chain) family of cytokine receptors. CCRs that permit private cytokine signaling were introduced into known CAR-T cell therapies and their impact on CAR-T cell phenotype, function, and anti-tumor activity was tested. First, CCR-containing T cells were tested for activation-induced changes in phenotype and expansion following stimulation with small molecule conjugates in vitro. The most promising results were obtained using either the IL-7Rα- or IL-2Rβ/γ-containing CCR’s. CCR-containing constructs were then transduced into one of two different anti-BCMA CAR-T constructs, ide-cel or cilta-cel, using a lentiviral transduction system. CAR-T cells containing each CCR were then compared with CAR-T cells lacking CCRs for phenotype and function in vitro. Results indicated that engineering the IL-7Rα CCR with both of these CAR-T’s led to increased expansion, cytokine secretion and improved tumor cell killing. Next, CAR-T cells expressing the IL-7Rα CCR were compared to CAR-T cells lacking CCR for efficacy and durability in a mouse model of human multiple myeloma. IL-7Rα CCR-expressing CAR-T cells demonstrated improved long-term tumor control compared to ide-cel and cilta-cel alone. In addition, IL-7Rα CCR-expression increased T cell numbers in blood, spleen and bone marrow of tumor-bearing mice. These increases were particularly evident in the CD8+population of CAR-T cells in vivo. These results demostrate that private cytokine signaling using small-molecule activatable CCRs can improve efficacy and durability of CAR-T cells. We are testing these CCRs in mouse models of human solid tumors to assess their ability to overcome the immunosuppressive microenvironment. Citation Format: John R. Newcomb, Jun Ren, Sakeena Syed, Zachary Caruolo, Lindsay Webb, Craig McKay, Stephanie Kwei, Carl Novina, Fred Mermelstein. Chimeric cytokine receptors increase memory phenotypes and improve expansion and efficacy of CAR-T cell therapies [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 3609.

Chimeric Antigen Receptor T-Cell Therapy in Acute Myeloid Leukemia: State of the Art and Recent Advances.

Chimeric antigen receptors (CAR)-T-cell therapy represents the most important innovation in onco-hematology in recent years. The progress achieved in the management of complications and the latest generations of CAR-T-cells have made it possible to anticipate in second-line the indication of this type of treatment in large B-cell lymphoma. While some types of B-cell lymphomas and B-cell acute lymphoid leukemia have shown extremely promising results, the same cannot be said for myeloid leukemias-in particular, acute myeloid leukemia (AML), which would require innovative therapies more than any other blood disease. The heterogeneities of AML cells and the immunological complexity of the interactions between the bone marrow microenvironment and leukemia cells have been found to be major obstacles to the clinical development of CAR-T in AML. In this review, we report on the main results obtained in AML clinical trials, the preclinical studies testing potential CAR-T constructs, and future perspectives.

Integrating Immune Therapies for the Treatment of Multiple Myeloma.

Patients with relapsed or refractory multiple myeloma (RRMM) that is refractory to a proteasome inhibitor, an immunomodulatory drug (IMiD), and an anti-CD38 antibody (triple-class refractory MM) have poor outcomes. Recently, 2 classes of T-cell engaging therapies-CAR T-cell therapy and bispecific T-cell engaging antibodies (BsAbs)-have resulted in unprecedented response rates and survival outcomes in these heavily pretreated patients. The most common targets are BCMA and GPRC5D, with other targets in development. The main classes of adverse effects include cytokine release syndrome, neurotoxicity, infections, and cytopenias, as well as adverse effects unique to specific products. As of September 2023, 2 BCMA-targeting CAR-T cell products, 2 BCMA-targeting BsAbs, and 1 GPRC5D-targeting BsAb, are FDA-approved for standard-of-care use in patients with RRMM who received at least 4 prior lines of therapy, including prior treatment with a proteasome inhibitor, an IMiD, and an anti-CD38 antibody. Earlier-line use is under investigation and has shown promising results. Several other investigational CAR-T constructs and bispecific antibodies are in clinical development. As these therapies become more widely used, including in earlier-line setting, efforts to understand optimal sequencing and mitigate toxicities remain critical.

Two Players, One Goal: BiTE ® Vs CART Targeting FLT3 in AML

Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 presents with a rather favorable expression profile due to broad AML expression and limited expression within the normal hematopoietic compartment (Brauchle et al., 2020). Importantly, expression on AML bulk cells and leukemic stem cells is observed independent of FLT3 mutational status. Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR-mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. On-target off-leukemia toxicity was evaluated in mixing assays of pAML cells and healthy donor bone marrow (hBM). The impact of positive costimulation on both approaches was tested using our previously established Ba/F3 model system (Marcinek et al., 2023), which is devoid of any human costimulatory molecules. As a surrogate for synapse formation, conjugate formation as well as consecutive T-cell degranulation were studied. Further, we studied the cell death pathway mediating cytotoxicity against AML target cells, e.g. FAS/FASL vs perforin/granzyme B. The impact of continuous stimulation by BiTE®-and CART on evolving T-cell exhaustion was tested using our previously established 28-day long-term culture assay (Philipp et al., 2022). Finally, we compared BiTE® and CAR in an in vivo xenograft AML model. In a second in vivo experiment, all mice were harvested 18 days after T-cell injection, followed by T-cell isolation from the bone marrow and subsequent bulk RNA sequencing. BiTE®- and CAR-redirected T cells led to comparable effector-to-target (E:T) ratio-dependent specific lysis of various AML cell lines and pAML cells. Using our in vitro mixing experiments, we could show that BiTE®- and CAR-redirected T cells efficiently killed pAML cells while mostly sparing the hBM. Using the Ba/F3 model system, we observed that the expression of the costimulatory molecule CD86 significantly increased the cytotoxicity of BiTE®-redirected T cells while CAR-mediated lysis was unaltered. Furthermore, we were able to show that the percentage of T cell-AML conjugates formed as well as degranulation of CD107a was significantly higher when using CART compared to BiTE® molecules. Interestingly, when we added a FAS-blocking antibody to our cocultures, we could show that CART-mediated lysis of the target cells was significantly reduced. This was not observed when using the BiTE® construct. Using our long-term culture system, we found that T-cell proliferation and cytokine secretion decreased with both constructs over time. The cytotoxic capacity of BiTE®-redirected T cells seemed to decrease faster compared to the CART construct. Moving to an in vivo AML xenograft model, we observed that CART-treated mice had an improved OS compared to the BiTE®-treated mice (Fig.1). This was accompanied by enhanced T-cell proliferation and splenic homing of the CAR T cells. Bulk RNA sequencing of T cells isolated from the murine bone marrow 18 days post T-cell injection revealed signs of exhaustion in BiTE®-redirected T cells compared to CAR T cells. We found an upregulation of several inhibitory immune receptors and transcription factors (Fig.2) that have been well described to promote T-cell exhaustion. Furthermore, gene set enrichment analysis revealed that genes upregulated in effector vs exhausted were also upregulated in CAR T cells. This was accompanied by an upregulated glycolysis and fatty acid metabolism in CAR T cells. We conclude, that FLT3 is a promising target antigen with limited on-target off-leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR construct provided an advantage in T-cell activation and might lead to better T-cell homing, resulting in improved efficacy and delayed T-cell exhaustion in vivo. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell-based immunotherapy platforms and identify suitable combination partners.

First Fever between Lymphodepletion and Day 30 after CAR-T Cell Therapy: Patterns, Origins, and Implications in Lymphoma Patients

Introduction: Fever is a common adverse event associated with chimeric antigen receptor-T cell (CAR-T cell) therapy and serves as the hallmark of cytokine release syndrome (CRS). However, limited data are available on competing and contributing etiologies of fever. Thus, we aimed to investigate the causes, risk factors, and implications of fever in CAR-T cell recipients in the period with the most profound immunosuppression: between lymphodepletion (LD) and day 30 after infusion. Methods: In this single-center retrospective analysis, we studied 304 non-Hodgkin lymphoma patients (80% large B-cell lymphoma, 11% mantle cell lymphoma, 4% follicular lymphoma) treated with CD19-CAR-T cells (48% axi-cel, 23% tisa-cel, 23% liso-cel, 6% brexu-cel) between 2016 and 2023. First fever was defined as a temperature of 38°C or higher between LD and day 30 after CD19-CAR-T cells. Patients experiencing fever were routinely cultured, following institutional guidelines. Additional workup, including respiratory virus PCR panel, stool gastrointestinal PCR panel, Clostridioides difficile PCR, urine culture, chest x-ray, and fungal infection assessment, was done at the medical team's discretion. A contributing infectious source for the fever was considered if there was a positive test in the workup within 72 hours of the initial fever. Results: Fever occurred in 232/304 (76%) of patients between LD and day 30 after CAR-T cell infusion. The median time from LD to first fever was 5 days (range 2-13) and from CAR-T infusion to first fever was 2 days (range: LD initiation to 16). Of patients who had not developed a fever up to the day of infusion (n=271), 199 (73%) experienced a fever between infusion (day 0) and day 30. The primary etiology of fever between LD and day 30 was CRS (n=159/232, 68.5%), with 54% experiencing grade 2 or higher. An infectious source was identified in 52/232 (22%) of all patients and 35/194 (18%) of the subgroup developing CRS. Leading infectious etiologies were gastrointestinal (GI), bloodstream, and respiratory infections in 20 (38%), 14 (27%), and 11 (21%) of patients, respectively (Figure A). The most frequent pathogens identified were Clostridioides difficile for GI tract infections (10/20), Enterobacteriaceae for bloodstream infections (8/14), and common upper respiratory viruses, such as Rhinovirus and Parainfluenzavirus (10/16). Importantly, in patients developing fever after infusion, an infectious source was identified in 42/199 (21%). Among them, 12/42 (29%) were bloodstream infections, and 10/42 (24%) were Clostridioides difficile infections requiring rapid antibiotic management. Of an array of potential risk factors, only pre-apheresis metabolic tumor volume was associated with an increased risk for an infectious cause of fever in a univariable logistic-regression model (p=0.029). Furthermore, an infectious source of fever, compared to a non-infectious cause, was associated with an increased risk of mortality (HR 1.60, 95%CI 1.02-2.51, p=0.039) in a multivariable Cox regression model adjusted for LDH range before lymphodepletion, age and CAR-T construct type (Figure B). Conclusions: This study represents one of the first and largest investigations into fever patterns and origins in CAR-T cell therapy recipients. Although fever is common, treatable infectious sources are infrequent. Our findings may have implications for antibiotic stewardship and fever management in CAR-T patients. Finally, the association between infectious fever and inferior survival warrants further investigation on the biological processes driving mortality.

Discovery and Development of CD70 As a Cellular Therapy Target in High Risk Multiple Myeloma

Background: Even in the BCMA CAR-T era, multiple myeloma (MM) patients with high-risk genotypes have the poorest outcomes. We thus leveraged MMRF CoMMpass data to identify potential novel surface immunotherapeutic targets in high-risk MM. As described below, we focused on CD70, an immunotherapeutic target known to be upregulated on many hematologic and solid tumors but minimally expressed on normal tissue. While CD70 was previously explored in MM (McEarchern et al, Clin Cancer Res (2008)), it was not further investigated due to heterogeneous expression in newly diagnosed samples. Here we revisit this target to suggest cellular therapies against CD70 could be highly beneficial in high-risk MM patients, currently the greatest unmet need in the field. Methods: Primary patient samples were obtained under an IRB-approved protocol. CAR-T lentiviral transduction was performed on paired CD4/CD8 human T cells from at least 3 healthy donors. Effector function was assessed by cytotoxicity against MM cell lines and CAR-T profiling for memory and exhaustion markers. In vivo efficiency was assessed using bioluminescence. ATAC-seq peaks were called using Promo and HOMER (data from Jin et al. Blood (2018)). Machine learning was performed using XGBoost approach applied to CoMMpass RNA-seq data with 80% of samples for training and 20% as test set. Results: Analyzing CoMMpass mRNA data (release IA19) we first evaluated surface protein encoding-genes upregulated in tumors at relapse compared to diagnosis. Among &amp;gt;100 significantly upregulated genes, we focused on CD70 given known promise as an immunotherapy target. Further analysis across genomic subtypes revealed a specific, marked upregulation of CD70 in t(4;14) and gain 1q ( Fig. 1), as well as R-ISS 3 at diagnosis. Flow cytometry profiling of 8 cell line models, including t(4;14) models KHM11, KMS26, and LP1, revealed consistent CD70 protein expression with antigen density similar to BCMA. Profiling of relapsed primary MM samples confirmed that 19 of 28 samples had CD138+ plasma cells positive for CD70 expression. Of 5 patients in whom both BCMA and CD70 were measured, antigen densities were also similar. To target CD70, we then designed 10 unique anti-CD70 CAR-T constructs. 9 of these were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. By a structure-guided design based on the CD70:CD27 co-crystal structure (PDB: 7KX0), we also derived a unique truncated fragment of CD27 as an alternative CD70 binding element. All CD70 CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI8226. We then selected the top 3 performing scFv based-CARs as well as our natural ligand (CD27) CAR-T for in vivo studies. All 4 CD70 CAR-Ts prolonged survival of MM.1S-bearing mice compared to empty CAR ( n=5 mice/arm, p&amp;lt;0.005 by log-rank), similar to BCMA CAR-T controls. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p&amp;lt; 0.0001 by ANOVA) and persistence compared to scFv-based ( Fig. 2). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high risk MM. We further investigated why CD70 may be upregulated in high risk MM. Analysis of ChIP-seq in ENCODE and primary MM tumor ATAC-seq was used to nominate a set of transcription factors that bind the CD70 locus. Extending from CoMMpass data, we built a predictive machine learning model for CD70 expression as a function of transcription factor gene expression, nominating key transcription factors TFAP2A, LEF1, MYB, and PAX5 as CD70 regulators; validation is now underway. Additionally, we showed that knockout of NSD2 in t(4;14) models KMS34 and KMS11 led to significantly decreased CD70 surface expression, implicating this central epigenetic driver in CD70 overexpression in this high-risk MM subtype. Finally, we also demonstrated that treatment with the hypomethylating agent azacytidine can drive increased CD70 expression, similar to that shown in AML models. Conclusion: Taken together, targeting CD70 appears to be a highly promising cellular therapeutic strategy for MM, with particularly high utility in high-risk patients after BCMA CAR-T relapse. We are continuing preclinical and future clinical development of this CAR-T strategy, using our natural ligand CD27 design with extended in vivo persistence.

Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies

BackgroundApproximately 50% of patients who receive anti-CD19 CAR-T cells relapse, and new immunotherapeutic targets are urgently needed. We recently described CD72 as a promising target in B-cell malignancies and developed...

TSHR-based chimeric antigen receptor T cell specifically deplete auto-reactive B lymphocytes for treatment of autoimmune thyroid disease

Graves' disease (GD) is a prominent antibody-mediated autoimmune disorder characterized by stimulating antibodies (TRAb) that target the thyroid-stimulating hormone receptor (TSHR). Targeting and eliminating TRAb-producing B lymphocytes hold substantial therapeutic potential for GD. In this study, we engineered a novel chimeric antigen receptor T cell (CAR-T) therapy termed TSHR-CAR-T. This CAR-T construct incorporates the extracellular domain of the TSH receptor fused with the CD8 transmembrane and intracellular signal domain (4-1BB). TSHR-CAR-T cells demonstrated the ability to recognize and effectively eliminate TRAb-producing B lymphocytes both in vitro and in vivo. Leveraging this autoantigen-based chimeric receptor, our findings suggest that TSHR-CAR-T cells offer a promising and innovative immunotherapeutic approach for the treatment of antibody-mediated autoimmune diseases, including GD.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients’ own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

CAR-T State of the Art and Future Challenges, A Regulatory Perspective.

This review is an outlook on CAR-T development up to the beginning of 2023, with a special focus on the European landscape and its regulatory field, highlighting the main features and limitations affecting this innovative therapy in cancer treatment. We analysed the current state of the art in the EU and set out a showcase of the field's potential advancements in the coming years. For this analysis, the data used came from the available scientific literature as well as from the European Medicines Agency and from clinical trial databases. The latter were investigated to query the studies on CAR-Ts that are active and/or relevant to the review process. As of this writing, CAR-Ts have started to move past the "ceiling" of third-line treatment with positive results in comparison trials with the Standard of Care (SoC). One such example is the trial Zuma-7 (NCT03391466), which resulted in approval of CAR-T products (Yescarta™) for second-line treatment, a crucial achievement for the field which can increase the use of this type of therapy. Despite exciting results in clinical trials, limitations are still many: they regard access, production, duration of response, resistance, safety, overall efficacy, and cost mitigation strategies. Nonetheless, CAR-T constructs are becoming more diverse, and the technology is starting to produce some remarkable results in treating diseases other than cancer.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

CAR T-Cell Persistence Correlates with Improved Outcome in Patients with B-Cell Lymphoma.

Chimeric antigen receptor (CAR) T-cell therapy has led to profound and durable tumor responses in a relevant subset of patients with relapsed/refractory (r/r) B-cell lymphomas. Still, some patients show insufficient benefit or relapse after CAR T-cell therapy. We performed a retrospective study to investigate the correlation between CAR T-cell persistence in the peripheral blood (PB) at 6 months, assessed by droplet digital PCR (ddPCR), with CAR T-cell treatment outcome. 92 patients with r/r B-cell lymphomas were treated with CD19-targeting CAR T-cell therapies at our institution between 01/2019-08/2022. Six months post-treatment, 15 (16%) patients had no detectable circulating CAR-T constructs by ddPCR. Patients with CAR T-cell persistence had a significantly higher CAR T-cell peak (5432 vs. 620 copies/ug cfDNA, p = 0.0096), as well as higher incidence of immune effector cell-associated neurotoxicity syndrome (37% vs. 7%, p = 0.0182). After a median follow-up of 8.5 months, 31 (34%) patients relapsed. Lymphoma relapses were less frequent among patients with CAR T-cell persistence (29% vs. 60%, p = 0.0336), and CAR T-cell persistence in the PB at 6 months was associated with longer progression-free survival (PFS) (HR 2.79, 95% CI: 1.09-7.11, p = 0.0319). Moreover, we observed a trend towards improved overall survival (OS) (HR 1.99, 95% CI: 0.68-5.82, p = 0.2092) for these patients. In our cohort of 92 B-cell lymphomas, CAR T-cell persistence at 6 months was associated with lower relapse rates and longer PFS. Moreover, our data confirm that 4-1BB-CAR T-cells have a longer persistence as compared to CD-28-based CAR T-cells.

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Harnessing the Potential of Chimeric Antigen Receptor T-Cell Therapy for the Treatment of T-Cell Malignancies: A Dare or Double Dare?

Historical standard of care treatments of T-cell malignancies generally entailed the use of cytotoxic and depleting approaches. These strategies are, however, poorly validated and record dismal long-term outcomes. More recently, the introduction and approval of chimeric antigen receptor (CAR)-T cell therapy has revolutionized the therapy of B-cell malignancies. Translating this success to the T-cell compartment has so far proven hazardous, entangled by risks of fratricide, T-cell aplasia, and product contamination by malignant cells. Several strategies have been utilized to overcome these challenges. These include the targeting of a selective cognate antigen exclusive to T-cells or a subset of T-cells, disruption of target antigen expression on CAR-T constructs, use of safety switches, non-viral transduction, and the introduction of allogeneic compounds and gene editing technologies. We herein overview these historical challenges and revisit the opportunities provided as potential solutions. An in-depth understanding of the tumor microenvironment is required to optimally harness the potential of the immune system to treat T-cell malignancies.

A Phase I Clinical Trial of Point-of-Care Manufactured Fresh Anti-CD19/20/22 Chimeric Antigen Receptor T Cells for Treatment of Relapsed or Refractory Lymphoid Malignancies (Non-Hodgkin Lymphoma, Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia, B Prolymphocytic Leukemia)

Background: CD19-targeted CAR-T cells have transformed the treatment of relapsed/refractory B-cell malignancies. However, relapse occurs in over 50% of patients treated with commercial CD19 directed CAR-T due to antigen escape from selective immune pressure either on the target antigen or due to exhaustion and lack of persistence of CAR-T cells. Hence there is an unmet need for next-generation CAR-Ts that have broad antigen recognition and novel costimulatory domains to promote survival and decrease exhaustion of CAR-Ts. Targeting three B-cell antigens simultaneously is a novel approach to prevent antigen-negative relapse while inclusion of the OX40 domain will promote CAR-Ts persistence and expansion. Whether targeting three commonly expressed antigens is feasible or safe and will result in durable remissions is not known. We hypothesize that this trispecific targeting of CD19/20/22 CAR-T construct will be safe and effective in patients with relapsed, refractory B-cell malignancies. Study Design and Methods: This parallel group phase 1 study follows a 3+3 phase 1 design to determine the safety of the infusion of point-of-care manufactured fresh CAR-T cells targeting CD19/20/22 and to find the recommended phase II dose for this cellular therapy. CAR-T cells will be manufactured over 6 days. Patients will be enrolled in one of the two cohorts: Cohort A (single infusion): Non-Hodgkin's Lymphoma (NHL) with lesions <= 5 cm; Indolent lymphomas; B-PLL (B-Pro lymphocytic leukemia); Chronic Lymphocytic Leukemia -CLL (without Richter's transformation), Cohort B (split infusion): B-ALL (B-Acute lymphoblastic leukemia); Lymphoid blast crisis of Chronic myeloid leukemia; CLL (with Richter's transformation); NHL with lesions > 5 cm and/or lymphoblastic lymphoma, or NHL with circulating lymphoma cells. Patients who are CAR-naïve will be enrolled in this study. The study involves infusion of anti-CD19/20/22 CAR-T cells administered after an immunosuppressive conditioning regimen of fludarabine and cyclophosphamide. Cyclophosphamide 60 mg/kg will be infused intravenously over 60 minutes on day -6. Fludarabine 25 mg/m2 will be infused intravenously daily over 30 minutes on days -5 to -3. In each group, patients will be enrolled sequentially to each dose level, starting with dose level 1. Three CAR-T dose-levels: 5x105, 1x106 & 2x106 cells/kg/dose will be explored to determine MTD. For cohort B patients, the total CAR-T cell dose infused, will be divided in two doses, dose 1 (day 0) is 40% of the total dose and dose 2 (day 7) is 60% of total dose. The minimum number of subjects enrolled in the complete study will be 18 (9 per cohort) and the maximum number in dose escalation will be 36 (18 per cohort). Patients in each cohort will be monitored for 30 days before enrollment of next patientTwo patients have been enrolled in the trial and received fresh infusion of Anti CD19/20/22 CAR-T cells and accrual is ongoing. Updates on the initial cohort of patients will be presented at the meeting. ClinicalTrials.gov NCT: NCT05418088 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 5575: Pre-clinical validation of a LGR5-targeting CAR-T against colorectal cancer

Abstract Chimeric antigen receptor T cells (CAR-T) have demonstrated robust clinical activity against B-cell malignancies, and rapid translation to approval for use in man has inspired the search for CAR-T cells against solid tumors. Cancer stem cells are a small population of cells within a tumor that have the capacity to self-renew, differentiate, and initiate new tumors. Cancer stem cells are known to be resistant to chemotherapy and radiotherapy and are enriched in residual disease which allows relapse. Targeting cancer stem cells will reduce the tumors’ ability to generate new cells resulting in tumor remission. We have tested a novel CAR-T cell that has the potential to target cancer stem cells. Leucine-rich G protein-coupled receptor 5 (LGR5) expression is restricted to stem cell populations in multiple tissues in adults. In addition, LGR5 is a marker of cancer stem cells and LGR5+ cells are implicated in tumor progression and metastasis. We designed six lentiviral CAR-T constructs targeting the extra cellar domain of LGR5 with differing linker lengths and binding domain orientation, and tested gene delivery to purified human CD3 T cells and in vitro cytotoxicity. All constructs were expressed robustly (&amp;gt;90%), and four of six LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity against colorectal cancer cell lines in vitro. We confirmed the on-target specificity of the LGR5 targeting CAR-T using a cell line over-expressing the LGR5 protein and antibody competition assays, which blocked cytotoxicity. Using optimized gene delivery and CAR-T manufacturing protocols, we generated pre-clinical scale batches of the four lead constructs to evaluate their potency in a pre-clinical colorectal cancer xenograft mouse model. Post expansion our cell products expressed markers of self-renewing naïve-like and central memory phenotype and three out of four LGR5-targeting CAR-T cells were able to significantly inhibit the growth of human colorectal tumors. These results suggest a CAR-T targeting LGR5 may provide long-term protection against tumor growth. Our data positions LGR5-targeting CAR-T therapy as a viable therapeutic for human metastatic colorectal cancer types with minimal off-target effects, which we now aim to test in human clinical trials. Citation Format: Veronika Bandara, Stuart Mills, Emma Thompson, Lih Tan, Batjargal Gundsambuu, Silvana Napoli, Jade Foeng, Dylan McPeake, Justin Coombs, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon Barry. Pre-clinical validation of a LGR5-targeting CAR-T against colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5575.