CD19-directed Chimeric Antigen Receptor T-cell Research Articles

Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Is There Still a Role for Autologous Stem Cell Transplantation in the CAR T-Cell Era?

Recently, CD19-directed chimeric antigen receptor (CAR) T-cell therapies have revolutionized treatment strategies for diffuse large B-cell lymphoma (DLBCL). CAR T-cell therapy is increasingly used as a second-line therapy for patients with DLBCL with early relapse or refractoriness to initial chemoimmunotherapy and displaced high-dose chemotherapy, followed by autologous stem cell transplantation (ASCT) as the standard of care for these patients. However, patients with late relapse or chemosensitive disease still benefit from autologous stem cell transplantation. We will review practice-changing studies in early relapse (ZUMA-7 and TRANSFORM) under consideration of the negative BELINDA trial, with a focus on register data, comparing CAR T-cell therapy and ASCT for patients responding to salvage therapy.

Circulating Tumor DNA as a Complementary Prognostic Biomarker during CAR-T Therapy in B-Cell Non-Hodgkin Lymphomas.

The emergence of CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment paradigm for R/R B-cell NHLs. However, challenges persist in accurately evaluating treatment response and detecting early relapse, necessitating the exploration of novel biomarkers. Circulating tumor DNA (ctDNA) via liquid biopsy is a non-invasive tool for monitoring therapy efficacy and predicting treatment outcomes in B-NHL following CAR-T therapy. By overcoming the limitations of conventional imaging modalities, ctDNA assessments offer valuable insights into response dynamics, molecular mechanisms of resistance, and early detection of molecular relapse. Integration of ctDNA monitoring into clinical practice holds promise for personalized therapeutic strategies, guiding the development of novel targeted therapies, and enhancing patient outcomes. However, standardization of assay methodologies and consensus on clinical response metrics are imperative to unlock the full potential of ctDNA in the management of B-NHL. Prospective validation of ctDNA in clinical trials is necessary to establish its role as a complementary decision aid.

Sequencing of anti-CD19 therapies in the management of diffuse large B-cell lymphoma.

Several second- and third-line immunotherapeutic options for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) ineligible for autologous stem cell transplant are directed against the B-cell antigen CD19. The anti-CD19 monoclonal antibody tafasitamab, paired with the immunomodulator lenalidomide, mediates antibody-dependent cellular toxicity and cellular phagocytosis; the antibody-drug conjugate loncastuximab tesirine delivers the DNA-cross-linking agent tesirine via CD19 binding and internalization; and CD19-directed chimeric antigen receptor T-cell therapy (CAR-T) products are engineered from autologous T cells. While CD19 expression is assessed at diagnosis, clinically relevant thresholds of CD19 expression-which may not be detectable with current routine methodologies-have not been defined and may vary between CD19-directed treatment modalities. Determining optimal treatment sequencing strategies with CD19-directed therapy has been hampered by the exclusion of patients with prior CD19-directed therapies from major clinical trials. Antigen escape, attributed to mechanisms including epitope loss and defective cell-surface trafficking of CD19, is an important cause of CAR-T failure. Limited data suggest that CD19 expression may be maintained after non-CAR-T CD19-directed therapy and retrospective analyses indicate that some patients with disease that relapses after CAR-T may benefit from subsequent CD19-directed therapy. To date, clinical evidence on the effect of anti-CD19 therapy prior to CAR-T is restricted to small case series. Prospective studies and detailed analyses to understand how pre- and post-treatment CD19 expression correlates with clinical responses to subsequent CD19-directed therapy are needed to fully maximize treatment strategies.

Reducing and controlling metabolic active tumor volume prior to CAR T-cell infusion can improve survival outcomes in patients with large B-cell lymphoma

Bridging therapy before CD19-directed chimeric antigen receptor (CAR) T-cell infusion is frequently applied in patients with relapsed or refractory Large B-cell lymphoma (r/r LBCL). This study aimed to assess the influence of quantified MATV and MATV-dynamics, between pre-apheresis (baseline) and pre-lymphodepleting chemotherapy (pre-LD) MATV, on CAR T-cell outcomes and toxicities in patients with r/r LBCL. MATVs were calculated semi-automatically at baseline (n = 74) and pre-LD (n = 68) in patients with r/r LBCL who received axicabtagene ciloleucel. At baseline, patients with a low MATV (< 190 cc) had a better time to progression (TTP) and overall survival (OS) compared to high MATV patients (p < 0.001). High MATV patients who remained stable or reduced upon bridging therapy showed a significant improvement in TTP (p = 0.041) and OS (p = 0.015), compared to patients with a high pre-LD MATV (> 480 cc). Furthermore, high MATV baseline was associated with severe cytokine release syndrome (CRS, p = 0.001). In conclusion, patients with low baseline MATV had the best TTP/OS and effective reduction or controlling MATV during bridging improved survival outcomes in patients with a high baseline MATV, providing rationale for the use of more aggressive bridging regimens.

Bendamustine as Lymphodepletion for Brexucabtagene Autoleucel Therapy of Mantle Cell Lymphoma

Brexucabtagene autoleucel (brexu-cel) is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for treatment of relapsed/refractory mantle cell lymphoma (MCL). During a fludarabine shortage, we used bendamustine as an alternative to standard cyclophosphamide/fludarabine (cy/flu) lymphodepletion (LD) prior to brexu-cel. We assessed MCL patient outcomes as well as CAR T-cell expansion and persistence after brexu-cel following bendamustine or cy/flu LD at our center. This was a retrospective single institution study that utilized prospectively banked blood and tissue samples. Clinical efficacy was assessed by 2014 Lugano guidelines. CAR T-cell expansion and persistence in peripheral blood were assessed on day 7 and at ≥month 6 for patients with available samples. Seventeen patients received bendamustine and 5 received cy/flu. For the bendamustine cohort, 14 (82%) received bridging therapy and 4 (24%) had CNS involvement. Fifteen patients (88%) developed CRS with 4 (24%) ≥grade 3 events. Six (35%) patients developed ICANS with 4 (24%) events ≥grade 3. No patient had ≥grade 3 cytopenias at day 90. Best objective (BOR) and complete response (CRR) rates were 82% and 65%, respectively. At 24.5 months median follow-up, 12-month progression-free survival (PFS) was 45%, 24-month PFS was 25%, and median duration of response was 19 months. Median OS was not reached. BOR was 25% (1/4) for patients with CNS involvement. CAR transgene expansion after bendamustine LD was observed on day 7 in all (4/4) patients tested and persisted at ≥6 months (2/2), regardless of response. Bendamustine LD before brexu-cel for MCL is feasible and safe with a lower frequency and shorter duration of cytopenias than reported for cy/flu. Both CAR T-cell expansion and persistence were observed after bendamustine LD. Outcomes appear comparable to the real world outcomes reported with cy/flu LD.

Reinfusion of CD19 CAR T cells for relapse prevention and treatment in children with acute lymphoblastic leukemia

AbstractRelapse after CD19-directed chimeric antigen receptor (CAR)–modified T cells remains a substantial challenge. Short CAR T-cell persistence contributes to relapse risk, necessitating novel approaches to prolong durability. CAR T-cell reinfusion (CARTr) represents a potential strategy to reduce the risk of or treat relapsed disease after initial CAR T-cell infusion (CARTi). We conducted a retrospective review of reinfusion of murine (CTL019) or humanized (huCART19) anti–CD19/4-1BB CAR T cells across 3 clinical trials or commercial tisagenlecleucel for relapse prevention (peripheral B-cell recovery [BCR] or marrow hematogones ≤6 months after CARTi), minimal residual disease (MRD) or relapse, or nonresponse to CARTi. The primary endpoint was complete response (CR) at day 28 after CARTr, defined as complete remission with B-cell aplasia. Of 262 primary treatments, 81 were followed by ≥1 reinfusion (investigational CTL019, n = 44; huCART19, n = 26; tisagenlecleucel, n = 11), representing 79 patients. Of 63 reinfusions for relapse prevention, 52% achieved CR (BCR, 15/40 [38%]; hematogones, 18/23 [78%]). Lymphodepletion was associated with response to CARTr for BCR (odds ratio [OR], 33.57; P = .015) but not hematogones (OR, 0.30; P = .291). The cumulative incidence of relapse was 29% at 24 months for CR vs 61% for nonresponse to CARTr (P = .259). For MRD/relapse, CR rate to CARTr was 50% (5/10), but 0/8 for nonresponse to CARTi. Toxicity was generally mild, with the only grade ≥3 cytokine release syndrome (n = 6) or neurotoxicity (n = 1) observed in MRD/relapse treatment. Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may reduce relapse risk in a subset of patients, and can reinduce remission in CD19+ relapse.

Monocytes in leukapheresis products affect the outcome of CD19–targeted CAR T-cell therapy in patients with lymphoma

AbstractCD19–directed chimeric antigen receptor (CAR) T cells can induce durable remissions in relapsed/refractory large B-cell lymphomas (R/R LBCLs), but 60% of patients do not respond or relapse. Biological mechanisms explaining lack of response are emerging, but they are largely unsuccessful in predicting disease response at the patient level. Additionally, to maximize the cost-effectiveness of CAR T-cell therapy, biomarkers able to predict response and survival before CAR T-cell manufacturing would be desirable. We performed transcriptomic and functional evaluations of leukapheresis products in 95 patients with R/R LBCL enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel and axicabtagene ciloleucel. A signature composed of 4 myeloid genes expressed by T cells isolated from leukapheresis products is able to identify patients with a very short progression-free survival (PFS), highlighting the impact of monocytes in CAR T-cell therapy response. Accordingly, response and PFS were also negatively influenced by high circulating absolute monocyte counts at the time of leukapheresis. The combined evaluation of peripheral blood monocytes at the time of leukapheresis and the 4-gene signature represents a novel tool to identify patients with R/R LBCL at very high risk of progression after CAR T-cell therapy and could be used to plan trials evaluating CAR T cells vs other novel treatments or allogeneic CAR T cells. However, it also highlights the need to incorporate monocyte depletion strategies for better CAR T production.

Transforming CLL Management with Immunotherapy: Investigating the Potential of CAR T-Cells and Bispecific Antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in three multiply relapsed CLL patients, with two of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence, and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

Determinants of outcomes and advances in CD19-directed chimeric antigen receptor therapy for B-cell acute lymphoblastic leukemia.

Relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive B-cell neoplasm associated with poor outcomes. Conventional multiagent chemotherapy and bispecific antibody therapy may induce remission; however, relapse rates remain high and overall survival is poor. Chimeric antigen receptor T-cell (CAR-T) therapy provides durable, deep complete remission, and long-term cures in relapsed and refractory B-ALL. However, with this new treatment modality, 10%-30% of patients do not achieve remission, and over 50% experience relapse after therapy. Currently, there are two approved CD19-specific CAR-T cell constructs in B-ALL, Tisagenlecleucel and Brexucabtagene Autoleucel by the United States Food and Drug Administration, and the European Medicines Agency (EMA). In this review, we discuss patients, disease, and CAR-T predictors of outcomes in B-ALL. We describe the two approved CD19-directed CAR-T cell products, review the current literature, and discuss factors associated with high risks of therapy failure and future direction in CAR-T cell therapy for B-ALL.

Infections in children following chimeric antigen receptor T-cell therapy for B-cell acute lymphoblastic leukemia.

CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy is transforming care for pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). There are limited pediatric-specific data concerning the infection risks associated with CD19 CAR-T therapy and the adequacy of current antimicrobial prophylaxis guidelines for these patients. We describe the antimicrobial prophylaxis used and the types of infectious occurring in the first 100 days following CAR-T therapy for relapsed or refractory B-cell ALL in children and adolescents (≤18 years) at our centre. Twenty-seven patients received their first CAR-T infusion (CTI) during the study period. Almost all patients (96%) had a comprehensive Infectious Diseases review prior to CTI, which informed a personalised prophylaxis or fever/sepsis plan in six (22%). Overall, six (22%) patients had one or more infections during the study period including five (19%, 0.9 per 100 days-at-risk) from days 0-30 and three (n = 20, 15%, 0.6 per 100 days-at-risk) from days 31-100. Bacterial blood stream infections were the most common type of infection encountered during both time periods, and one patient had probable pulmonary aspergillosis. There were no infection-related deaths. Our study contributes important information on the spectrum of infections encountered in pediatric patients with B-ALL post CAR-T therapy. Overall, the burden of infectious complications post CAR-T therapy in our cohort is lower than previously reported in the literature. Results suggest that our prophylaxis recommendations are effective in this population.

Hypogammaglobulinemia and Infection Risk in Relapsed Refractory B Cell Malignancy Patients Treated with Varnimcabtagene Autoleucel (IMN-003A), a CD19-Directed Chimeric Antigen Receptor T (CAR-T) Cell Therapy, in the Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an anti-CD19 CAR-T cell immunotherapy for patients with relapsed refractory B cell malignancies (RR BCM) in the Phase 2 IMAGINE study. Hypogammaglobinemia can result from normal CD19+ B cell depletion (on-target, off-site effect) by CD19 directed CAR-T cells. RR BCM patients (pts) are already at risk for infections due to impaired immune function, lymphodepletion preparative regimen and prior immunosuppressive therapies. Intravenous immunoglobulin (IVIg) replacement is used to reduce the risk of infections due to hypogammaglobulinemia. We report the impact of var-cel on hypogammaglobinemia, infectious complications, risk factors, IVIg usage and clinical outcomes for RR BCM patients treated in first-in-India industry study (CTRI/2022/03/041162). Methods: RR BCM pts treated with var-cel in this study were reviewed. Hypogammaglobulinemia was defined as IgG &amp;lt;4 g/L. Infections reported during pre-apheresis (post screening), D0 to D+28 post CAR-T infusion and beyond D+29 for study duration were analyzed. IVIg usage for hypogammaglobulinemia was at investigator discretion. Patients were monitored for infection events. RR BCM response was assessed as per NCCN (B-ALL) and IWG (B-NHL) criteria. Appropriate statistical tools were used for data analysis. Results: At data cut-off, of 25 pts enrolled (median age 31 yrs, range 3 - 66) with RR BCM (n=13 B-ALL; n=12 B-NHL), 24 pts received var-cel (1 withdrawal). Median follow-up after IMN-003A administration was 205 days (range 12 - 434). Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%; B-NHL 91.7%) and 80.9% (17/21) at D+90 (B-ALL 80%; B-NHL 81.8%). Treatment related mortality was 4.2% (n=1/24); 3 pts died of disease progression. Of the 24 pts, none had hypogammaglobulinemia (IgG &amp;lt;4g/L) at baseline. Eleven pts (45.8%) developed hypogammaglobulinemia post var-cel infusion. Of these, 10 pts (90.9%) received prophylactic intravenous immunoglobulin (IVIg). In total, 70.8% (n=17/24) pts received IVIg infusions (Table 1). The median time to initiation of IVIg after var-cel infusion was 58 days (range 15 to 189 days). Of evaluable pts, IVIg was used in 15 of 17 (88.2%) responders at D+90 (CR / PR) vs 0 of 4 (0%) in non-responders (SD / PD) (p=0.06). 52.9% (n=9/17) pts on prophylactic IVIg reported infections. Patients who received IVIg had more infections (30 events; p=0.76). The mean number of infections with and without IVIg was 1.8 and 1.6 respectively. The median overall survival was significantly higher in pts who received IVIg prophylaxis (p=0.02). There was no difference in survival observed based on presence or absence of hypogammaglobulinemia post var-cel infusion (p=0.46). A total of 41 infections were reported in 13 pts. Of these, the incidence from screening to D-1; D0 to D+28 and beyond D+29 were 9 events (4 pts); 12 (11) and 20 (7) respectively. Bacterial, viral and fungal infections reported were 28 events (11 pts); 12 (8) and 1 (1) respectively. Bacterial infections were sepsis (n=7); urinary tract infections (n=5), respiratory (n=14) and central line (n=2). There were 12 viral events, 11 of respiratory origin and one involving conjunctiva. There was one episode of probable fungal candidemia. Stool surveillance detected 27 positive events in 13 pts. Table 2 summarizes the reported infectious organisms. There were 23 infections documented in 13 patients who did not develop hypogammaglobulinemia (median 2 infections; mean 1.8; range 1 - 6). Of 11 pts with hypogammaglobulinemia, there were 18 infections (median 3.5; mean 1.6; range 1 - 9). Complete and partial responders had more infections compared to non-responders (p = 0.05). Conclusions: This is the first-in-India report evaluating hypogammaglobulinemia and infection risk post CAR-T cell infusion. The IVIg usage and documented infections was more in responders. The safety and efficacy outcomes of varnimcabtagene autoleucel (IMN-003A) are consistent with reported outcomes of approved CD19 directed CAR-T cell therapies. The use of prophylactic IVIg, often led by individual practice, to reduce the number of infections and its impact on efficacy outcomes remains uncertain. Potential strategies to reduce infection risk may help improve outcomes.

Manufacturing of a Subsequent Autologous CAR-T Product after Prior CAR-T Is Safe and Feasible

Introduction: Patients with large B-cell lymphoma frequently fail to achieve a durable remission following CD19-directed chimeric antigen receptor T-cell therapy (CAR-T). Unfortunately, the outcomes for patients who progress after CAR-T are dismal with the median overall survival of approximately 6 months. Trials are now demonstrating the possibility of a subsequent autologous CAR-T for treating these patients. We demonstrated a CD22-directed CAR-T (CAR22) achieved a 68% and 53% overall (ORR) and complete response (CR) rate in 38 subjects who progressed after a CD19 CAR-T (CAR19) (NCT04088890). Of the 20 patients who achieved CR, 17 never relapsed after a median follow up of 22.8 months. The safety and feasibility of manufacturing a second (or more) CAR-T from patients has largely not been addressed and some concerns have been raised regarding the theoretical risk of malignant transformation due to the repeatedly introduction of new genomic material through serial CAR-T manufacturing. Here we address the impact of residual CAR19 on CAR22 production and patient outcomes. Method: We evaluated the patients who progressed after CAR19 and underwent manufacturing for CAR22. Single-cell RNA-sequencing (scRNAseq; 10X) was used to quantify the frequency of axi-cel transcripts in CAR22 products. Patients' peripheral blood mononuclear cells (PBMCs) were collected on day 7, 14, 21, and 28 after CAR22 infusion. The FMC63 and CD22.BB.z sequence (with albumin as a control) were quantified using quantitative polymerase chain reaction (qPCR) to determine the presence of CAR19 and CAR22 within the PBMCs. The assay's limit of detection and quantification (LOQ) were calculated as 1.68 and 5.1 copies per 50ng of DNA reaction with 95% confidence, respectively. Results: Among 40 patients underwent manufacturing for CAR22, 2 failed manufacturing due to insufficient T cells in the apheresis material. One of the two patients received prior alemtuzumab in combination with a CAR19 product, likely contributing to the failure. Among the 38 who were successfully manufactured, 37 patients had received prior CAR19, including axi-cel (n = 28), tisa-cel (n = 3), liso-cel (n = 3), autologous CAR19 in clinical trial (n = 1), bispecific CD19/CD22 CAR-T (n = 1), and bispecific CD19/CD20 CAR-T (n = 1). The median progression-free survival (PFS) from the previous CAR19 infusion was 3.3 months (range from 1.0 to 40.4 months), with a median age of 64.4 years old (ranging from 24.9 to 83.9 years old) at the time of relapse. The median time from CAR19 infusion to leukapheresis for CAR22 manufacturing was 6.8 months (ranging from 1.2 to 42.9 months). Thirteen CAR22 products were available for scRNAseq; all from patients who had received axi-cel previously. The median frequency of CAR19 positive cells was approximately 68 (ranging from 0 to 2146) per million cells per CAR22 product. Residual CAR19 cell proportions were not statistically significantly different between patients who underwent CAR22 manufacturing within or after 6 months of CAR19 infusion. Thirty-one patients had available PBMCs post-CAR22 infusion for analysis. Twelve patients (39%) exhibited an increase in CAR19 qPCR transgene levels above the LOQ during CAR22 treatment. Most of these patients (n = 8) underwent leukapheresis for CAR22 manufacturing within 6 months after CAR19 infusion. The median CAR19 qPCR transgene levels increased by 1.2 log (ranging from 0.2 to 2.1 log; equivalent to 1.4 to 133.7 copies per 50ng of DNA), while CAR22 transgene levels increased by a median of 3.7 log (ranging from 0.7 to 3.9 log; equivalent to 4.7 to 9027.1 copies per 50ng DNA). In the comparison between patients who underwent leukapheresis within or beyond 6 months after CAR19 infusion, there were no statistically significant differences in the maximal CAR19 and CAR22 qPCR transgene levels. The ORR, cytokine release syndrome severity, immune effector cell-associated neurotoxicity syndrome severity and PFS after CAR22 were not statistically significantly different between patients with detectable CAR19 qPCR or not. Conclusion: We found that CAR19 was detectable in the majority of CAR22 products, but the levels of CAR19 transcripts were rare. While CAR19 cells were detected post-CAR22 infusion, the levels were frequently just at the LOQ and did not appear to contribute to toxicity or clinical outcomes. Overall, we find that serial manufacturing of autologous CAR-T products is safe and feasible.

A Novel Composite Endpoint of Toxicity-Free, Progression-Free Survival (TPFS) after CD19-Directed Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory Large B-Cell Lymphoma

Background: CD19-directed chimeric antigen receptor T-cell (CAR T) therapy with axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), or lisocabtagene maraleucel (liso-cel) are FDA-approved for relapsed and/or refractory (r/r) large B-cell lymphoma (LBCL) after ≥2 prior lines of systemic therapy (all 3) or earlier after 1 line (axi-cel and liso-cel) based on durable remissions of 30-40%. Pivotal trials and real-world data demonstrated notable heterogeneity in toxicity and efficacy between CAR T products. To account for such differences, we have established a novel composite endpoint of toxicity-free, progression-free survival within 6 months (TPFS6) after CAR T-cell therapy. We also studied factors associated with TPFS6 across CAR T-cell products and its impact on long-term success of CAR T-cell therapy. Methods: This retrospective cohort analysis included 282 consecutive adult patients with r/r LBCL and its variants who received axi-cel (n=228), tisa-cel (n=47), or liso-cel (n=7) at Moffitt Cancer Center (05/2015-01/2022). TPFS6 was defined as absence of severe (≥Grade 3) cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS), progressive disease (PD)/stable disease (SD) and non-relapse mortality (NRM) within 6 months of CAR T-cell infusion. CRS and ICANS were graded according to CTCAE or ASTCT Consensus grading. Factors associated with TPFS6 were assessed via logistic regression multivariate analysis (MVA). Landmark analysis at the 6-month point examined the impact of TPFS on overall survival (OS). All analyses were performed utilizing SAS and R. Results: For the entire cohort (median age=64 years [range, 19-86]; 60.3% males), the TPFS6 was 37% (95% confidence interval [CI] 31-43, Figure 1A) with no difference between the recipients of axi-cel, tisa-cel, or liso-cel ( p=0.97). Individual components of TPFS6 such as severe CRS/ICANS, NRM, and PD/SD accounted for 6.8%/30.3%, 1.7%, and 61.2%, respectively. Patients with TPFS6 differed from their counterparts by gender (males, 51% vs 65.7%, p =0.014), presence of bulky (8.7% vs 17.4%, p =0.045) or extra nodal (≥2 sites, 30.8% vs 46.6%, p=0.009) disease, ECOG performance (≥2, 7.7% vs 20.8%, p=0.004), prior autologous transplant (25% vs 13.5%, p=0.015), C-reactive protein (CRP) (&amp;gt;4 mg/dL, 10.6% vs 35.4%, p&amp;lt;0.001), ferritin (≥400 ng/mL, 37.5% vs 62.4%, p&amp;lt;0.001), lactate dehydrogenase (LDH) (≥225 U/L, 51% vs 76.4%, p&amp;lt;0.001) at lymphodepletion (LD). TPFS6 patients were comparable to those experiencing at least one of the TPFS6 endpoints according to age, race, number of prior lines or use of bridging therapy, LBCL variants, Ann Arbor staging, double/triple hit status, prior CNS involvement, and vein-to-vein time (all p&amp;gt;0.1). In logistic regression MVA, lower levels of baseline LDH (odds ratio [OR]=0.44, p=0.004), CRP (OR=0.33, p=0.004), ECOG&amp;lt;2 (OR=0.43; p=0.05) and female gender (OR=0.5; p=0.01) were associated with TPFS6. In the landmark Cox MVA of OS, TPFS6 was strongly associated with 5.2-fold survival difference ( p&amp;lt;0.001, Figure 1B) after adjustment for age (hazard ratio [HR]=1.24 per 10-year increment, p=0.077) male gender (HR=1.92, p=0.013), ECOG≥2 (HR=2.34, p=0.004), high LDH at LD (HR=2.63, p&amp;lt;0.001), and CAR T product (HR=1.67 for tisa-cel vs axi-cel, p=0.079). At 6 months, overall response and complete response rates were 48% and 45%, respectively. With median follow up of 15.4 months for surviving patients, the estimated 2-year OS and PFS were 54.5% and 39.3%, respectively. Conclusions: Only 37% of patients were alive at 6 months without experiencing a TPFS-defining event. TPFS6 was associated with known clinical risk factors such as CRP, LDH, ECOG and gender but not the type of CAR T product. TPFS6 encompasses clinically meaningful components associated with OS and it therefore represents an ideal recovery outcome by measuring initial treatment success without progression, major morbidity, and mortality. Thus, TPFS6 needs to be further explored as a uniform endpoint for evaluating CAR T-cell safety and efficacy in future clinical trials of r/r LBCL.

Evaluating the Safety of HCAR19 (Now Actalycabtagene Autoleucel- Actaly-Cel™), a Novel Humanized CD19-Directed Chimeric Antigen Receptor T-Cells in Pediatric, Adolescents, and Young Adults with Relapsed/ Refractory B- Acute Lymphoblastic Leukemia (ESHA) - Report of Phase-1B

Background and aims Chimeric Antigen Receptor (CAR) T-cell therapies for relapsed-refractory Pediatric B-cell Acute Lymphoblastic Leukemia (r/r-B-ALL) show remarkable success but remain prohibitively expensive. We developed and reported a Humanized CD19-directed novel CAR T-cell- HCAR19 (now -Actalycabtagene autoleucel- Actaly-cel™) with robust activity and low toxicity in pre-clinical studies. This led to regulatory approval for the first CART-cell trial in India as a Pilot Phase-1 study (CTRI/2021/05/033348), and it's encouraging safety profile and responses led to approval for a roll-on Phase 1b/2 Trial (CTRI/2023/03/050689) to monitor safety, identify Phase-2 Dose (P2D) and assess efficacy. Trial design permitted pooling of data of both trials for all objectives. We report here the Phase-1b analysis of Actaly-cel in r/r-B-ALL in Pediatric, Adolescents and Young Adults 3-25 years of age. Methods Patients with r/r B-ALL not in remission after 2 or more lines of therapy ineligible for Allogenic-Stem Cell Transplant (Allo-SCT) were included if no CD19-negative blasts population were detected on flow-cytometry records. Those with isolated extra-medullary disease, active neurological involvement/ sequelae, genetic syndromes/prior malignancy, or prior CD19-directed therapy were excluded. Bridging therapy was at physician discretion. Lymphodepletion was with Fludarabine (30mg/m 2) x 3-days and cyclophosphamide (500 mg/m 2) x 2-days. There were 3 Target Doses, TD-1, 2 &amp; 3 CART-cells at 1 x10 6, 3-5 x10 6 and 10-15 x10 6/kg recipient weight respectively. Escalation and de-escalation followed 3+3 design with Coherence-guided dose-decision for bivariates- efficacy and toxicity, allowing all patients to be evaluable. Primary end-point was Overall Response Rates (ORR) at Day-30 on bone-marrow flow-cytometry. Patients were monitored for toxicities, in-vivo dynamics of Actaly-cel, and cytokine profiles. Results Of 12 patients enrolled Actaly-cel was infused in 9 at time of analysis as 2 were withdrawn prior to apheresis (1-progression, 1-withdrawl of consent) while 1 is awaiting infusion. Three patients were assigned TD-1 and 6, TD-2. In TD-2 group, 1 received lower dose (2.13x10 6/kg) due to obesity and weight gain from severe-Covid19 prior to infusion. Median age was 16-yrs (range:8-24), M:F-3:2, median prior lines of therapy were 3 (range:2-5), 6 had high-risk cytogenetics, and median pre-infusion bone-marrow blasts were 19.5%(range:0-88%). Eight patients were evaluable for response and toxicities. All had Grade 3 leukopenia and thrombocytopenia. Peak cytokine release syndrome (CRS) was Grade-3 in 1(12.5%), Grade-2 in 2(25%), and Grade-1 in 5(62.5%). Those with ≥ Grade-2 CRS received tocilizumab with full recovery. Only 1 patient had Immune effector cell associated neurotoxicity syndrome (ICANS) peak-Grade 3, and was also the only one with Grade-3 CRS and had severe-Covid19 prior to infusion. ICANS resolved with steroids in 4 weeks, but recurred in the second month post-infusion, this time associated with leukemia-progression. Toxicities corroborated with IL-6 (median peak: 25pg/ml, range:2.8-3604), but not with Ifn-γ, which remained low (median peak:6.25pg/ml, range:0-68.1) (Fig-1). ORR was 87.5% (1 no-response-NR), 3 Morphological Remission with MRD-positive (Partial Response-PR), and 4 MRD-negative (Complete Response-CR). Responses correlated with Actaly-cel expansion dynamics (Fig-2) the only non-response being in a patient with poor expansion. At median follow-up of 3 months (range:1-15) 3 patients with no or partial Day-30 response expired from progression within few weeks. All 4 with CR had B-cell aplasia needing intravenous immunoglobulin. Two of them underwent subsequent Allo-SCT, 1 relapsed post-transplant and died from disease progression 15 months later, while the other is alive in remission at 15 months. Two patients remain in CR at 3 and 12 months without further therapy, while 1 with PR is on follow-up. Patients receiving TD-2 had ORR of 100% (CR-3, PR-1) with peak toxicity of Grade-2 CRS in only 1. Conclusion In a Phase-1/1b trial, Actaly-cel had good safety profile with low toxicity and robust activity resulting in high ORR that improved at higher doses. In-vivo dynamics and toxicity profile of Actaly-cel showed robust activity and low cytokines corroborating with pre-clinical studies. A median dose of 5x10 6/kg Actaly-cel is proposed as the P2D.

A Phase 1 Dose Escalation Trial of Third-Generation CD19-Directed CAR T-Cells Incorporating CD28 and Toll-like Receptor 2 (TLR2) Intracellular Domains for Relapsed or Refractory B-Cell Non-Hodgkin Lymphomas (ENABLE)

Introduction: Chimeric antigen receptor (CAR) T cell therapies directed against CD19 and incorporating either CD28 or 4-1BB intracellular co-stimulatory domains are a standard of care for relapsed or refractory (r/r) B-cell lymphomas. CD19-directed CAR T-cell products employing CD28 co-stimulation yield high response rates, but have been associated with higher rates of immune effector cell-associated neurotoxicity syndrome (ICANS) and severe cytokine release syndrome (CRS) than 4-1BB co-stimulated products. There is a need for CAR T-cell products that combine high efficacy with low toxicity. Toll-like receptor 2 (TLR2) is expressed by T-cells, and its engagement enhances T-cell expansion, modulates cytokine production, and promotes long-lived T-cell memory. In preclinical studies, interposition of an intracellular domain from TLR2 between CD28 and CD3ζ resulted in reduced CAR T-cell production of ICANS-associated cytokines GM-CSF and IFN-γ, while maintaining production of the homeostatic cytokine IL-7. We investigated the safety and efficacy of a novel third generation CD19-directed CAR T-cell product, which combines CD28 and TLR2 co-stimulatory domains ( Figure). Methods: We completed a first-in-human phase 1 dose escalation trial of WZTL-002, comprising autologous 1928T2z CAR T-cells (‘ENABLE’, NCT04049513) for patients with r/r B-cell non-Hodgkin lymphomas (B-NHL). A 3+3 dose escalation design was used, with doses from 5 × 10 4 to 1 × 10 6 viable CAR T-cells/kg body weight. Eligible participants had radiologically assessable disease, satisfactory organ function and no central nervous system (CNS) involvement by lymphoma. Bridging therapy was permitted after leukapheresis and pending CAR T-cell manufacture and release. WZTL-002 CAR T-cells were administered intravenously after 3 days of fludarabine (30mg/m 2/day) and cyclophosphamide (500mg/m 2/day) lymphodepletion. Adverse events (AEs) were graded by CTCAE 5.0 except CRS and ICANS (graded by American Society for Transplantation and Cellular Therapy criteria). Response assessment was by PET/CT at month 3, per Lugano 2014 criteria. Pharmacokinetic analyses were by droplet digital PCR for CAR transgenes in blood mononuclear cells. Results: Of 21 patients treated within the dose escalation trial, median age was 57 years (range 23 - 70); 10 (48%) were female; 4 (19%) Māori. Lymphomas were of large cell histology in 17 (81%, Table). Participants had received a median of 4 prior lines of therapy, including autograft in 11 (52%) and allograft in 1. Product phenotyping showed WZTL-002 CAR T-cells were median 49% CD4 +, 41% CD8 + and 34% CD62L +. As of June 14 2023, all patients had reached the primary follow-up timepoint (3 months after WZTL-002 administration). Grade ≥ 3 AEs occurring in ≥ 10% of recipients were: neutropenia (95%), lymphopenia (57%), hypogammaglobulinemia (57%), anaemia (43%), febrile neutropenia (43%), thrombocytopenia (24%) and tumor pain (19%). Grade 1 - 2 CRS occurred in 13 patients (62%); 6 received tocilizumab and 3 dexamethasone. No CRS of grade ≥ 3, and no ICANS of any grade, occurred. Two grade 4 cytopenia dose limiting toxicities occurred at day 21 (one each at 5 × 10 5 and 1 × 10 6 cells/kg), both resolved to grade 2 by day 90. Maximum tolerated dose was not reached. Responses were seen at all dose levels with complete metabolic response in 11 (52%) at month 3. WZTL-002 CAR T-cells expanded at all dose levels. Among recipients of 0.5 - 1.0 × 10 6 CAR T-cells/kg (n = 12), median peak CAR T-cell level (C max) was 93,950 transgene copies/μg genomic DNA (reached at median day 10) and CAR T-cells persisted in 9 of 11 assessed (82%) at day 90. A recommended phase 2 dose (RP2D) range of 0.5 - 1 × 10 6/kg was selected. Conclusions: In this dose escalation trial of a novel third-generation CD19-directed CAR T-cell product that combines CD28 and TLR2 co-stimulatory domains for r/r B-NHL, we observed no severe CRS, no ICANS of any grade and complete responses at all dose levels. At RP2D, in vivo CAR T-cell expansion was similar to or greater than other CD19-directed products. In conjunction with preclinical findings, this study suggests interposition of a TLR2 domain between CD28 and CD3ζ domains may reduce CAR T-cell-related ICANS risk while retaining efficacy. Enrolment to a dose expansion cohort has commenced and will assess outpatient management and the safety and efficacy of WZTL-002 CAR T-cells manufactured using a closed automated process.

Loss of p53 Impairs Death Receptor Expression and Confers Resistance to CD19-Directed CAR T-Cell Therapy in B-Cell Precursor Acute Lymphoblastic Leukemia

Background In recent years, new therapy strategies such as the antibody-drug conjugate inotuzumab ozogamicin, the bi-specific T-cell engager blinatumomab, and chimeric antigen receptor (CAR) T-cells have been introduced to salvage relapsed precursor B acute lymphoblastic leukemia (ALL). However, recent evidence indicates that these new therapies are ineffective in TP53-aberrant ALL, although the mechanism remains unclear. TP53 is the most frequently mutated gene in cancer, but in ALL, mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse, TP53 aberrations are found in approximately 12% of the patients and predict a very poor outcome. Consequently, relapsed TP53 deleted ALL is now classified as ‘very high risk’. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance to classical chemotherapeutics. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wild type and knockout models in TP53 WT ALL cell lines Nalm6 and RCH-ACV. Additionally, we prepared CD19-directed CAR T-cells with a 4-1BB co-stimulatory domain from three healthy donors by isolating, transducing, and expanding CD4+ and CD8+ T-cells separately. With flow cytometry, we assessed survival of p53 WT and p53 KO leukemic cells in the presence of CD4+ and CD8+ CAR T-cells at a 1:1 CD4:CD8 ratio. Moreover, we performed mRNA profiling by qRT-PCR and cell membrane profiling by flow cytometry to determine how loss of p53 affects CAR T mediated killing. Initial findings in cell lines were then validated in patient-derived xenografts. Results We observed that loss of p53 led to up to a 35% decrease in killing by overnight CAR T treatment in both cell line models (Figure 1A). Although loss of p53 has been linked to reduced cell surface expression of CD19 (Pan et al., 2020, Leukemia), no changes in CD19 expression were observed in response to p53 loss. In search for an explanation for the diminished killing, we performed mRNA profiling and found a reduced expression of death receptors CD95/Fas and DR5/TRAILR2 in p53-deficient cells, which was confirmed at the protein level with flow cytometry (Figure 1B). Indeed, CRISPR/Cas9 mediated loss of CD95/Fas expression in p53 wild type cells reduced CAR T mediated cell killing, consistent with a key role for CD95/Fas in T-cell mediated killing. Conversely, treatment of p53 proficient cells with MDM2 inhibitors induced expression of CD95/Fas and DR5/TRAILR2 (Figure 1B), which translated into enhanced killing of target cells. Most of these observations have already been recapitulated in a diagnosis-relapse pair of patient-derived cells in which the diagnosis was p53wild typeand the relapse was p53 deficient (Figure 1A-B). We observed lower baseline mRNA expression of CD95/Fas and DR5/TRAILR2 in p53-deficient cells, accompanied by a 50% reduction in killing relative to the p53 wild type controls. Conversely, MDM2 inhibition increased death receptor expression only in p53-proficient cells. The ongoing experiments validating enhanced killing after MDM2 inhibition in patient-derived cells will be presented during the meeting. Conclusions Loss of p53 impairs expression of CD95/Fas and DR5/TRAILR2, thereby protecting ALL cells from CAR T induced cell death. In addition, our results indicate that the p53-controlled expression of these receptors may be exploited to increase the efficacy of CAR T-cell therapy in p53-proficient patients.

The Impact of Inotuzumab Ozogamicin (InO) Treatment on Brexucabtagene Autoleucel (Brexu-cel) Outcomes in Adults with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Cellular Immunotherapies: Late Phase and Commercially Available Therapies The Impact of Inotuzumab Ozogamicin (InO) Treatment on Brexucabtagene Autoleucel (Brexu-cel) Outcomes in Adults with Relapsed/Refractory B-cell Acute Lymphoblastic Leukemia (B-ALL) Introduction: Brexu-cel is the first approved CD19-directed chimeric antigen receptor (CAR) T-cell therapy for adult patients (pts) with relapsed/refractory (r/r) B-ALL. InO, an anti-CD22 antibody drug conjugate, is also approved for the same indication. With the accessibility to several targeted therapies in r/r B-ALL, the optimal sequence remains uncertain. The effect of prior treatment with InO on brexu-cel outcomes remains underreported, especially as a bridging therapy, as well as the effect of previous response to InO on post brexu-cel outcomes. Methods: This is a retrospective multicenter analysis from 25 U.S. institutions of adults (≥18 years) with r/r B-ALL treated with commercial brexu-cel from 2021 to 2023 post FDA approval. Methodologies for assessing minimal residual disease (MRD) (minimal threshold of 10 -4) included flow cytometry, NGS, or qPCR depending on institutional practice. Progression-free survival (PFS) and overall survival (OS) were calculated from day of brexu-cel infusion and were not censored for hematopoietic cell transplant (HCT) or maintenance. All living patients were censored at the time of last follow-up prior to data lock, which occurred on June 30, 2023. Results: Among 152 infused, 83 (54.6%) had pre-CAR InO therapy (InO-exposed), with a median of 3 administered doses (range: 1-22). Within the InO-exposed cohort, 23 (28%) pts received InO as a CAR T-cell bridging therapy (ie, between apheresis and lymphodepletion) with or without pre-apheresis and 60 (72%) pts received InO only during pre-apheresis. Baseline characteristics for InO exposed and InO-naïve pts are shown in Table 1. InO-exposed pts had higher median prior lines of therapies (4 vs. 3; p= 0.05), more frequently had active disease (≥5% marrow blasts) at the time of apheresis (67% vs. 44%, p= 0.02), and had lower incidence of Ph+ disease (19% vs. 45%, p=0.003), compared to InO-naïve pts, respectively. The incidences of cytokine release syndrome (CRS) (85% vs. 85%, p= 0.26) and ICANS (58% vs. 57%, p= 0.36) post infusion were similar, and post-infusion death in remission occurred in 17% and 12% among InO-exposed and InO-naïve pts, respectively. Morphological complete remission (CR) and MRD- rates following brexu-cel infusion were 89% and 77% for InO-exposed pts, and 92% and 70% for InO-naive pts, respectively. Post CAR therapy, more InO-naïve pts underwent consolidation/maintenance therapy (transplant, chemotherapy, or TKIs) compared to InO-exposed pts (41% vs. 19%, p= 0.004). The median follow-up after brexu-cel was 8.4 (range) months. Median OS (not reached (NR) vs. 12 months; p= 0.033) and median PFS (NR vs. 7 months; p= 0.029) were superior in InO-naïve pts compared to InO-exposed pts, respectively. However, after adjusting for pre-apheresis disease burden and post-CAR maintenance therapy, there were no longer significant differences in OS (HR= 1.25;95%CI: 0.62-2.53; p= 0.53) or PFS (HR= 1.24;95%CI:0.71-2.16, p=0.45) based on pre-CAR InO exposure. When InO-exposed pts were stratified based on prior InO-response (CR vs. no response), InO-responsive pts had superior estimated 12-month OS (64%) and PFS (38%) relative to InO-refractory pts (OS: 33%; PFS: 34%), but inferior to InO-naïve pts (OS: 75%; PFS 56%), with p-values of 0.0001 and 0.021 for OS and PFS, respectively (Figure 1). The timing of pre-CAR InO therapy did not impact brexu-cel survival outcomes, with comparable estimated 12-month OS (43% vs. 58%, p= 0.35) and PFS (46% vs. 38%, p= 0.57) for InO-exposed pts during bridging therapy and patients who received InO as a therapy prior to apheresis. Conclusion: After adjusting for pre-CAR disease burden and post-CAR consolidation/maintenance, we found that prior InO exposure does not significantly associate with PFS or OS following brexu-cel. However, relative to InO-responsive patients, patients who were InO-refractory appear to have worse post-CAR survival outcomes. Finally, timing of InO administration (ie as a prior line of therapy or as CAR bridging) did not influence outcomes brexu-cel outcomes.

Modified Endothelial Activation and Stress Index (mEASIX) Score and Immune Effector Cell Associated Haematotoxicity (ICAHT) Following Varnimcabtagene Autoleucel (IMN-003A), a CD19-Directed Chimeric Antigen Receptor T (CAR-T) Cell Therapy, in the Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an anti-CD19 CAR-T cell immunotherapy for patients (pts) with relapsed refractory B cell malignancies (RR BCM) in first-in-India Industry Phase 2 IMAGINE study, administered in a fractionated (10%/30%/60%) infusion protocol. Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) are commonly associated adverse events with CAR-T cell therapy. The modified Endothelial Activation and Stress Index (mEASIX) score [lactate dehydrogenase (LDH) (U/L) × CRP (mg/dL) / platelets (PLT) (10 9 cells/L)] is a prognostic predictor of outcomes for patients in post-allogeneic transplant setting. Elevated LDH and low PLT levels have been associated with severe ICANS, and high IL-6 level is a recognized marker for CRS. Immune Effector Cell Associated Hemato-Toxicity (ICAHT) represents the most common toxicity after CAR-T cell therapy. CAR-HEMATOTOX score is used to risk stratify patients for cytopenia. This abstract reviews the mEASIX score and ICAHT for pts in the IMAGINE study, prognostic value of mEASIX for CRS, ICANS and responses, and correlation of ICAHT score with outcomes. Methods: mEASIX scores were calculated for each pt at lymphodepletion (LD), D0, D+1 and D+4. CRS and ICANS were graded as per American Society for Transplantation and Cellular Therapy (ASTCT) criteria. Pts were monitored for ICAHT post CAR-T infusion and risk stratified using CAR-HEMATOTOX score. ICAHT was graded using CTCAE v5.0. Appropriate statistical tools were used for analysis. Results: Twenty-four RR BCM pts (12 B-ALL; 12 B-NHL) were treated with var-cel in this study. Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%; B-NHL 91.7%) and 80.9% (17/21) at D+90 (B-ALL 80%; B-NHL 81.8%). AESIs reported were CRS (Grade [G] 1 62.5%; G3+ 4.2%; overall 66.7%); ICANS (G1 4.2%; G3+ 0%; overall 4.2%); neutropenia (G3+ 91.7%; overall 100%); anemia (G3+ 29.2%; overall 95.8%); and thrombocytopenia (G3+ 20.8%; overall 91.7%). CRS median onset was D+5 and duration 3 days. No G3+ ICANS was reported. Treatment related mortality was 4.2% (n=1/24, CRS); 3 pts died of disease progression. The mEASIX score at LD, D0, D+1 and D+4 and its correlation with CRS, ICANS and responses is shown in Table 1. At D+1, mEASIX score &amp;lt;1 was predictive for responses (entire cohort, p=0.05; OR 15.0). At D+4, mEASIX score &amp;gt;1 was nearing statistical significance (p=0.07) for CRS (all grades; OR 6.0). However, mEASIX score at other timepoints was not predictive for CRS, ICANS or responses. The incidence of ICAHT was 100% (all grades; n=24), 100% neutropenia (G3+ 91.7%); 95.8% (n=23) anemia (G3+ 29.2%) and 91.7% (n=22) thrombocytopenia (G3+ 20.8%). The median time (range) to resolution of anemia, neutropenia and thrombocytopenia was 90 (11-270), 17.5 (1-181) and 9 (1-181) days respectively. Classical, biphasic and prolonged cytopenia pattern was seen for Neutropenia in 6, 2 and 16 pts respectively; for Anemia in 2, 0 and 21 patients respectively and for Thrombocytopenia in 4, 1 and 17 patients respectively. Overall, all patients, except two, were pancytopenic during their treatment course. Figure 1 shows the temporal course of var-cel mediated haematotoxicity (ICAHT). Two pts (8.3%) each received red cells, platelets, G-CSF and romiplostim; majority did not need supportive treatment. CAR-HEMATOTOX score was low risk in 18 pts and high risk in 6 pts. There was no statistical correlation between low risk (18 pts) and high risk (6 pts) with responses (17 pts, p=0.92) or with relapse / disease progression (9 pts, p=0.21). Conclusions: This is the first-in-India industry study evaluating mEASIX and ICAHT in patients treated with CAR-T cell therapy. mEASIX score &amp;lt;1 was predictive of responses at D+1 and score &amp;gt;1 at D+4 was mildly predictive of CRS. All patients developed ICAHT but only a small number of patients needed supportive treatment. Overall, var-cel was well tolerated with manageable cytopenia. The CAR-HEMATOTOX score was not predictive of relapse / disease progression in the IMAGINE study. mEASIX score may be used as an early predictor of CRS for permissive use of Tocilizumab before onset of severe symptoms. The ICAHT profile was heterogeneous, probably reflective of hematopoietic reserve, lines of treatment and downstream immune responses. The predictive role of mEASIX score and ICAHT profile in a real-world setting needs further study.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.

Blood-Brain Barrier and Neuronal Damage during Icans in Patients Treated with Anti-CD19 CAR-T Cells

Background Immune effector cell-associated neurotoxicity syndrome (ICANS) is the most frequent neurological toxicity occurring after treatment with chimeric antigen receptor T-cells (CAR-T), with a reported incidence of around 30-50% according to different settings and products. Clinical manifestations may widely range from dizziness to seizures, and tend to achieve response with high-dose steroids. The pathogenesis of ICANS is unclear; despite some data suggest a role for endothelial activation and impairment of the blood-brain barrier (BBB). Glial fibrillary acidic protein (GFAP) and neurofilament light chain (NFL) are reliable astroglial and axonal neuronal damage biomarkers, respectively. NFL provides structural support to neurons, while GFAP is crucial for astrocyte function and BBB maintenance. Elevated levels of NFL and GFAP in cerebrospinal fluid indicate BBB damage, aiding the diagnosis and monitoring of neurological conditions like multiple sclerosis and traumatic brain injury. Recently, preliminary studies reported that NFL serum levels correlate with the severity of neurotoxicity after CAR T-cell treatment. Aim and Methods In our study, we measured GFAP and NFL both at baseline and at day 7 in 34 patients with R/R B-cell malignancies treated with CD19 CAR-T cells (15 Axi-cel, 12 Tisa-cel, 7 Brexu-cel), and explored their association with clinically relevant neurological toxicities and markers of endothelial impairment. We have collected peripheral blood samples from patients treated with CD19-directed CAR-T cells at baseline and at every other day up to 14 days after CAR-T infusion. We have measured GFAP and NFL levels and compared them with clinical neurotoxicity and serum markers of endothelial damage or inflammatory activity. Results Elderly patients tended to have higher values of GFAP at day 0 and to raise those levels after CAR-T infusion. When measured after infusion, GFAP positively correlated with endothelial biomarkers such as von Willebrand factor (p=0.041) and sST2 (p=0.015) and got some trends with IL-2R (p=0.050) and SUPAR (p=0.074). NFL positively correlated with the vWF (p=0.021) and with the endothelial activation score mEASIX (p=0.015) (Figure 1A). Overall, 11/34 patients (33%) developed ICANS. Baseline GFAP and NFL values did not impact the subsequent development of CRS or ICANS. When compared to the baseline, an increase of NFL was observed in 50% of cases. Patients experiencing an increase in NFL values after CAR-T infusion were more likely to develop ICANS of higher grades (p=0.046, OR 4.14) and to receive treatment with steroids (p=0.023). The median increase of NFL in patients with ICANS was of 25% of baseline levels. When representing values measured after CAR-T infusion in a heatmap, it emerges that a profile with higher GFAP and NFL may describe patients with ICANS of a higher grade (Figure 1B). Conclusion To sum up, our data confirm a correlation between severe ICANS and elevated NFL and G-FAP serum levels. Interestingly, these data also correlate with markers of endothelial activation, suggesting an active role of BBB impairment - considered in both its components- and concomitant neuronal damage in the pathogenesis of ICANS.