CD19-targeted Chimeric Antigen Receptor Research Articles

Long-term survival outcomes in patients with relapsed/refractory large B-cell lymphoma after treatment of relma-cel: 4-year follow-up in RELIANCE study.

e19001 Background: RELIANCE study (NCT04089215) demonstrated the efficacy and safety of relma-cel, a CD19-targeted chimeric antigen receptor T-cell product, in patients with heavily pre-treated relapsed/refractory large B-cell lymphoma (r/r LBCL). We previously reported efficacy of relma-cel, noting objective response rate (ORR) in 77.59% of patients (CRR 53.45%), 2-year progression-free survival (PFS) rate and overall survival (OS) rate were 38.8% and 69.3%, respectively. Herein, we aimed to update the long-term survival results for 4-year follow-up (FU). Methods: Study design was previously published. Eligible patients were adults with histologically confirmed r/r LBCL and had failed at least two prior lines of therapy, including an anti-CD20 monoclonal antibody and an anthracycline-based regimen. Patients were enrolled at 10 clinical sites in China and underwent lymphodepletion prior to receiving relma-cel at a single dose of 100 × 106 (n=27) or 150 × 106 (n=32) CAR+ T cells. The primary endpoint was ORR at 3-month, as assessed by investigators. Secondary endpoints were duration of response (DOR), PFS, OS and safety profiles. For this abstract, we collected the survival status and updated the survival outcomes. Results: From Nov 2017 to Dec 2019, a total of 68 patients were enrolled, and 59 received relma-cel infusion. Among 58 evaluable efficacy patients with 2-year FU, 31 achieved CR (CR group), 14 achieved PR (PR group) and 13 had no response (Non-responder group). As of Dec 18, 2023, with 48.1 months (95% CI, 37.75 to 49.38) of median FU, 25 patients were still alive, 18 were died (5 deaths in CR group, 5 in PR group and 8 in Non-responder group), and 15 patients were censored due to lost to FU including 7 lost within 2 years and 8 after 2 years. The median OS were not reached (NR), and 1-year and 2-year OS rate were reported previously. The update estimated landmark OS rates were 69.3% at 3-year and 66.7% at 4-year. The OS rates of CR group at each year were numerically higher than those of PR group and Non-responder group (87.1% v.s. 59.3% v.s. 30.8% at 3-year and 82.5% v.s. 59.3% v.s. 30.8% at 4-year; Table). Conclusions: Four-year FU results in RELIANCE Study suggested that, although heavily pretreated, the patients who had response, especially who achieved CR, may experience greater survival benefits, from relma-cel therapy. Clinical trial information: NCT04089215 . [Table: see text]

Malnutrition and cachexia are associated with poor CAR T-cell therapy outcomes including survival

Background & AimsChimeric Antigen Receptor (CAR) T-cell therapy has emerged as a revolutionary treatment for patients with refractory or relapsed B-cell malignancies. However, a significant proportion of patients experience negative outcomes, including severe inflammatory toxicities and relapse. Cachexia and malnutrition are known secondary syndromes in many cancer patients, attributed to the effects of active malignancy, systemic inflammation, and cumulative treatment burden; however, further research is required to accurately characterise these issues in CAR T-cell patients. The aims of this service evaluation were to explore the changes in nutritional status (malnutrition and cachexia) in CAR T-cell therapy patients and the potential impact on patient outcomes including survival. Additionally, we describe the utilisation of dietetic resources in this specific patient population in a London tertiary referral centre. MethodsAdult haematology patients receiving licensed CD19-targeting CAR T-cell therapy at University College London Hospital between 01/04/19 and 01/09/21 were included. Data were collected from the time of treatment consent, and throughout admission to day of discharge: body weight (BW), C-reactive protein, albumin, lactate dehydrogenase, nutrition-risk screening scores (hospital-specific) and dietetic input. Clinical outcomes such as 12-month all-cause mortality, intensive care unit (ICU) admission, high-grade toxicities, and length of hospital stay (LoS) were also recorded. Cachexia and malnutrition were defined using the modified Glasgow Prognostic Score (mGPS) and Global Leadership Initiative on Malnutrition (GLIM) consensus, respectively. Results114 patients (55.6±15.1 years; 57% males) with B-cell non-Hodgkin’s lymphoma (n=109) and B-cell acute lymphoblastic leukaemia (n=5), receiving axicabtagene ciloleucel (n=89) and tisagenlecleucel (n=25) were included. Median LoS for treatment was 34 (27-38) days. Prior to treatment, 31.5% of patients developed malnutrition, with pre-cachexia/refractory cachexia (mGPS) identified in 43.6% of patients. This altered nutritional status pre-treatment was significantly associated with adverse patient outcomes post-infusion; mGPS was independently associated with inferior overall survival (HR=3.158, CI=1.36-7.323, p=0.007), with malnutrition and mGPS associated with increased LoS (p=0.037), sepsis (p=0.022) and ICU admission (p=0.039). During admission, patients experienced significant BW loss (-5.6% (-8.8 to -2.4); p=<0.001), with 68.4% developing malnutrition. Malnutrition screening during admission identified 57% patients at-risk, with 66.6% of patients referred to dietetics; however, there was a lack of malnutrition screening and dietetic referrals prior to treatment. ConclusionPre-treatment malnutrition and cachexia was significantly associated with adverse CAR T patient outcomes, including mGPS cachexia status independently associated with inferior overall survival. Further research in this novel space is essential to confirm the extent and impact of nutritional issues, to assist with implementing dietetic pathways, and to identify potential interventions with a view to optimising outcomes.

The immunoglobulin superfamily ligand B7H6 subjects T cell responses to NK cell surveillance.

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

Treatment of concomitant myasthenia gravis and Lambert-Eaton myasthenic syndrome with autologous CD19-targeted CAR T cells

Myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS) are autoimmune disorders affecting neuromuscular transmission. Their combined occurrence is rare, and treatment remains challenging. Two women diagnosed with concomitant MG/LEMS experienced severe, increasing disease activity despite multiple immunotherapies. Anti-CD19 chimeric antigen receptor (CAR) Tcells have shown promise for treating autoimmune diseases. This report details the safe application of anti-CD19 CAR Tcells for treating concomitant MG/LEMS. After CAR Tcell therapy, both patients experienced rapid clinical recovery and regained full mobility. Deep B cell depletion and normalization of acetylcholine receptor and voltage-gated calcium channel N-type autoantibody levels paralleled major neurological responses. Within 2months, both patients returned to everyday life, from wheelchair dependency to bicycling and mountain hiking, and remain stable at 6 and 4months post-CAR Tcell infusion, respectively. This report highlights the potential for anti-CD19 CAR Tcells to achieve profound clinical effects in the treatment of neuroimmunological diseases.

Patients with aggressive B-cell lymphoma receiving CAR T-cell therapy have a low rate of severe infections despite lack of universal antibacterial and antifungal prophylaxis.

Our aim was to describe the frequency and severity of infectious complications after chimeric antigen receptor (CAR) T-cell therapy in patients with large B-cell lymphoma (LBCL). We retrospectively reviewed clinical records of LBCL patients treated with CD19-targeted CAR T-cell therapy from July/2018 to December/2021 at our institution, and identified all infectious episodes from CAR T-cell infusion until disease progression, death or last follow-up. Overall, 137 patients were included. Thirty six percent had received ≥3 previous lines of therapy and 26% an autologous hematopoietic cell transplantation (auto-HCT). Cytokine release syndrome occurred in 87 (64%) patients. Antibacterial prophylaxis was not used in any patient; only 38% received antifungal prophylaxis. Sixty three infectious events were observed in 41 (30%) patients. Fifty two (83%) of the infectious events had at least one pathogen identified (bacteria [n = 38], virus [n = 11], and fungi [n = 3]). Most of the infectious events occurred during hospitalization for CAR-T treatment. Infection-related mortality was observed in two patients. Independent risk factors for infection included male gender, previous auto-HCT, ≥3 lines of treatment and pre-lymphodepletion neutropenia. Infections after CAR T-cell therapy in patients with lymphoma are frequent but generally not severe. A conservative and tailored antimicrobial prophylaxis seems to be a safe approach.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Oncotherapy resistance explained by Darwinian and Lamarckian models.

Cell and antibody therapies directed against surface molecules on B cells, e.g., CD19-targeting chimeric antigen receptor T cells (CD19 CAR-T), are now standard for patients with chemorefractory B cell acute lymphoblastic leukemias and other B cell malignancies. However, early relapse rates remain high. In this issue of the JCI, Aminov, Giricz, and colleagues revealed that leukemia cells resisting CD19-targeted therapy had reduced CD19 but also low CD22 expression and were sensitive to Bruton's tyrosine kinase and/or MEK inhibition. Overall, their observations support the evolution of resistance following a Lamarckian model: the oncotherapy directly elicits adaptive, resistance-conferring reconfigurations, which are then inherited by daughter cells as epigenetic changes. The findings prompt reflection also on the broader role of epigenetics in decoupling of replication from lineage differentiation activation by the B cell lineage master transcription factor hub. Such oncogenesis and resistance mechanisms, being predictable and epigenetic, offer practical opportunities for intervention, potentially non-cross-resistant and safe vis-à-vis present cytotoxic and CAR-T treatments.

IL-18-secreting multiantigen targeting CAR T cells eliminate antigen-low myeloma in an immunocompetent mouse model

AbstractMultiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T cells in leukemia and lymphoma, CAR T cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to weak expression of BCMA on myeloma cells, suggesting that novel approaches to better address this antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the proinflammatory cytokine interleukin-18 (IL-18) and multiantigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T cells targeting the myeloma-associated antigens BCMA and B-cell activating factor receptor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18–secreting CAR T cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type-I/II interferon signaling, and activated macrophages to mediate antimyeloma activity. Simultaneous targeting of weakly-expressed BCMA and BAFF-R with dual-CAR T cells enhanced T-cell:target-cell avidity, increased overall CAR signal strength, and stimulated antimyeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multiantigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

Abstract CT129: A phase 1 dose escalation study of a novel coupled CAR T cell therapy, GCC19CART, for patients with metastatic colorectal cancer

Abstract Background: GCC19CART, the first clinical candidate from the CoupledCAR solid tumor platform, is designed to overcome the limitations of conventional CAR T-cells in solid tumor malignancies by pairing solid tumor CAR T-cells with CD19-targeting CAR T-cells to amplify the proliferation and activation of the solid tumor CAR T component. GCC19CART targets guanylate cyclase-C (GCC), which is reported to be expressed in the metastatic lesions of 70%-80% of subjects with colorectal cancers and largely restricted to the intestinal tract. A Phase 1 investigator-initiated clinical trial conducted in China showed preliminary efficacy with an overall response rate (ORR) at dose level 1 (1x106 CAR T-cells/kg) of 15.4% ORR (2/13) and 50% ORR (4/8) in dose level 2 (2x106 CAR T-cells/kg). Common side effects included cytokine release syndrome (CRS) and diarrhea/colitis. Immune effector cell-associated neurotoxicity syndrome (ICANS) was seen in a smaller proportion of patients. A phase 1 study was initiated in the United States in August 2022 for patients with metastatic colorectal cancer refractory to standard of care chemotherapy. As of a data cutoff on ​November 1, 2023, 4 subjects have been treated in the initial dose level. Methods: Subjects were initially screened for GCC expression; this requirement was later discontinued when immunohistochemistry studies revealed that the frequency of positive GCC expression exceeded 95%. Eligible subjects underwent leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m2 and cyclophosphamide 300mg/m2) 3 days prior to infusion, and then administration of a single infusion of GCC19CART. Responses were assessed by RECIST v1.1 by site investigator review and an independent third-party imaging contract research organization. Results: 4 subjects have been dosed at level 1 (1x106 cells/kg) and completed the DLT period at the time of data cutoff. The most common adverse events were CRS in 4/4 subjects (Grade 1: 2/4 (50%) or Grade 2: 2/4 (50%)) and diarrhea in 3/4 subjects (Grade 1: 1/4 (25%), Grade 2: 1/4 (25%), or Grade 3: 1/4 (25%)). Three out of four patients had a mild tremor (Grade 1) that resolved with corticosteroids and no other evidence of neurotoxicity. The combined overall response rate (ORR) was 50% (2/4) by independent review. Two subjects demonstrated a partial response (PR) while 1 additional subject had partial metabolic response (PMR) on PET/CT with stable disease (SD) and 1 patient had progressive disease per RECIST 1.1. Per site investigator review, the ORR was 25%, with 1 confirmed PR and 3 SD assessed as best response. The median PFS was 3.8 months in the dose level 1 group. Conclusions: Preliminary results demonstrate that GCC19CART has meaningful clinical activity and an acceptable safety profile in refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented, including from patients treated at dose level 2 (2x106 CAR T-cells/kg). Clinical trial information: NCT05319314. Citation Format: Bridget P. Keenan, Christopher H. Lieu, Marwan Fakih, Dongqi Chen, Lucy Lu, Eric Rowinsky, Victor Lu, Lei Xiao, Alan P. Venook, Benjamin Schlechter. A phase 1 dose escalation study of a novel coupled CAR T cell therapy, GCC19CART, for patients with metastatic colorectal cancer [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 CT129.

CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis

Progressive multiple sclerosis (MS) is characterized by compartmentalized smoldering neuroinflammation caused by the proliferation of immune cells residing in the central nervous system (CNS),including B cells. Although inflammatory activity can be prevented by immunomodulatory therapies during early disease, such therapies typically fail to halt disease progression. CD19 chimeric antigen receptor (CAR)-T cell therapies have revolutionized the field of hematologic malignancies. Although generally considered efficacious, serious adverse events associated with CAR-T cell therapies such as immune effector cell-associated neurotoxicity syndrome (ICANS) have been observed. Successful use of CD19 CAR-T cells in rheumatic diseases like systemic lupus erythematosus and neuroimmunological diseases like myasthenia gravis have recently been observed, suggesting possible application in other autoimmune diseases. Here, we report the first individual treatment with a fully human CD19 CAR-T cell therapy (KYV-101) in two patients with progressive MS. CD19 CAR-T cell administration resulted in acceptable safety profiles for both patients. No ICANS was observed despite detection ofCD19 CAR-T cells in the cerebrospinal fluid. In case 1, intrathecal antibody production in the cerebrospinal fluid decreased notably after CAR-T cell infusion and was sustained through day 64. CD19 CAR-T cell administration in progressive MS resulted in an acceptable safety profile. CAR-T cell presence and expansion were observed in the cerebrospinal fluid without clinical signs of neurotoxicity, which, along with intrathecal antibody reduction, indicates expansion-dependent effects of CAR-T cells on CD19+ target cells in the CNS. Larger clinical studies assessing CD19 CAR-T cells in MS are warranted. Both individual treatments as well the generated data were not based on external funding.

Regulatory T cells expressing CD19-targeted chimeric antigen receptor restore homeostasis in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a progressive disease leading to immune-mediated tissue damage, associated with an alteration of lymphoid organs. Therapeutic strategies involving regulatory T (Treg) lymphocytes, which physiologically quench autoimmunity and support long-term immune tolerance, are considered, as conventional treatment often fails. We describe here a therapeutic strategy based on Tregs overexpressing FoxP3 and harboring anti-CD19 CAR (Fox19CAR-Tregs). Fox19CAR-Tregs efficiently suppress proliferation and activity of B cells in vitro, which are relevant for SLE pathogenesis. In an humanized mouse model of SLE, a single infusion of Fox19CAR-Tregs restricts autoantibody generation, delay lymphopenia (a key feature of SLE) and restore the human immune system composition in lymphoid organs, without detectable toxicity. Although a short survival, SLE target organs appear to be protected. In summary, Fox19CAR-Tregs can break the vicious cycle leading to autoimmunity and persistent tissue damage, representing an efficacious and safe strategy allowing restoration of homeostasis in SLE.

Abstract 47: Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy

Abstract Introduction: Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown substantial anti-tumor efficacy in hematological malignancies during recent years. As the field evolves to novel target epitopes, longer CAR structures are needed to reach membrane proximal epitopes for improved efficacy. Our approach to reach such epitopes is to have (non-functional) SIRPalpha based extracellular extension, spacer, to our CAR structure. We have shown, in this proof-of-concept study, that stability of the extracellular spacer of a CAR plays a pivotal role to improve in vivo anti-tumor efficacy. Various SIRPalpha-based CARs, FiCARs, targeting CD19 was studied for their function. Protein stability of the CD19 CAR structures were characterized and subsequently the killing efficacy of target cells and cytokine release in in vitro as well as in vivo anti-tumor efficacy was evaluated. Methods &amp; Results: In vitro co-culture assays with primary CD19 CAR T and CD19 positive cancer cells had shown no significant differences between the different spacer containing FiCAR structures. A dose dependent killing efficacy was observed in all the tested structures with up to &amp;gt;90% killing of NALM6 and Raji cells. Comparable results for IL-2, IFN-gamma, TNF-alpha, IL-4, IL-13 and granzyme B response were seen in co-cultured assay. In the protein stability studies, modifications in the extracellular part of the CAR structure led to an increase in thermal stability (over 2 °C) and reduced/halted multimer formation. To study the FiCAR T cell function in an in vivo tumor model, NSG mice were injected i.v with Raji cells and few days later mice were injected i.v with FiCAR T cells. Anti-tumor efficacy and overall survival were studied at the end of the study (60 days), the enhanced stability of FiCAR T structure translated into improved anti-tumor efficacy and survival. Interestingly, none of the mice survived in the least stable structure, 40% survival with intermediate stability structures and over 70% survival was observed in most stable structures. Conclusion: A stabilized SIRPalpha-based CD19 FiCARs dramatically enhance anti-tumor efficacy and survival in Raji tumor model. The stabilization is critical for improved efficacy, especially in the in vivo setting. This data suggests that, in addition to the length of the CARs stability of the CAR structure appear to play a crucial role in the efficacy of the CAR T cells. The modifications presented here enhanced CD19-targeting CAR T cell efficacy. This approach requires further investigation to improve the efficacy of currently approved CAR T cells, especially CD19-CAR T cells. Citation Format: Henrik Paavilainen, Jan Koski, Veera Nikoskelainen, Manar Elmadani, Endrit Elbasani, Can Hekim, Emmi-Leena Ihantola, Virginie Kergourlay, Pirjo Vehmaan-kreula, Marie Nyman, Kiira Kalke, Markus Nurmi, Hector Monzo, Paivi Ojala, Anu Autio, Matti Korhonen, Anil Thotakura. Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy [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 47.

Abstract 1335: Generating functional CAR-NK cells for cancer immunotherapy

Abstract Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells are rapidly emerging as a promising cellular therapeutic overcoming the potential life-threatening adverse events that may result from CAR-T therapy, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Due to their intrinsic characteristics, NK cells can kill tumor cells without the requirement for recognition of antigen in the context of MHC. This removes the constraint of HLA matching and supports antigen independent cytotoxicity through NK cell intrinsic mechanisms. As such CAR-NK cells kill via both CAR-dependent and -independent mechanisms. For these reasons, they offer the advantage of being an “off-the-shelf” product; satisfying the requirement for large-scale production of cancer immunotherapies, with the potential to reach more patients at a reduced cost. The frequency of NK cells in the blood is low (~ 2-5%), therefore improving their in vitro expansion is key to generating sufficient material to offer a viable CAR-NK cell therapy. Here we outline an in-vitro method to generate, expand, and test primary human CAR-NK cells using engineered feeder cells, and go on to demonstrate the efficacy of the CAR-NK against target cancer cells. Primary human NK cells were cultured and expanded in the presence of irradiated K562 cells (iK562) modified to express 4-1BBL and membrane bound IL-21. Following initial expansion, NK cells were transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV; as an empty vector transduced control. Following transduction, CD19 CAR-NK and GFP-NK were enriched by FACS sorting to reach &amp;gt;80% purity and further expanded in the presence of 4-1BBL/mbIL21 iK562 feeder cells. Following sufficient expansion, cytotoxicity of fresh or cryopreserved CD19 CAR-NK cells was tested in vitro in a Tumor Killing Assay (TKA) against CD19+ or CD19- target cells. As expected, CD19 CAR-NK cells showed increased on-target cytotoxicity, increased degranulation and IFNꝩ secretion compared to controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting CD19 CAR engagement of the target antigen to mediate killing. Taken together these data show the generation, expansion, and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This approach provides a platform for in vitro characterization of novel CAR-constructs, allied with in-vitro safety assessment and in vivo pharmacology testing. Together, this offers the potential to further develop new and more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Henry Leonard, Dan Rocca, Sabrina de Munnik, Gemma Moiset, Robert Nunan, Lauren Schewitz-Bowers, Louise Brackenbury. Generating functional CAR-NK cells for cancer immunotherapy [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 1335.

Abstract 3610: Utilizing fructose metabolism to fuel anti-tumor immunity

Abstract Consumption of fructose in the form of high fructose corn syrup is elevated in the western diet, but it is not fully understood how fructose metabolism influences anti-tumor immunity. Fructose is canonically metabolized in the intestine and the liver and can shunt into glycolysis. In certain tumor types, fructose metabolism becomes deregulated, allowing tumor cells to metabolize fructose to power their biosynthetic processes. Similarly, reprogramming immune cells to be able to efficiently metabolize fructose may help us power immunotherapy to work in difficult to treat cancers, especially in individuals who have high fructose in their blood or in the tumor microenvironment (TME). Imparting effector T cells with the ability to use fructose for fuel can overcome one of the major metabolic limitations of the TME: limited nutrient supply. As T cells become activated, they alter their metabolism to support growth and proliferation. Activated CD8+ T cells upregulate their glycolysis which results in increased biosynthetic processes, such as synthesis of macromolecules and effector cytokines. However, tumor cells also consume substantial amounts of glucose, resulting in a glucose-depleted microenvironment, which could be one of the major barriers to immunotherapy working in solid tumors. Therefore, we engineered T cells to overexpress a fructose transporter, GLUT5, which is normally not expressed in the immune compartment at high levels. We hypothesized that increased fructose uptake by CD8+ T cells will ameliorate exhaustion caused by the glucose-low tumor microenvironment. Indeed, we determined that GLUT5 T cells are more efficient in killing in low glucose supplemented with fructose. We also observed that GLUT5 expression restores T cell glycolytic capacity under fructose to that of unexhausted T cells under glucose replete conditions. Similarly, GLUT5-expressing T cells proliferate faster in vitro and exhibit an improved ability to control B16 tumor growth in vivo. Additionally, combining GLUT5 cell infusion with checkpoint inhibition resulted in durable tumor control and prolonged mouse survival. Further, engineered CD19-targeted CAR T cells with expression of GLUT5 were more metabolically active and cytotoxic in low glucose conditions, mimicking the TME. In conclusion, GLUT5 expression in effector CD8+ and CAR T cells may allow immunotherapy to function in solid tumors under low glucose conditions. Citation Format: Tanya Schild, Rachel Chirayil, Lyric Haughton, Patrick Wallisch, Marjan Berishaj, David A. Scheinberg, Kayvan R. Keshari. Utilizing fructose metabolism to fuel anti-tumor immunity [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 3610.

Dynamic monitoring of circulating tumor DNA reveals outcomes and genomic alterations in patients with relapsed or refractory large B-cell lymphoma undergoing CAR T-cell therapy

BackgroundOver 50% of patients with relapsed or refractory large B-cell lymphoma (r/r LBCL) receiving CD19-targeted chimeric antigen receptor (CAR19) T-cell therapy fail to achieve durable remission. Early identification of relapse...

Transient acute kidney injury after chimeric antigen receptor T-cell therapy in patients with hematological malignancies.

Acute kidney injury (AKI) occurs in 30% of patients infused with chimeric antigen receptor (CAR) T-cells. The purpose of this study was to identify risk factors and long-term outcomes after AKI in patients who received CAR T-cell therapy. Medical records of 115 adult patients with R/R hematological malignancies treated with CD19-targeted CAR T-cells at Vall d'Hebron University Hospital between July 2018 and May 2021. Baseline demographic data including age, gender, ethnicity, body mass index (BMI), and co-morbidities, as well as the type of hematological neoplasia and prior lines of therapy were collected. Laboratory parameters including serum creatinine and whole blood hemoglobin were retrospectively reviewed and values were gathered for days +1, +7, +14, +21, and +28 post-infusion. A total of 24/115 (21%) patients developed AKI related to CAR T-cell therapy; 6/24 with AKI over chronic kidney disease (CKD). Two patients had AKI in the context of lymphodepleting (LD) chemotherapy and the other 22 after CAR T-cell infusion, starting at day+1 in 3 patients, day+7 in 13 patients, day +14 in 1 patient, day+21 in 2 patients, and day+28 in 3 patients. Renal function was recovered in 19/24 (79%) patients within the first month after infusion. Male gender, CKD, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) were associated with AKI. Male gender, CKD, ICANS grade ≥3 and CRS grade ≥2 were identified as independent risk factors for AKI on multivariable analysis. In terms of the most frequent CAR T-cell related complications, CRS was observed in 95 (82%) patients and ICANS in 33 (29%) patients. Steroids were required in 34 (30%) patients and tocilizumab in 37 (32%) patients. Six (5%) patients were admitted to the intensive care unit (1 for septic shock, 4 for CRS grade ≥2 associated to ICANS grade ≥2, and 1 for CRS grade ≥3). A total of 5 (4.4%) patients died in the first 30 days after CAR T-cell infusion for reasons other than disease progression, including 4 cases of infectious complications and 1 of heart failure. Our results suggest that AKI is a frequent but mild adverse event, with fast recovery in most patients.

Streamlined measurement of chimeric antigen receptor T-cell concentration, size, viability and two-color phenotyping during manufacturing

Background aimsThe successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype). MethodsIn this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer). ResultsOur results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression. ConclusionsThese measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples.

Treatment of secondary CNS lymphoma using CD19-targeted chimeric antigen receptor (CAR) T cells

BackgroundAggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported.MethodsWe prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity.ResultsTwo of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient’s CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells.ConclusionsOur results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of “fit” and highly tumor-reactive CAR-T with low off-target reactivity into the CNS.

Immunometabolic Adaptation of CD19-Targeted CAR T Cells in the Central Nervous System Microenvironment of Patients Promotes Memory Development.

The spatiotemporal immunometabolic landscape of CD19-targeted CAR T cells from patients reveals metabolic adaptations in specific microenvironments that can be exploited to maximize the therapeutic efficacy of CAR T cells.

Early evaluation of CAR-T cell therapy response in R/R DLBCL patients using 18F-FDG PET/CT.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy provides a durable response in patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). The role of fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for early evaluation of response in patients with that immunotherapy was evaluated. Three separate 18F-FDG PET/CT examinations of 53 patients (29 males, 24 females; median 62 years old) with R/R DLBCL were conducted; before bridging therapy [time of decision (TD)], before CAR-T (tisagenlecleucel, n=37; lisocabtagenemaraleucel, n=16) infusion [time of CAR-T infusion (IT)], and one month (M1) after CAR-T infusion. Response was evaluated based on the Deauville 5-point scale and Lugano criteria. Among 21 patients (39.6%) with complete metabolic response (CMR) at IT-PET, 20 were able to continue CMR, while one showed progression at M1-PET. Among 32 patients (60.4%) with non-CMR at IT-PET, 12, 8, 4, and 8 showed CMR, partial metabolic response (PMR), (non-metabolic response (NMR), and progressive metabolic disease (PMD), respectively, at M1-PET as compared with IT-PET. Evaluations of M1-PET as compared with baseline TD-PET indicated 32, 7, 5, and 9 patients with CMR, PMR, NMR, and PMD, respectively. After a median 10.1 months, 26 patients showed progression and 13 had died from DLBCL. The 32 who achieved CMR showed significantly longer progression-free (P<0.0001) and overall survival (P<0.0001) periods as compared to the 21 non-CMR patients. Fluorine-18-FDG PET/CT findings obtained one month after CAR-T cell therapy showed accuracy for early response evaluation and prediction of progression in patients with R/R DLBCL.