Techniques to study chimerism at the tissue level in humanized mice.

Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

B-cell maturation antigen chimeric antigen receptor T-cell re-expansion in a patient with myeloma following salvage programmed cell death protein 1 inhibitor-based combination therapy.

CD84: A Novel Target for CAR T-Cell Therapy for Acute Myeloid Leukemia

A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [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 LB070.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Chimeric antigen receptor (CAR) T-cell therapy represents a revolutionary treatment for hematological malignancies. However, improvements in CAR T-cell therapies are urgently needed since CAR T cell application is associated with toxicities, exhaustion, immune suppression, lack of long-term persistence, and low CAR T-cell tumor infiltration. Major efforts to overcome these hurdles are currently on the way. Incrementally improved xenograft mouse models, supporting the engraftment and development of a human hemato-lymphoid system and tumor tissue, represent an important fundamental and preclinical research tool. We will focus here on several CAR T and CAR NK therapies that have benefited from evaluation in humanized mice. These models are of great value for the cancer therapy field as they provide a more reliable understanding of sometimes complicated therapeutic interventions. Additionally, they are considered the gold standard with regard to assessment of new CAR technologies in vivo for safety, efficacy, immune response, design, combination therapies, exhaustion, persistence, and mechanism of action prior to starting a clinical trial. They help to expedite the critical translation from proof-of-concept to clinical CAR T-cell application. In this review, we discuss innovative developments in the CAR T-cell therapy field that benefited from evaluation in humanized mice, illustrated by multiple examples.

Introduction: Chimeric antigen receptor (CAR) T cell immunotherapy has had only modest success in solid malignancies due in part to tumor antigen heterogeneity and the immunosuppressive tumor microenvironment. We have previously demonstrated that administration of cyclophosphamide (CTX) prior to CAR T infusion promoted the eradication of solid tumors that lacked homogenous expression of the CAR target antigen. Although CTX has multiple effects, we hypothesized that one potential mechanism was depletion of T-regulatory cells (Treg). To test this, we used a genetic model of Treg depletion involving mice with FOXP3-driven diphtheria toxin receptor expression (FOXP3-DTR) and an orally administered chemokine receptor 4 (CCR4) antagonist called CCR4-351. Treg express CCR4 and migrate toward CCL17 and CCL22 which are frequently upregulated by tumors. We have previously shown that the tool compound CCR4-351 decreases Treg accumulation in tumors. Methods: Transgenic FOXP3-DTR C57BL/6 mice were injected s.c. in the flank with a syngeneic murine mesothelioma cell line expressing human mesothelin. When tumors reached ~85 mm3, diphtheria toxin was given i.p. and 24 hours later, 2 doses of 107 transduced human anti-mesothelin CAR T cells (M11) were injected i.v. 2 days apart. In our pharmacologic study, wild type, tumor-bearing C57BL/6 mice were given CCR4-351 daily at 50 mg/kg by oral gavage. Mice were treated with M11 CAR T cells as above, starting one day after CCR4-351 therapy. Tumor growth and mouse survival were determined. Results: Genetic depletion of Treg alone did not significantly slow tumor growth compared to untreated control. However, the combination of M11 CAR T cells plus genetic depletion of Treg significantly slowed tumor growth compared to vehicle control (p<0.05, one-way ANOVA). Compared to untreated controls, treatment with CCR4-351 (p<0.04, log rank) or M11 CAR T cells (p<0.001, log rank) alone significantly improved survival, however there was no significant difference in survival between CAR and CCR4-351 only treated mice. In contrast, the combination of M11 CAR T cells with CCR4-351 resulted in enhanced antitumor activity with 4/9 mice cured and significantly increased survival compared to CAR (p<0.01, log rank) and CCR4-351 (p<0.006, log rank) monotherapies. Conclusions: Treg depletion via systemic ablation or inhibition of trafficking, resulted in augmentation of the antitumor effects of CAR T cells in our pre-clinical immunocompetent solid tumor model. If generalizable, these data support further investigation as to whether the orally bioavailable CCR4 antagonist currently in clinical development, FLX475, may be used to augment human CAR T cell therapy for solid tumors. Citation Format: Michael S. Leibowitz, Nicholas S. Olimpo, Liqing Wang, Aparna Jorapur, Deepa Pookot, Maria Liousia, Evguenia Arguiri, Jing Sun, Astero Klampatsa, Dirk G. Brockstedt, Wayne W. Hancock, Steven M. Albelda. T-regulatory cells impair CAR T cell-mediated antitumor activity in a murine solid tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1585.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

2539 Background: CAR-T cell therapy has been curative for many patients with refractory, progressive hematologic cancers, resulting in several FDA approvals. However, this therapy has not been successful for solid cancers, reflecting the need for suitable antigen targets for each disease and solutions to immunological barriers in solid tumors. Here, we have identified the desmosomal cadherin, desmoglein 2 (DSG2), as an effective CAR-T cell therapy target in epithelia-derived solid tumors. DSG2 contributes to cell proliferation, migration, and other emerging tumor-promoting pathways, resulting in its upregulation in nearly all solid cancers and correlating with poor prognosis. Moreover, we explored CRISPR-Cas9-mediated elimination of the inhibitory receptor CD5 to enhance in vivo CAR-T cell expansion and solid tumor efficacy. Methods: DSG2-directed CAR-T cells were generated from human T cells using a scFv derived from a murine hybridoma targeting the extracellular domain of DSG2 in a 3 rd generation CAR design with CD28, 4-1BB, and CD3ζ signaling domains. CD5 elimination employed electroporation of complexed gRNA-Cas9 ribonucleoprotein (RNP). DSG2 expression was characterized in human cancers and cell lines and CAR-T cell activity was examined in vitro by cytokine production and target cell cytolysis. In vivo efficacy studies employed cancer xenografts in NSG mice treated with CAR-T cells. Safety studies employed a human DSG2 transgenic mouse treated with syngeneic murine CAR-T cells for clinical, serum biomarkers, and histopathological evaluation. Results: In vitro studies revealed recognition and lysis of solid cancer cell lines and effector cytokine production. Administration of DSG2-directed CAR-T cells eliminated metastatic cell-derived xenografts, patient-derived xenografts, and orthotopic tumors derived from various solid cancers, including colorectal, pancreatic, lung, prostate, breast, and liver. Moreover, elimination of CD5 enhanced the expansion of DSG2-directed CAR-T cells in vivo, resulting in curative efficacy at sub-therapeutic CAR-T cell doses. Safety studies revealed no toxicity in any human DSG2 transgenic mouse tissues. Conclusions: These studies reveal the robust antitumor activity of DSG2-directed CAR-T cells in solid tumors, which is enhanced by CD5 deletion, without toxicity in a human transgenic mouse model. Thus, CD5-deleted DSG2-directed CAR-T cells are a promising therapeutic approach that may be safe and effective for all solid cancers.

Novel CAR T Modules (Tmod) to Target HLA-Class I Loss in Lymphoma

Introduction: Despite significant progress in the chimeric antigen receptor (CAR)-T cell therapy of B cell malignancies, the 5-year progression-free survival rate in large B cell lymphoma following CD19-targeting CAR-T cell treatments remains below 50%. While some treatment failures were attributed to CD19 antigen loss, immunogenicity of murine single-chain fragment variable (scFv) elements has been implicated in relapse and resistance to CAR-T cell re-treatment. In this study, we evaluated fully human CD20 and CD19 dual-targeting CAR constructs (h20.19 CARs) combining human scFvs and distinct co-stimulatory domains and architectures, to mitigate tumor antigen escape, reduce immunogenicity, and improve CAR functionality. Methods: The h20.19 CAR constructs were assembled from fully human scFvs against lymphoma antigens CD19 and CD20, with hinge, transmembrane and co-stimulatory domains derived from 4-1BB, CD28, OX40 or ICOS. Tandem second generation (T8/8BBz), tandem third generation (T28/28BBz, T8/28BBz), and bicistronic (D28z/BBz, DBBz/28z, DOXz/ICz) CARs were evaluated. CAR T cells were derived from healthy donors’ CD4+ and CD8+ T cells by lentiviral transduction, and CAR expression was evaluated by flow cytometry. CAR-mediated target cell killing was assessed by co-culture assays, and cytokine elaboration by ELISA. In vivo CAR-T cell function was evaluated in a Raji lymphoma NSG xenograft model using low CAR-T dose (2x106/mouse). Tumor-free survivors were rechallenged with Raji cells to compare CAR-T cell potency and persistence. Results: The hCD20.19 CAR-T cells lysed CD19+CD20+ tumor lines Raji, NALM-6 and REH, and single-antigen lines 293T CD19 and 293T CD20, but not the parental CD19-CD20- 293T cells, indicating antigen-specific efficacy. Cytokines TNFα, IL2 and IFNγ were strongly upregulated in response to Raji cells. During repeat co-culture with Raji cells, the third-generation tandem hCD20.19 CARs and the bicistronic CARs showed greater cytotoxicity than the second-generation CAR T8/8BBz. In vivo, all third-generation tandem and bicistronic CARs induced rapid and durable Raji tumor rejection by study day 7 post CAR-T, with no apparent toxicity. Upon long rest and rechallenge with Raji cells on study day 50, all h20.19 CARs attenuated tumor progression as compared to age-matched untreated mice cohort. CARs T8/28BBz and D28z/BBz afforded the least protection from Raji rechallenge, whereas CAR DOXz/ICz mediated rapid tumor rejection, but mice met euthanasia criteria by study day 71 due to weight loss. The tandem CAR T28/28BBz and bicistronic CAR DBBz/28z, demonstrated tumor rejection, persistence and safety after rechallenge, up to study day 86. Conclusions: In summary, the fully human dual-targeting h20.19 CARs exhibit robust anti-tumor function and persistence in vitro and in vivo, mediate long-term remissions and resist tumor rechallenge. Citation Format: Peirong Hu, Brittany Steimle, Ngoc Tran, Pradyot Dash, Dina Schneider. Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3198.

Tocilizumab is commonly used for the management of chimeric antigen receptor (CAR) T-cell therapy-associated cytokine release syndrome (CRS). However, it remains unknown whether tocilizumab or its dosage affects the efficacy and safety of CAR T-cell therapy. The objective of this multicenter retrospective study was to explore the impact of tocilizumab on CAR T-cell therapy. In total, 93 patients with B-cell acute lymphoblastic leukemia (B-ALL) receiving humanized anti-CD19 CAR T cells were recruited from May 2016 to November 2022. Forty-five patients received tocilizumab (tocilizumab group), whereas 48 patients did not (nontocilizumab group). Thirteen patients received >1 dose of tocilizumab. The primary end point was the effect of tocilizumab on the efficacy and safety of CAR T cells. Additionally, proliferation, killing, and cytokine assays of CAR T cells were performed in vitro in the presence of tocilizumab. The median age of the patients was 33 years, with 47 males and 46 females. Patients in the tocilizumab group showed similar complete response (CR) rate, overall survival (OS), and event-free survival (EFS) compared with the nontocilizumab group. Compared with patients who received ≤1 dose of tocilizumab, receiving >1 dose of tocilizumab did not affect their CR rate, OS, or EFS. In the tocilizumab group, all patients experienced CRS and 26.7% experienced immuneeffector cell-associated neurotoxicity syndrome (ICANS). In the nontocilizumab group, 64.6% of patients experienced CRS and 8.3% experienced ICANS. Up to 75% of ICANS and 87.5% of grade ≥3 ICANS occurred in the tocilizumab group. In vitro, tocilizumab did not impair the proliferation and killing effects of CAR T cells. Tocilizumab does not affect the efficacy of CAR T cells but may increase the likelihood of ICANS.

BackgroundAlthough most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their...

Chimeric Antigen Receptor (CAR) T cell therapy has achieved high response rates in patients with relapsed or refractory hematologic malignancies. However, comparable efficacy in solid tumors remains limited, partly due to poor CAR T cell persistence and immune-mediated rejection. A major contributor, which has hampered the clinical efficacy of CAR T cells in clinical practice, is the immunogenicity of the murine-derived single-chain variable fragments (scFvs) commonly used in CAR constructs. Cell and humoral immune responses to the murine parts of CARs have been implicated in CAR T cell rejection. Here, we describe the generation and in vitro characterization of humanized CAR T cells targeting prostate-specific membrane antigen (PSMA) on prostate cancer cells, based on two distinct murine scFvs (A5 and D7). Humanization improved the germinality index and successfully preserved CAR surface expression. Functional assays demonstrated that humanized PSMA-CAR T cells retained antigen-specific binding, activation and cytotoxicity, differentiation, exhaustion and cytokine secretion profiles comparable to their murine counterparts. These results support the feasibility of humanization as a strategy to reduce immunogenicity without compromising CAR T cell capabilities, providing a foundation for further in vivo validation in solid tumor settings.

Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated >50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, >10 IgGs were identified that met the criteria for affinity and selectivity and had a >50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of >95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed >80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979