4-1BB Costimulatory Domain Research Articles

Regulation of the Transcriptional Repressor BACH2 Overcomes Tonic Signaling-Driven CAR T Cell Dysfunction

Unlike endogenous T cell receptors (TCRs), chimeric antigen receptors (CARs) have an intrinsic predisposition to initiate intracellular signaling in the absence of target antigen (Long AH, Nature Medicine 2015). This phenomenon, referred to as tonic signaling, has previously been shown to be a potent driver of T cell exhaustion when CARs contain the CD28 costimulatory domain (Lynn R, Nature 2019). We previously showed that, in contrast to CD28, CARs containing the 41BB costimulatory domain acquire enhanced functionality from tonic signaling (Singh N, Nature Medicine 2021). These data indicate that the impact of tonic CAR signaling is directly dependent on the CAR costimulatory domain. To understand the molecular regulators responsible for this divergent functionality, we generated CARs targeting CD22, an established immunotherapy target for B cell malignancies, that contained either CD28 or 41BB costimulatory domains and either did or did not signal tonically (22/28+, 22/BB+, 22/28-, 22/BB-). We confirmed that tonic signaling of CD28 drove impaired cytotoxic function, T cell expansion and cytokine secretion in response to antigen, while tonic 41BB improved all parameters of T cell function. Bulk RNA sequencing of CAR T cell products at the conclusion of manufacturing, when the impact of tonic CAR signaling was predicted to be most impactful, revealed significantly more transcriptional activity in both tonic CARs. Comparison of differentially expressed genes demonstrated that 22/28+ had high expression of targets of NFAT and AP1, transcription factors that promote T cell effector function but are also drivers of T cell exhaustion when persistently active (Martinez GJ, Immunity 2015; Lynn R, Nature 2019). 22/BB+ instead demonstrated high activity of BACH2, a transcriptional repressor that has recently been shown to preserve naïve and memory cell states by antagonizing effector programs (Yao C, Nature Immunology 2021; Roychoudhuri R, Nature Immunology 2016). We generated 22/28+ cells that overexpressed BACH2 (22/28+_BACH2) and found that this intervention completely rescued acute anti-tumor function, with cytotoxic activity of these cells equivalent to 22/BB+. Mass cytometry revealed that 22/28+_BACH2 cells were phenotypically very similar to 22/BB+ cells at the conclusion of manufacturing and after stimulation with target cells, expressing high levels of granzyme B, perforin, IFNγ and markers of memory differentiation. Intriguingly, 22/28+_BACH2 cells expressed very high levels of the transcription factor TCF7, known to play a role in maintaining memory, suggesting that transgenic BACH2 expression play a broad role in lineage regulation. To interrogate how BACH2 overexpression impacted long-term T cell function, we chronically stimulated these cells using our established in vitro protocol (Selli ME, STAR Protocols 2023) in which co-cultures of CAR T cells and tumors are continually replenished with additional tumor to maintain a persistent and high antigen burden. Similar to our previous studies, early function of 22/28+_BACH2 cells was significantly improved over 22/28+ but BACH2 overexpressing cells lost the ability to proliferate and kill tumor cells much earlier than either 22/28+ or 22/BB+. Phenotypic analysis revealed at time of cell failure revealed that almost all 22/28+_BACH2 cells expressed markers of a central memory phenotype (CD62L hiCD45RO hi). Collectively, these data suggest that BACH2 overexpression can overcome tonic CAR signaling-induced dysfunction by antagonizing exhaustion programs but simultaneously “locks” these cells into a memory state with restrained effector function, thus limiting their long-term efficacy.

Reinfusion of Varnimcabtagene Autoleucel (IMN-003A) in Patients with Relapsed Refractory B Cell Malignancies Is Feasible with Sustained Responses

Background: Varnimcabtagene autoleucel, a CD19 directed CAR-T cell therapy with a 4-1BB co-stimulatory domain and a non-FMC63 based murine scFv (A3B1 binder), has demonstrated remarkable responses in relapsed/refractory B cell malignancies (RR BCM) in the IMAGINE study. Reinfusion of var-cel is an option to improve functional persistence for CD19+ relapsed disease. This abstract describes the clinical outcomes following reinfusion of var-cel in patients (pts) who relapsed after first infusion. Methods: Patients (pts) aged 3 to 45 years (B-ALL) and ≥ 18 years (B-NHL) with RR BCM were eligible for IMAGINE study if they had measurable disease, as assessed by lymphoid blasts (B-ALL) or metabolic tumour bulk (B-NHL), received ≥1 prior regimen, refractory to the last line of treatment with good performance status (ECOG 0 to 1). The primary target dose was 1x10 6/kg CAR+ cells (B-ALL) and 5x10 6/kg CAR+ cells (B-NHL) (overall range 0.1x10 6 to 5x10 6) and was infused over 3 days (10%/30%/60% fractions). Primary objectives were overall response rate (ORR: CR + CRi in B-ALL and CR + PR in B-NHL) at day +90 after first infusion, and safety. Response was assessed as per NCCN (B-ALL) and IWG (B-NHL) criteria. Adverse events (AEs) were graded using CTCAE v5.0. CRS and ICANS were graded according to ASTCT criteria. Criteria for reinfusion was: 1) achieved response with subsequent disease progression and presence of CD19 positive malignant cells; 2) available second dose for retreatment from initial apheresis and manufacturing run; and 3) after primary endpoint at D+90. This abstract evaluates the outcomes at D+28 after var-cel reinfusion. Results: At data cut-off, 25 pts were 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) with majority of infusions on Days 0, +3, +7. ORR was 91.7% at D+28 and 80.9% at D+90. Median progression free survival (PFS, range 12-NR), duration of response (DOR, range 0-NR) and overall survival (OS) were not reached (range 12-NR). Nine pts had relapsed disease (37.5%) with median PFS of 178 days (range 28-319). Of these 9 pts, 7 had CD19+ relapse (77.8%) and 2 (with B-ALL) had CD19- relapse. Four of these pts received second infusion at full dose (same as primary infusion) fractionated over 3 days (10%, 30%, 60%). For these 4 pts, duration of B cell aplasia after primary infusion was 90 days to NR (range); var-cel was detectable in 1 pt (n=1/4) at relapse; and median PFS was 166 days (range 90-275). Tumour burden at relapse was: Ph+ B-ALL (n=1) with 22% blasts (p210 BCR ABL transcript 36.7%); B-NHL (n=3) with TMTV range 7.5 to 277 ml and SPD range 660.5 to 8686 mm 2 (Table 1). PDL1 exhaustion marker was expressed in one B-NHL pt only. Salvage regimen in B-NHL pts was involved field radiotherapy (n=2); 1 B-NHL pt was reinfused without salvage treatment. B-ALL pt received Dasatinib, Inotuzumab and CNS prophylaxis. Preparative conditioning regimen for these 4 patients were: Fludarabine-Cyclophosphamide (n=1), with Rituximab (n=2) and Rituximab with Nivolumab (n=1). Post reinfusion, var-cel was not detected in 1 pt (B-ALL) however B cell aplasia was persistent. In B-NHL pts, Cmax range was 7222 to 31846 CAR+ copies / ug genomicDNA; Tmax was D+2 after reinfusion and duration of var-cel persistence was range 2 days to NR (Figure 1). Post reinfusion, no CRS (0%) or ICANS (0%) was reported. Hematotoxicity was the most common AE (100%) with neutropenia (G3+ 100%); anemia (G3+ 66.7%); and thrombocytopenia (G3+ 33.3%;). Hypogammaglobulinemia (IgG <4g/L) was seen in 2 pts (50%, new onset 1 pt). Time to first response after reinfusion was 28 days. The D+28 ORR for evaluable pts (n=2) was 100%. Median PFS after reinfusion is NR. Post reinfusion 1 pt (Ph+ B-ALL) died of disease progression at 313 days (50 days after reinfusion). The evaluable B-NHL pts are in remission. Conclusions: Reinfusion of varnimcabtagene autoleucel (IMN-003A) is feasible, safe and well tolerated. Preparative conditioning regimen for reinfusion needs personalization guided by CAR persistence, disease biology (CD20, PDL1 expression) and haematological reserve. After reinfusion, prolonged B cell aplasia was observed with 100% ORR at D+28 in evaluable pts. Var-cel was detected post reinfusion in B-NHL patients with sustained responses. Reinfusion may be considered in preference for some pts. The ideal preparative regimen and pt characteristics to guide reinfusion outcomes remains an area of research.

Safety and Efficacy of a Locally Produced Novel Anti-BCMA Chimeric Antigen Receptor T-Cell (CART) (HBI0101) for the Treatment of Relapsed and Refractory Multiple Myeloma

BACKGROUND: Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CART) therapy shows remarkable efficacy in patients with relapsed and/or refractory (R/R) multiple myeloma (MM), with recent phase 3 studies showing clear advantage of idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel)over standard-of-care. However, access to these commercial CART products is a major barrier, and it is estimated that only around a quarter of eligible R/R MM patients will receive CART nowadays. HBI0101 is a novel second generation optimized anti-BCMA CART with 4-1BB co-stimulatory domain, that was developed in an academic setting, at Hadassah Medical Center and Bar-Ilan University. The phase Ia study evaluating HBI0101 (NCT04720313) demonstrated a manageable safety profile and an initial high efficacy (Asherie, Haematologica 2022). Here, we present the updated results of 50 patients receiving 800x10^6 CART cells in the NCT04720313 phases 1a-b / 2. METHODS: The phase 1a study with HBI0101 which first evaluated 20 R/R MM patients with ≥3 prior lines of therapy, including a PI, IMiD and anti-CD38 antibody, revealed a dose of 800x10^6 CART cells to be safe and effective. The phase Ib and phase 2 of the study evaluated this dose infusion in 43 additional patients. Inclusion criteria were relatively permissive with minimum creatinine clearance of 20ml/min, platelet count of 30x10 9/ml, left ventricular ejection fraction of 40%, and ECOG performance status of ≤2. Lymphodepletion included fludarabine 25mg/m 2 and cyclophosphamide 250mg/m 2 on days -5 to -3 before infusion (bendamustine 90mg/m 2 and on days -4 and -3 for patients with creatinine clearance <30ml/min). Planned manufacturing time was 10 days. RESULTS: Of 51 patients who underwent lymphocyte apheresis, 50 (98%) were treated with 800x10^6 CART cells; 7 patients in the phase Ia cohort and 43 in the phases 1b / 2 cohorts. HBI0101 production success rate was 100%, with manufacturing time of 10 days for all patients. The median age was 65y (range 40-84). The median number of prior lines was 4 (range 3-13) and most patients (47/50, 94%) were triple refractory to PI, IMiD and anti-CD38 antibody. Twenty patients (40%) were penta-refractory and 10 (20%) were refractory to the anti-BCMA antibody drug conjugate belantamab mafodotin. Forty-six patients (92%) were refractory to the last line of therapy. Twelve patients (24%) had high risk cytogenetics (t(4:14), t(14:16) or deletion 17p); 31 (62%) when 1q gain included. Sixteen patients (32%) had extramedullary involvement at study entry. Grade 3-4 hematological toxicities were common (anemia- 62%, thrombocytopenia- 54%, neutropenia- 98%, lymphopenia- 100%). Cytokine release syndrome (CRS) occurred in 48/50 (96%)- grade 1/2- 17 patients (82%); grade 3- 7 patients (14%). Tocilizumab was used in 40/48 patients with CRS (median of 1 dose, range 1-4) and corticosteroids in 8/48. Two cases of immune effector cell associated neurotoxicity syndrome (ICANS) were observed (grade 1 and 2). Two patients developed CART-associated hemophagocytic lymphohistiocytosis syndrome (HLH). No irreversible organ toxicities or treatment related deaths occurred. The overall response rate (ORR) was 45/50 (90%), including 29 patients (58%) with complete response (CR)/stringent CR, 10 (20%) with very good partial response and 6 (12%) with partial response. Thirty-five patients (70%) achieved minimal residual disease (MRD) negativity at day +30. At a median follow-up of 11.9 months (range: 0.6-19), the median progression-free survival was 10.6 months, and the median overall survival was not reached (Figure 1). Although the presence of extra-medullary disease and prior anti-BCMA therapy correlated with worse outcome, high response rates were still observed (ORR of 98%/75% for patients without/with extra-medullary disease, respectively, and 95%/75% for patients without/with prior anti-BCMA therapy, respectively). Updated results will be communicated at the presentation time. CONCLUSION: Our findings demonstrate the manageable safety and high efficacy profiles of HBI0101 also in high-risk patients. Fast production time enabled the timely treatment of 98% of apheresed patients. These favorable data are encouraging and support decentralization of CART production at an academic setting, ensuring a sufficient CART supply in the light of the increasing demand.

Varnimcabtagene Autoleucel (IMN-003A): Differences in T Cell Subset Phenotype in B-ALL and B-NHL Cohorts Did Not Influence Efficacy Outcomes in the Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies. A fractionated infusion of 1 x 10 6 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 10 6 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days (10%, 30%, 60%). Phenotypic data comparing the apheresis (AP) and final product (FP) from B-ALL and B-NHL cohorts, related manufacturing and clinical response observations are discussed in this abstract. Methods: IMN-003A was manufactured using the Miltenyi CliniMACS Prodigy®, a cGMP compliant closed system. The apheresis and final infusion product (FP) were analysed for percent CAR transduction and T cell subsets by flow cytometry. CAR copies in the final product were determined by ddPCR. Cell counts measured using automated Vi-cell counter were used to calculate fold expansion. Results: Apheresis PBMC from 25 patients in the IMAGINE study were selected for T-cells, transduced & expanded to achieve the FP, which was infused in 24 patients (1 patient withdrawn). Three patients needed a second apheresis and re-manufacturing to achieve the target dose. The FP comprised primarily of CD3+ T cells (median 99.38%, range 96.3 - 99.92%) with mean of 38.18% CAR+ T cells for B-ALL cohort (range: 20.28 - 58.35%) and significantly lower 27.53% CAR+ T-cells for B-NHL cohort (range: 8.5 - 51.07%, p=0.02). The vector copy number (VCN) however did not differ between the two cohorts (mean of 2.66 and 2.37 copies for B-ALL and B-NHL cohorts respectively; p=0.4). The fold expansion at Day 6 of manufacturing was also significantly lower for B-NHL cohort (6.93) compared to the B-ALL cohort (10.33, p=0.02). Consistent with the relatively more challenging manufacturing for the B-NHL cohort with 5 times higher target dose, it was observed that the mean proportion of naïve T-cells (CCR7 + CD45RA +) was lower in both AP (B-NHL 9.71%, B-ALL 27.24%, p<0.01) and FP (B-NHL 16.68%, B-ALL 48.94%, p<0.0001), while the terminally differentiated T EMRA and effector memory T EM subsets were higher in the AP (B-NHL 53.8%, B-ALL 45.08%, p=0.175) & FP (B-NHL 48.77%, B-ALL 29.54%, p=0.021), highlighting the T-cell phenotypic differences in the two cohorts. The mean CD4/CD8 ratio in AP & FP was 0.56 and 1.14 respectively. There was reversal of CD4/CD8 ratio from apheresis to final product (CAR+ T cells, ratio=3.37) which was statistically significant (p<0.01). The CD4:CD8 ratio did not differ between B-ALL and B-NHL cohorts. This could potentially be explained by the higher proportion of naïve T-cells observed within CD4 subset compared to CD8 subset as against the proportion of T EMRA cells. Despite the higher fraction of CD4 CAR-T cells within the final product infused, post-infusion, there was a statistically significant trend (p<0.01) towards reversal of CD4/CD8 ratio in the peripheral blood from D0 to D+90. Ongoing flow-cytometry based analysis shall help further characterise the CAR+ T-cells post-infusion. Interestingly, despite phenotypic differences in T-cell subsets between B-ALL and B-NHL, overall response rate (ORR) at D+90 was very similar for both cohorts [B-ALL 80% (n=8/10); B-NHL 81.8% (9/11)]. Updated results shall be presented at the meeting. Conclusions: Differences in the manufacturing outcomes (CAR % transduction, fold expansion) and T cell phenotype (naïve and effector T EMRA cell subsets) were observed in Apheresis and FP, between the B-ALL and B-NHL cohorts. A higher fraction of relatively more differentiated T cell phenotypes of effector (T EMRA) and effector memory (T EM) and a lower fraction of naïve T cells in the B-NHL cohort and within CD8 T cells, reflects a manufacturing conundrum for B-NHL patients with a higher target dose and the CD4:CD8 ratio in the final product. However, these differences in the final product did not adversely impact the clinical responses at D+90 with equivalent efficacy in both cohorts. The ideal T cell phenotype or their proportions within the final product for maximum efficacy thus remains uncertain. A deeper characterisation including exhaustion status of the apheresis, final product and post-infusion samples for the T cell subsets can provide further insights.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

Phase I Clinical Study of Humanized BCMA-Single-Domain Antibodies-Targeting CAR T (BCMA-CART) in Patients with Relapsed/Refractory Multiple Myeloma

Background:The CAR structure adopted by CART-BCMA is composed of a single-domain antibody targeting BCMA, CD8 hinge region, transmembrane region, 4-1BB co-stimulatory domain, and CD3-ζ T cell activation domain. In the phase I clinical study (NCT05346198) carried out in China, the safety and efficacy of CART-BCMA were initially evaluated through the dose escalation + dose expansion design, and the recommended dose (RD dose) for follow-up studies was determined as well. Method:Enrolled patients with relapsed and/or refractory multiple myeloma (RRMM) had previously received at least 3 lines of therapy including at least one proteasome inhibitor and one immune modulator. After lymphatic preconditioning chemotherapy (program: fludarabine 30mg/m 2/d and cyclophosphamide 300mg/m 2/d, -5, -4, -3, for 3 consecutive days), all patients received a single dose of CART-BCMA at 2.5×10 6, 5×10 6 and 7.5×10 6 CAR-positive T cells/kg (subject body weight), according to the dose level in which they were enrolled. R esults :Up to June 26, 2023, a total of 15 cases of RRMM received 2.5×10 6 CAR-positive T cells/kg (n=3), 5×10 6 CAR-positive T cells/kg (n=11) or 7.5×10 6 CAR-positive T cells/kg (n=1) dose level of CART-BCMA cell infusion therapy on the basis of the dose level in which they were enrolled. The median follow-up time was 14.42 months (range: 10.2-26.6 months). The median age of the subjects was 63.0 years old (range: 36-73 years old) (Table 1), 73.3% of the subjects (n=11) had previously received ≥4 lines of multiple myeloma therapy, and the median line of treatment was 5 lines (range: 3-9 lines), among which, 2 subjects (13.3%) had previously received autologous hematopoietic stem cell transplantation, and 4 subjects (26.7%) had relapsed/refractory diseases after previous treatment with proteasome inhibitors, immune modulators and CD38 monoclonal antibodies (three-drug relapsed/refractory). Nine subjects (60.0%) had high-risk cytogenetic abnormalities according to sMART 3.0 criteria, and 6 subjects (40.0%) had extramedullary plasmacytoma. The most common grade ≥3 adverse events were hematological toxicity, including decreased neutrophil count (n=13, 86.7%), decreased white blood cell count (n=12, 80.0%), decreased lymphocyte count (n=10, 66.7%), anemia (n=8, 53.3%), decreased platelet count (n=4, 26.7%). The first patient in the 7.5×10 6 CAR-positive T cells/kg dose group had a dose-limiting toxicity (DLT) event of grade 4 platelet count decrease, and no DLT event occurred in the other low-dose groups (2.5 and 5×10 6 CAR-positive T cells/kg). All 15 patients (100.0%) developed grade 1-2 cytokine release syndrome (CRS), no grade ≥3 CRS occurred, and the median time to CRS was 2.0 days (range: 1-13 days) , with a median duration of 7.0 days (range: 2 to 17 days). After the occurrence of CRS, all patients recovered without sequelae after supportive treatment, and 6 patients (40%) received tocilizumab (3 subjects received 2 doses, 3 subjects received 1 dose), 4 patients (26.7%) used glucocorticoids. There was no event of immune effector cell-associated neurotoxicity syndrome (ICANS), and no death event occurred. The median time to the first response (TTR) of the patients was 0.953 months (range: 0.92 to 2.23 months). All 15 patients who received CART-BCMA infusion achieved remission, and the overall remission rate (ORR) ( At least achieve PR or better efficacy) was 100% (95%CI, 78.20%-100.00%), as shown in Figure 1, among which, the best response of 9 patients (60%) was strict complete remission ( sCR), 5 patients (33.3%) achieved very good partial response (VGPR), and 1 patient (6.7%) achieved PR. All sCR patients achieved MRD negativity. The ORR of 6 patients with extramedullary plasmacytoma was 100%, of which 2 achieved sCR and 4 achieved VGPR. Notably, gradual deepening of remission was observed in 12 patients (80%), of which 10 patients (66.7%) further deepened the depth of remission on days 60-90 after infusion, and 2 patients (13.3%) were assessed sCR at day 360. Conclusion:Data from this phase 1 clinical study showed that CART-BCMA is well tolerated and highly efficacious in patients with RRMM. Patients with extramedullary plasmacytoma had the comparable response to those without extramedullary diseases. Notably, 12 patients (80%) patients elicted deeper response after 3 months of CART-BCMA infusion

Varnimcabtagene Autoleucel (IMN-003A): Pharmacokinetic Profile with Predominant Naïve and Central Memory Phenotype Demonstrates Sustained In Vivo Persistence and Durable Responses in a First-in-India Industry Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies (RR BCM). A fractionated infusion of 1 x 10 6 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 10 6 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days as 10%, 30% and 60% fractions after Fludarabine-Cyclophosphamide lymphodepletion regimen (LR). Here we present var-cel pharmacokinetic data and correlation with efficacy. Methods: Varnimcabtagene autoleucel was manufactured using a cGMP compliant closed system (CliniMACS Prodigy ®). The manufacturing data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). Peripheral blood (PB) samples were obtained after consent at screening and as per patient schedule after infusion. Persistence of IMN-003A was evaluated in PB after infusion by ddPCR. Safety and efficacy data were collected for analysis. Bone marrow (BM) MRD (B-ALL) and PET-CT (B-NHL) was assessed at screening, D+28 and D+90 for efficacy. Minimal residual disease (MRD) was performed by FC with 10 -4 sensitivity. Apheresis, FP, and PB/BM samples on D+28 were analyzed for T-cell subpopulations and surface markers by FC. Statistical analysis was calculated using appropriate tests. Results: Of 25 patients (pts) enrolled and apheresed, 24 pts received var-cel (1 withdrawal) with majority of infusions on Days 0, +3, +7. Median product doubling time and manufacturing time was 1.04d (range 0.74-2.12) and 14 days (range 10 - 27) with 100% success with T cell purity median 99.4% (range 96.3-99.9). The mean % transduction of var-cel was 33.12% (range 8.50 - 58.35) with median transgene copies / genome of 2.57 (range 0.76 - 4.16). The mean CD4/CD8 ratio at apheresis and final product was 0.47 and 2.58 respectively. There was reversal of CD4/CD8 ratio from apheresis to final product (CAR+ T cells) which was statistically significant (p<0.01). There was statistically significant increase (p<0.01) in the number of naïve (CCR7+ RA+) and central memory (CCR7+ RA-) T cell subsets between apheresis and final product (CAR+ T cells). There was predominance of naïve and central memory CAR+ T cell subsets in the FP (58.19%; Figure 1A). Median Apheresis to Infusion time was 25d (range 16 - 95). There was a statistically significant trend (p<0.01) towards reversal of CD4/CD8 ratio in the PB from D0 to D+90. Var-cel showed maximum PB expansion (T max) at D+10 (range 7 - 28 days) with median C max 125,242 CAR copies / µg gDNA (range 18256 - 413,968). Among pts with 28-day and 90-day and last follow-up, 79.2%, 29.2% and 20.8% respectively had evidence of var-cel in PB, with median persistence of 28 days (range 10 - NR). Post-infusion, peak interleukin-6 level was median 42.3 pg/mL (range 6.71 - 17,481). B cell aplasia was observed in all pts concurrent with CAR-T expansion (Figure 1B). Median loss of B cell aplasia was not reached (range 42 - NR), with no apparent association with response at D+28 and D+90. Hypogammaglobulinemia was noted in 11 pts and 16 pts received IVIg. Of evaluable patients with CD4+ T-cell counts below 200/μL, recovery (>200/μL) was seen at median of 12 days (range 2 - NR) after var-cel infusion. Overall response rate (ORR) was 91.7% (22/24) at D+28 (B-ALL 91.7%, MRD neg 83.3% (n=10/12); B-NHL 91.7%, CR 66.7%) and 80.9% (17/21) at D+90 [B-ALL 80%, MRD neg 80% (n=8/10); B-NHL 81.8% (9/11), CR 63.6%]. One B-NHL pt in PR at D+28 achieved CR at D+90. Three pts relapsed by D+90. Updated results will be presented at the meeting. Conclusion: Varnimcabtagene autoleucel manufacturing process results in a drug product characterized by highly pure T cells, with high proportion of naïve and central memory polyfunctional cells. In this industry-led first-in-India phase-2 study, var-cel resulted in a cytokine response with sustained in vivo proliferation and persistence resulting in robust and durable responses.

PBMC-Humanized Mouse Model to Assess Individual Variability in Efficacy and Toxicity of Universal CD19 Chimeric Antigen Receptor T Cell Immunotherapy

Chimeric antigen receptor T cell (CART) immunotherapy has been accumulating extraordinary breakthroughs in fighting against hematological malignancies. Particularly, universal, “off-the-shelf”, CARTs (uCARTs) are emerging as an attractive alternative to patient-derived CARTs, offering cost-effectiveness, immediate availability for treatment, and independence from patients' lymphopenic conditions. However, uCARTs share the same safety concerns with autologous CARTs while the efficacy and persistence of uCARTs vary by each product and remain uncertain. Further, T cells sourced from healthy donors for uCARTs may manifest individual-specific immune characteristics upon interaction with cancer cells and the patient's immune system. These characteristics often remain latent when assessed in vitro and complicate the prediction of uCART therapy's toxicity and efficacy on an individual basis. We previously demonstrated that the PBMC-humanized mouse model can capture efficacy and toxicity of autologous and allogeneic CD19 CARTs. We showed that the CART-induced toxicity varied by the combination of PBMC donors and CART cell donors. Here, we extend the use of PBMC-humanized mice to assess the potential toxicity, efficacy, and persistence of uCARTs. Female NSG™-DKO mice (n = 19-20 mice per donor) were irradiated and reconstituted with human PBMCs sourced from four selected donors (Donor A, B, C, and D) out of 58 available in our PBMC bank. The selection criteria were based on their pre-characterized in vivo human cytokine induction profiles, specifically IFN-gamma response levels to OKT3 treatment (Donor A: 268 pg/ml; B: 1936 pg/ml; C: 4862 pg/ml; D: 13538 pg/ml). PBMC-humanized mice were grouped into three different CD19 CART treatments; a) Allogeneic CARTs were manufactured from a healthy donor's T cells containing CD28 costimulatory domain (19-28z) and no genetic modification was made for “off-the-shelf” availability; b) uCART 1 cells (19-28z) were knocked out of TCRab via CRISPR-cas9 (19-28z); c) uCART 2 cells were knocked out of CD3 and TCRab using TALENs and magnetically depleted for remaining CD3/TCRab, and contain 4-1BB costimulatory domain. Body weight change and clinical scores were measured throughout the experiments. We measured the levels of human CD45, human CD19, and CART expansion from blood collected 8 days post-treatment using flow cytometry. We measured 54 human cytokine levels from serum collected 48 hours post-treatment using a multiplex assay. CART-induced toxicity, measured by body weight loss, dramatically varied by PBMC donors and the treatments (Figure 1). uCART 2 induced significant body weight loss in Donor B and D reaching the lowest 6 days post-treatment. Notably, all mice humanized with Donor C and treated with uCART 2 reached a humane endpoint within 12 days post-treatment. We only observed noticeable body weight loss from uCART 1 in Donor C. Allogeneic CART did not induce body weight loss across the four donors. Efficacy, measured by the % of elimination of human CD19 cells in peripheral blood compared to the PBS-treated group, also varied by PBMC donor and the treatments (Figure 2). Overall, Allogeneic CART showed the best elimination of CD19 cells. Mice humanized with Donor D responded with the poorest efficacy of the three CART treatments. CART expansion was observed only in uCART 2 treated mice that reached a humane endpoint within 6 days post-treatment. CART persistence measured 16 days post-treatment was not observed across three CARTs and this corresponds to the clinical data of uCARTs. Principal component analysis of human cytokine data revealed that uCART 2 induced the most distinct cytokine responses compared to the other CARTs, driven by IFN-gamma, IL-10, IL-5, IL-3, and GM-CSF. In Donor-A humanized mice, the same set of human cytokines (IFN-gamma, IL-10, IL-5, IL-3, GM-CSF, IL-13) was induced in response to the three CARTs, while the degree of induction varied by treatment. In contrast, Donor D-humanized mice showed vastly different responses to each treatment; uCART 2 induced significant levels of IL-6, TNF-beta, MIG/CXCL9, MDC, MCP-3, IL-13, and IL-2. In sum, we show that PBMC-humanized mice capture individual variations in the toxicity and efficacy of uCARTs. This platform can be used to preclinically assess efficacy and persistence of uCARTs as well as used to predict toxicity from the interaction between the host and healthy T cell donor of uCARTs.

Comparative Analysis of Immune Effector Cell-Associated Hematotoxicity (ICAHT) and Infection Incidence Following CD19-Targeted CAR-T Therapy Versus CD7-Targeted CAR-T Therapy

Introduction CD19 CAR-T cell therapy has been revolutionizing the treatment of refractory /relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) over the past decade. While CD7 CAR-T therapy, an innovative approach, has only recently been applied over the last three years in our center to treat R/R T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). While there are studies and guidelines from EBMT addressing hematologic toxicity, specifically Immune Effector Cell-Associated Hematotoxicity (ICAHT), following CD19 CAR-T treatment, the literature is still scarce on ICAHT occurrence post-CD7 CAR-T therapy. Here we conducted a retrospective study aiming at comparing ICAHT and infection incidences following the two distinct CAR-T therapies. Methods Data were extracted from two separate clinical trials. The CD19 CAR is structured with CD19 scFv, CD28-hinge, 4-1BB costimulatory region, and CD3ζ activation domains. T cells were activated by CD3/CD28 microbead 2 days before CD19-CAR lentivirus infection. “Naturally selected” anti-CD7 CAR-T cells (NS7CAR) were transduced an anti-CD7 CAR into bulk peripheral T lymphocytes via a lentivirus vector. NS7CAR is a 2nd generation murine-based CAR-T containing 4-1BB and CD3ζ co-stimulatory domains. All patients received lymphodepletion regimens of fludarabine (30 mg/m 2/d) and cyclophosphamide (300 mg/m 2/d) from Day -5 to Day -3 before CAR-T infusion. A single infusion of either CD19 CAR-T or CD7 CAR-T cells was administrated at a medium dose of 1×10 6/kg. Fitted patients undergo consolidation allogeneic hematopoietic stem cell transplantation (Allo-HSCT) within 1-3 months after CAR-T cell infusion. The hematology toxicity and infection were analyzed during the period from CD19 CAR-T or CD7 CAR-T cell infusion to Allo-HSCT. Hematology toxicity is graded according to “ICAHT: EHA/EBMT Consumption Grading and Best Practice Recommendations” (Table 1). Infection diagnosis includes both etiological diagnosis and imaging diagnosis. Results Between June 1, 2019, and September 22, 2022, 60 patients with R/R B-ALL patients received CD19 CAR-T therapy (https://clinicaltrials.gov NCT04546893, and NCT04792593). Meanwhile, between December 1, 2020, and July 16, 2022, 60 patients with R/R T-ALL/LBL received CD7 CAR-T therapy (NCT04572308, NCT04916860). The CD19 CART group had 19 patients ≤ 14 years old, with a male/female gender ratio of 33:27. The CD7 CART group had 20 patients ≤ 14 years old, with a gender ratio of 49:11. The incidence of severe early ICAHT (Grade III+IV, day 0-30) from CD19 CAR-T cells infusion to allo-HSCT was observed to be 18.3%, which was lower than the incidence of the early ICAHT post CD7 CAR-T therapy at 33.3%, p =0.035. However, the incidence of severe late ICAHT (after day+30) following CD19 CAR-T and the CD7 CAR-T therapy was similar: 27.8% vs. 28.6%, with no statistical difference( p=0.57). There was no statistical difference between CD19 CAR-T and CD7 CAR-T therapy groups in the infection rates with 28.3%vs. 36.7%, p=0.33. In the CD19 CAR-T group, bacterial infections were noted in 10 patients: 6 with Gram-positive bacteria (1 Enterococcus, 1 Streptococcus agalactis, and 4 Staphylococcus), and 4 with Gram-negative bacteria (1 Pseudomonas aeruginosa, 2 Acinetobacter baumannii, and 1 Klebsiella pneumoniae). Viral infections were observed in 7 patients (EBV 2, CMV 2, BKV 3), and 1 had fungal infection. Infection sites included pulmonary infections (7 cases), sepsis (10 cases), and other areas (4 cases). Post-CD7-CAR-T, bacterial infections were observed in 9 cases, 7 with Gram-positive bacteria (6 Staphylococcus, and 1 Streptococcus bradycardia) and 2 with Gram-negative bacteria (1 Pseudomonas aeruginosa, and 1 Klebsiella pneumoniae). Six cases had viral infections (EBV 2, CMV 2, HHV1 type 1, parainfluenza virus 1), and 6 with fungal infections. Infection sites included pulmonary infection (12 cases), sepsis (10 cases), and other areas (5 cases). Conclusions Our study noted a slightly higher incidence of ICAHT within 30 days following CD7 CAR-T infusion compared to CD19 CAR-T therapy. However, there was no statistical difference in the incidence of ICAHT beyond the 30-day. There was no statistical difference for infection rate after CD19 CAR-T and CD7 CAR-T. In both groups, Gram-positive bacteria were primary responsible for bacterial infections.

Targeting Normal Myelopoiesis Negatively Affects CAR T Cell Activity

Chimeric antigen receptor (CAR) T cells have not been successful for the treatment of acute myeloid leukemia (AML) to date, and the reason for this is not clear. While CAR T cells have been traditionally studied as independent agents targeting tumor, in reality they work in the context of the endogenous host immune system, which may influence outcomes of therapy. We hypothesized that targeting normal myelopoiesis is detrimental to CAR T cell activity, and this creates a unique obstacle to effective CAR T cell therapy for AML. We generated prototypical CAR T cells targeting B cell (CD19) and myeloid (CD33) antigens, using a standard 2 nd generation CAR design with 4-1BB and CD3ζ costimulatory domains. In parallel we created tumor cell lines expressing both targets by adding CD33 expression to a CD19+ B cell line (Ramos +33), and by adding CD19 expression to a CD33+ myeloid cell line (THP1 +19). For ease of tracking tumor cells were labeled with GFP, and CAR T cells were labeled with RFP. CD19-targeting CAR T cells (CART19) and CD33-targeting CAR T cells (CART33) exhibited similar anti-tumor activity in standard in vitro cytotoxicity assays and in vivo NSG mouse models. We then proceeded to add PBMCs to CAR T cell and tumor co-culture and measured tumor cell clearance and CAR T cell expansion using flow cytometry and the Incucyte Live-Cell analysis system. Addition of PBMCs enhanced the tumor clearance and CAR T cell expansion of both CART19 and CART33 in short-term co-culture assays (7-10 days). Fractionation of PBMCs revealed that CD33+ classical monocytes were able to replicate the effect of PBMCs, while the presence of CD19+ B cells did not have any effect. Monocytes are viable in vitro for less than 7 days. To assess the long-term effects of monocytes on CAR T cells, we performed serial replating assays, in which fresh tumor cells and monocytes were added to CAR T cells every 7 days. Ongoing monocyte support further improved CART19 expansion and tumor clearance. However, the continuous presence of monocytes was detrimental to CART33 activity upon serial replating, as evidenced by suboptimal T cell expansion and loss of tumor control. To study the effects of normal myelopoiesis on CAR T cell activity in vivo, NSGS mice (NOD/SCID/IL2Rg null mice with human SCF, IL-3 and GM-CSF expression) were injected with human cord blood CD34+ cells to engraft human hematopoiesis. Mice were then injected with THP1 +19 tumor cells, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from the same cord blood product as the CD34+ cells. Consistent with our in vitro data, CART19 and CART33 showed better anti-tumor activity in the presence of human hematopoiesis. Notably, while the anti-tumor activity of CART19 and CART33 was equivalent in non-humanized mice, CART19 had better long-term activity in humanized mice, as mice treated with CART33 had late tumor relapses. To directly compare the effects of myeloid cells as bystanders versus targets, we lentivirally transduced cord blood CD34+ cells with human CD19 so that they would be targets for CART19. We then differentiated these cells into myeloid progenitors (MP) by culturing in IMDM with 20% FBS and the following cytokines: SCF (100ng/ml), FLT3L (50ng/ml), IL-3 (10ng/ml), and GM-CSF (10ng/ml). After 10 days of culture >95% of MP expressed CD33, and were selected for >95% CD19 expression (Fig A). Addition of non-transduced MP to CAR T cell and Ramos +33 co-culture enhanced CART19-mediated tumor clearance without affecting CART19 expansion. However, the presence of MP CD19+ had a detrimental effect on both tumor control and expansion of CART19, confirming that CAR T cells are less effective when targeting myeloid cells. In contrast, both MP and MP CD19+ had similar effects on CART33 that mirrored the negative effects of MP CD19+ on CART19 (Fig B). To summarize, we show that normal myeloid cells have beneficial effects on CAR T cell function, and targeting these same myeloid cells is detrimental to CAR T cell activity. These findings provide insight into the reason for suboptimal CAR T cell activity in AML, and show that independent of target antigen and CAR construct, the nature of the cells being targeted can change the effectiveness of this therapy.

Efficacy and Safety of a Third Generation CD20 CAR-T (MB-106) for Treatment of Relapsed/Refractory Indolent B-Cell Non-Hodgkin Lymphoma: Phase-1 Results from a Multicenter Trial

Background: Indolent B-cell non-Hodgkin lymphomas (NHL) remain incurable despite multiple therapeutic options, including chemoimmunotherapy, targeted therapies, bispecific antibodies, and CD19-targeted chimeric antigen receptor T (CAR-T) cells. MB-106, a fully human 3 rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains, had high overall response rate (100%) and complete response (CR) rate (91%) in follicular lymphoma (FL) patients at doses ≥ 3.3×10 6 cells/kg in a single institution trial (Shadman et al., 17-ICML, 2023). We seek to replicate the favorable efficacy and safety profile of MB-106 in a multicenter trial with centralized manufacturing. Methods: MB-106 is being evaluated in a Phase 1/2, open label, multicenter study utilizing a 3+3 dose-escalation design in R/R B-cell NHL patients with confirmed CD20 expression. Prior treatment with CD19-targeted CAR-T therapy is permitted. Following lymphodepletion (cyclophosphamide 300 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days), MB-106 is administered to patients with indolent B-cell NHL at one of two dose levels (DL): DL1, 3.3×10 6; and DL2, 1.0×10 7 cells/kg. MB-106 is infused in the outpatient setting if allowed by the institution, except for the first patient in each dose level cohort (overnight observation). Dose-limiting toxicities (DLT) are monitored through day 28. The treatment response is assessed on day 28 and on subsequent visits, and best overall response is reported here. CAR-T persistence is assessed by both flow cytometry and qPCR in peripheral blood mononuclear cells. Results: Three patients with indolent NHL (2 FL, 1 Waldenström macroglobulinemia [WM]) received MB-106 at DL1. In addition, one patient with hairy cell leukemia variant (HCL-v) received non-conforming material (drug product that failed to meet release criteria) at DL1, following FDA authorization. These four patients were heavily pre-treated with a median of 5.5 prior lines of treatment. High-risk features included progression of disease within 24 months of first-line treatment (POD24, n=1), prior autologous stem cell transplant (n=1) and prior CD19-targeted CAR-T therapy (n=1). No DLTs were observed at DL1. Three patients experienced Grade 1 cytokine release syndrome. There have been no occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS). The 2 FL patients achieved CR by both PET-CT and bone marrow biopsy, one of whom had been previously treated with CD19-targeted CAR-T. The WM patient, who had nine prior treatments, including autologous stem cell transplant, and high disease burden, achieved very good partial response, with complete metabolic response by PET-CT, morphologic clearance of lymphoma in bone marrow, and resolution of the IgM paraprotein. The patient with HCL-v, who had been transfusion dependent, continues to have stable disease with decreased disease in the bone marrow, and achieved complete transfusion independence which is ongoing at 6 months. All patients displayed MB-106 expansion with peak levels between 7-14 days post-infusion. Persistence of CAR T-cells is ongoing at 6 months. Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in responses, including CRs, and CAR-T persistence in patients with R/R indolent NHL and was associated with favorable safety profile with no occurrence of Grade 3 or Grade 4 cytokine release syndrome and no ICANS of any grade. CRs have been observed in patients previously treated with CD19-targeted CAR-T in both this multicenter trial and the original single institution trial. Dosing is ongoing in the final dosing level (1.0×10 7 cells/kg) to establish the recommended Phase 2 dose for a planned pivotal trial in WM. It is anticipated that the complete data set from the Phase 1 indolent NHL arm will be presented.

Evaluation of the T-Cell Metabolic State of Starting Material and Manufactured CAR T Cell Products and Clinical Outcome of CAR T-Cell Therapy for Large B Cell Lymphoma

Background Although chimeric antigen receptor (CAR)-T cells are a viable treatment option for patients with relapsed or refractory (r/r) aggressive B-cell lymphomas, more than half of patients experience relapse within the first two years after product infusion. Successful autologous cell-based anti-cancer therapies require persistence of effector cells, features that highly depend on complex metabolic processes. We earlier demonstrated in chronic lymphocytic leukemia, that expansion and persistence of CD19-CAR T-cell infusion in vivo was correlated with increased mitochondrial mass and enhanced biogenesis(van Bruggen et al, Blood 2019). Metabolic features might therefore serve as early biomarker tools to predict efficacy and outcome of CAR T-cell therapy and might allow therapeutic targeting but did was never studied in a prospective clinical trial. Aims Perform extensive phenotyping and state-of-the-art metabolic state analyses of T cells prior and post CAR T cell production and investigate correlations with clinical outcomes and patient characteristics. Methods Samples were obtained from large B cell lymphoma (LBCL) patients that were part of the TRANSCEND-NHL-001 trial (NCT02631044, Abramson et al, Lancet 2020).Thirty-two patients with r/r B-cell lymphoma who received CAR-T cell treatment lisocabtagene maraleucel (16 responders (R) and 16 non-responders (NR)) were included in our analyses and (metabolically) analyzed by flow cytometry and extracellular flux analysis. Metabolic phenotyping was performed on 53 samples at baseline and 44 samples after an in vitro stimulation. Extracellular flux analysis was done on 14 DP samples. The cohort was split evenly between Responders and Non-responders, and balanced for a score calculated taking key clinical baseline characteristics that were found to be associated with response into account (NCT02631044, Abramson et al, Lancet 2020). Both T cells obtained at time of leukopheresis (starting material (SMAT)) and CAR-T drug products (DP), (following transduction of a CD19-targeted CAR T cell product with a defined CD4 and CD8 composition bearing a 4-1BB costimulatory domain and expansion) were analyzed directly after thawing and after stimulation with anti-CAR-antibody in case of CDP, or αCD3/αCD28 antibodies for SMAT samples. Patients consented to these studies and researchers were blinded for clinical outcomes at time of analysis. Results We found that the baseline clinical factors that associate with clinical response are related to specific metabolic cellular phenotypes, both in SMAT and DP, and with and without T cell stimulation. SMAT samples from patients with higher baseline scores show signs of an increased exhausted phenotype with regards to cellular ROS and exhaustion marker expression, whilst a more plentiful memory-like phenotype was seen in patients with a lower baseline score. When clustering by clinical response status, analysis of glucose and fatty-acid uptake, neutral lipid content, cellular ROS, mitochondrial mass and potential and flux data showed that responders, in both SMAT and DP samples, have an all-round increased metabolic activity upon αCD3/αCD28 or CAR T-receptor triggering, respectively, when compared to non-responders (Figure 1A). Furthermore, responding patients show enrichment of CD27 expression in the SMAT material, indicating a more abundant memory phenotype. Summary/Conclusions This is the first evidence of an association between a specific immune-metabolic phenotype of (CAR-) T cells and outcome of CAR-T cell therapy in patients with relapsed/refractory LBCL. More specifically, a higher abundance of memory phenotypes, metabolic activity and mitochondrial biogenesis of both harvested T cells and CAR-T product associates with improved clinical response. Combining metabolic, phenotypical and clinical characteristics and biomarkers of patient T cells (both pre- or post-production) shows the importance of the metabolic phenotype and fitness of T cell starting material, underlying the importance of early line CAR T therapy following diagnosis.

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models

BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo.MethodsFour different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models.ResultsCD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice.ConclusionsCD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

A Phase I Clinical Trial of Fully Human Anti-CD19 Chimeric Antigen Receptor T Cells for Relapsed or Refractory Lymphoid Malignancies

Background: Chimeric antigen receptor (CAR) T-cells have revolutionized the standard of care for numerous lymphoid malignancies but continue to present challenges related to toxicity, long-term efficacy, cost, and accessibility. Novel CAR-T cell constructs hold promise for improving desired outcomes. CD19 CAR-T cells with fully humanized chimeric antigen receptors have demonstrated excellent preclinical activity and the potential for lower rates of cytokine release syndrome (CRS) and severe immune effector associated neurotoxicity syndrome (ICANS). To evaluate safety, we conducted a phase 1 clinical trial evaluating fully human Hu19-CD841BBZ CAR-T cells with a 4-1BB costimulatory domain against relapsed or refractory (R/R) CD19-positive lymphoid malignancies, utilizing a single lymphodepletion regimen and local CAR-T cell manufacturing using the Prodigy® Device. Methods: We designed a parallel-group phase I trial with two clinical groups. Group A enrolled subjects with relapsed and refractory CD19+ lymphomas and chronic lymphocytic leukemia (CLL), while Group B comprised patients with acute lymphoblastic leukemia/lymphoma (ALL). The primary endpoint was safety and toxicity assessment, along with the identification of the recommended phase 2 dose in each clinical group. Secondary endpoints included efficacy evaluation. Dose escalation in each group followed a “3 + 3” phase I study design, with three dose levels: 5e5 CAR-T cells/kg, 1e6 CAR-T cells/kg, and 2e6 CAR-T cells/kg. Lymphodepletion consisted of cyclophosphamide 60mg/kg on day -6 and Fludarabine 25mg/m2/day on days -5 to -3. Dose-limiting toxicities included prolonged CRS grade 3 or higher, ICANS grade ≥ 3, and grade ≥ 3 non hematologic toxicities with the exception of self-limited (<7 days) laboratory abnormalities without associated symptoms or clinical consequences. Results: As of 7/1/2023, a total of 13 patients were enrolled in Group A (NHL), and two patients were enrolled in Group B (B-ALL). Three patients in Group A withdrew consent or were withdrawn at the investigator's discretion due to new comorbid conditions before cell collection. Additionally, three patients were unable to receive CAR-T cell infusions due to disease progression, including two with Burkitt lymphoma. Among the remaining patients, 7 in NHL and 2 in B-ALL, received CART cells. The average age was 59 (range: 40-73), and 2 patients were female. In the NHL group, diseases treated included CLL (3), mantle cell lymphoma (2), follicular lymphoma (1), and Waldenstrom's (1). The median number of prior treatments was 4 (range: 3-10). CAR-T cell product manufacturing was successful in all patients to achieve the target dose, with a mean transduction efficiency of 43.8% (29.7% - 54.6%). One potential dose-limiting toxicity, nodular regenerative hyperplasia of the liver, considered possibly related to CAR-T cells, was observed in the NHL group at the 1e6 cells/kg dose level, leading to an expansion of this dose level. Other observed toxicities were as expected and manageable. Six patients experienced CRS, with grades 1-2 in the NHL group. Both patients in the B-ALL arm experienced CRS, one grade 1 and one grade e, with observed severity congruent with known disease burden before receiving CAR-T cells. Two cases of CRS required no intervention, while the remaining cases received tocilizumab (n=4), steroids (n=2), and/or anakinra (n1). There were three occurrences of ICANS, two in Group A and one in Group B. Treatments included corticosteroids (n=3) and anakinra (n=2). Among the 7 NHL patients evaluable for response, 2 achieved complete remission (follicular, mantel cell), three had a partial response (CLL), and one exhibited stable disease (Waldenstrom's). Two NHL patients (CLL and Mantel Cell) had subsequent disease progression. One B-ALL patient evaluable for response achieved a CR but experienced disease progression at one year and subsequently died. Discussion: In this parallel-group phase 1 study, fully human Hu19-CD841BBZ CAR-T cells with a 4-1BB costimulatory domain were successfully manufactured in all patients undergoing leukapheresis. Human CAR-T cells demonstrated preliminary safety and clinical activity in the setting of heavily pre-treated patients with R/R B cell malignancies. A platform of point-of-care manufacture permits access to novel CAR-T cell products, and may facilitate the development of novel immune effector cell products.

Primary Analysis of Varnimcabtagene Autoleucel (IMN-003A) in Phase 2 Study (IMAGINE), a First-in-India Industry CD19-Directed CAR-T Cell Therapy for Patients with Relapsed Refractory B Cell Malignancies

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous second-generation CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 based murine single chain variable fragment targeting CD19 (A3B1 binder), manufactured in India. Preclinical and Phase 1 study was conducted at HCB / IDIBAPS Spain. Here, we present the primary analysis of IMAGINE, a Phase 2, multicenter, single-arm study of var-cel for patients in India with relapsed/refractory B cell malignancies (RR BCM) (CTRI/2022/03/041162). Methods: Patients (pts) aged 3 to 45 years (B-ALL) and ≥ 18 years (B-NHL) with RR BCM were eligible if they had measurable disease, as assessed by lymphoid blasts (B-ALL) or metabolic tumour bulk (B-NHL), received ≥1 prior regimen, refractory to the last line of treatment with good performance status (ECOG 0 to 1). Bridging therapy was allowed after apheresis. Cyclophosphamide (300 mg/m 2) and fludarabine (30 mg/m 2) on days -5 to -3 were used as preparative lymphodepletion regimen. The target dose was 1x10 6/kg CAR+ cells (B-ALL) and 5x10 6/kg CAR+ cells (B-NHL) (overall range 0.1x10 6 to 5x10 6) and was administered in a fractionated manner (10%/30%/60%) over 3 days with at least 24h between infusions. Primary objectives were overall response rate (ORR: CR + CRi in B-ALL and CR + PR in B-NHL) at day +90 after first infusion, and safety. Response was assessed as per NCCN (B-ALL) and IWG (B-NHL) criteria; bone marrow minimal residual disease (MRD) for B-ALL was analyzed by flow cytometry at 10 -4 sensitivity and PET-CT for B-NHL. Adverse events (AEs) were graded using CTCAE v5.0. CRS and ICANS were graded according to the American Society for Transplantation and Cellular Therapy (ASTCT) criteria. Results: At data cut-off, of 25 pts enrolled (median age 31 yrs, range 3 - 66) with RR BCM [n=13 B-ALL, median blasts 0.34% (range 0.01 - 27.2); n=12 B-NHL, median TMTV 67.9 ml (range 27.1 - 1177), median SPD 3846 mm 2 (range 1001 to 35849], 24 pts received var-cel (1 withdrawal) with majority of infusions on Days 0, +3, +7 and 12 pts (50%) needed bridging therapy. Median CAR-T cell manufacturing time was 14 days (range 10 - 27) with 100% manufacturing success. 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%, MRD neg 83.3% (n=10/12); B-NHL 91.7%, CR 66.7%) and 80.9% (17/21) at D+90 [B-ALL 80%, MRD neg 80% (n=8/10); B-NHL 81.8% (9/11), CR 63.6%]. One B-NHL pt in PR at D+28 achieved CR at D+90. Three pts relapsed by D+90. Of MRD evaluable pts, response was 100% at day+28 (n=11/11) and 88.9% at D+90 (n=8/9). Median time to first response was 28 days. Median progression free survival (PFS, range 12-NR), duration of response (DOR, range 0-NR) and overall survival (OS) were not reached (range 12-NR). 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. Tocilizumab, steroids and anakinra usage was in 37.5% (n=9/24, majority for persistent G1 CRS), 8.3% and 4.2% respectively. Treatment related mortality was 4.2% (n=1/24); 3 pts died of disease progression. IMN-003A cells demonstrated peak expansion on D+10 (range 7 - 28 days). 79.2% at D+28 and 29.2% at D+90 had measurable CAR+ T cells in peripheral blood; median D+28 (range D+10 - NR). Updated results will be presented in the meeting. Conclusions: Varnimcabtagene autoleucel (IMN-003A), a First-In-India Industry CD19-directed CAR-T Cell Therapy for RR BCM, has demonstrated manageable safety profile and durable efficacy outcomes with deep responses including absence of severe neurotoxicity. This offers a significant benefit over standard treatment options for patients with RR BCM.

Naturally Selected CD7-Targeted Chimeric Antigen Receptor (CAR)-T Cell Therapy for Refractory/Relapsed Acute Myeloid Leukemia: Phase I Clinical Trial

Introduction Refractory or relapsed (r/r) acute myeloid leukemia (AML) is associated with a relatively poor prognosis, even in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), emphasizing the critical need for novel therapies. Approximately 30% of AML patients express CD7 on their leukemic blasts and malignant progenitor cells. Naturally selected CD7 CAR-T (NS7CAR-T) therapy has shown significant efficacy with a favorable safety profile in T-cell lymphoid malignancies. In a phase I clinical study (https://clinicaltrials.gov NCT04938115), we investigated the safety and efficacy of CAR-T therapy for treating r/r AML patients with CD7-positive disease. Methods Peripheral blood mononuclear cells were obtained from either the patients themselves (n=9) or the transplant donor (n=1) in cases of relapse post-transplant. T-cells were then purified using CD3+ magnetic beads. The second-generation CD7CAR with a 4-1BB costimulatory domain was manufactured following the manufacturer's protocol. Before the CAR-T cell infusion, bridging therapies were permitted for patients with rapid disease progression. All patients received intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) lymphodepletion chemotherapy for three consecutive days (Day -5 to Day -3). The median time from leukapheresis to CAR-T cell infusion was 15 days. Results Between June 2021 and January 2023, we enrolled 10 patients with CD7-positive r/r AML (CD7 expression >50% with good intensity) and administered NS7CAR-T cell infusions, with 4 receiving a low dose (5×10 5/kg) and 6 receiving a medium dose (1×10 6/kg). Table 1 displays the characteristics of the enrolled patients, revealing a median age of 34 years (7-63 years) and median bone marrow (BM) blasts percentage by flow cytometry (FCM) of 17.0% (2.0-72.7%) at enrollment. One patient presented with diffuse extramedullary disease (EMD). Before enrollment, patients had undergone a median of 9 (range: 3-17) prior lines of therapy. Seven patients had a history of allo-HSCT and the median interval period from the prior transplant to relapse was 12.5 months (3.5-19.5 months). Following infusion, the median peak of circulating NS7CAR-T cells was 2.72×10 5 copies/μg (0.671~5.41×10 5 copies/μg) genomic DNA, which occurred around Day 21 (Day 14 - Day 21) based on q-PCR, with 64.68% (40.08%~92.02%) occurring on Day 17 (Day 11 - Day 21) according to FCM. The median transduction efficiency of the products was 95.6% (70.4%-98.5%). At four weeks post NS7CAR-T cell infusion, 7/10 (70%) patients achieved complete remission (CR) in BM, and 6 of them attained minimal residual disease (MRD)-negative CR. Three patients showed no remission (NR), including 1 with EMD who had partial remission (PR) based on PET-CT evaluation on Day 35. All NR patients were found lost CD7. The median observation time was 178 days (28-752 days). Among the 7 patients who achieved CR, 3 who relapsed from prior transplants underwent consolidative 2 nd allo-HSCT about 2 months after CD7 CAR T-cell infusion. Two patients remained in leukemia-free survival on day 752 and day 315, respectively, while 1 patient died on day 241 due to transplant-related mortality. Among the other 4 patients without consolidative allo-HSCT, 3 relapsed on day 47, day 83, and day 115, respectively (all 3 patients were found to have CD7 loss), and 1 patient died from lung infection. Post-infusion, the majority of patients (80%) experienced mild cytokine release syndrome (CRS), with 7 displaying grade I and 1 having grade II CRS, while 2 patients (20%) experienced grade III CRS. None of the patients had neurotoxicity. Among the 7 patients with prior allo-HSCT, 1 who had a relapse approximately 100 days after prior allo-HSCT developed mild skin graft-versus-host disease following CAR-T therapy. Conclusion Our study highlights the NS7CAR-T therapy as a promising approach for achieving a favorable initial CR in CD7-positive AML patients, even in those who have undergone extensive prior treatments and experienced relapse post allo-HSCT. It potentially could serve as a bridging therapy before transplant. CD7 loss is a major issue either in NR patients or relapsed patients. The safety profile of NS7CAR-T therapy was manageable. However, to comprehensively assess the efficacy of NS7CAR-T in treating CD7-positive AML, further data from a larger cohort of patients and longer follow-up time are essential.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p<0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p<0.01).

Cytokine Profile Following Varnimcabtagene Autoleucel (IMN-003A) in Patients with Relapsed Refractory B Cell Malignancies in the First-in-India Industry Phase-2 Study (IMAGINE)

Background: Varnimcabtagene autoleucel (Var-cel, IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies. This abstract evaluates the cytokine profile and its correlation with safety outcomes and CAR-T expansion following var-cel infusion. Methods: Serum was collected pre-infusion and upto 13 timepoints post infusion as per study schedule for evaluating cytokine levels. Nine cytokines (IFN-g, TNF-α, IL-1β, IL-2, IL-7, IL-8, IL-10, IL-15 and MCP-1) were measured using a multiplexed kit on the Magpix platform. IL-6 level was measured using electrochemiluminescence immunoassay (ECLIA). Safety and adverse event data were collected for analysis along with measuring various cytokine levels in the serum. CAR-T persistence post infusion was monitored using ddPCR. Results: 24 patients (pts) were infused with var-cel in the IMAGINE study. Var-cel levels in peripheral blood post-infusion were measured at multiple timepoints for all infused pts using ddPCR platform. The var-cel levels peaked (Tmax) at a median of 10 days post infusion (D+10; range: 10 - 28 days) with median Cmax of 125,242 (range 18,256 - 413,968 copies/µg genomic DNA). The median duration of persistence was 56 days with persistent CAR seen in 3 pts at last follow-up. Adverse events of special interest (AESIs) reported were CRS (Grade [G] 1 62.5%; G3+ 4.2%; overall 66.7%) and ICANS (G1 4.2%; G3+ 0%; overall 4.2%). The median onset of CRS at D+5, 5 days before the median var-cel Tmax of D+10. The median duration for CRS was 3 days. No G3+ ICANS was reported. Serum IL-6 levels peaked at a median of D+7 post infusion and reached above 40 pg/mL for 13 pts, all of whom experienced CRS. All eight pts with no CRS had peak IL-6 levels below 40 pg/mL. Despite this significant association with CRS (p<0.001, Man-Whitney test) and published causation, IL-6 level peaked towards the end of clinical course of CRS manifestation, but usually at or before peak var-cel expansion (Tmax). Interestingly, the increase in IL-6 levels tended to begin early and persist longer for some B-NHL patients compared to B-ALL patients. Preliminary multiplexed cytokine analysis for evaluated pts (n=6) indicated that IFN-γ, TNF-α, IL-1β, IL-2, IL-7, IL-8 remained within published physiological limits for most patients at all timepoints (except one pt with highest levels at screening). One patient showed mild increase in IFN-γ (peak 50.88 pg/mL) and two pts showed mild increase in IL-8 around D+4 to D+7 (peak 47.71 pg/mL). IL-15 and MCP-1 levels were relatively higher (>2 fold) from D0 to D+7 post infusion, compared to the screening timepoint, consistent with the duration of CRS. Only IL-10, an anti-inflammatory and cytokine synthesis inhibitory factor stood out having a sharp peak (median: 230 pg/mL) for all six patients at D+4 to D+10, consistent with the timeline for end of CRS and before var-cel Tmax. Occasional pt with no clinical CRS also showed IL-10 peak. Updated cytokine profile data shall be presented at the meeting. Conclusions: This is the first-in-India industry study evaluating cytokine profile at multiple timepoints before and after var-cel infusion and its correlation with clinical CRS. As per published literature, IL-6 levels were significantly associated with clinical CRS and showed association with peak var-cel expansion. There was a sharp increase in both IL-6 and IL-10 levels in serum post infusion, concurrent with clinical CRS prior to peak var-cel expansion. A further detailed study of cytokine profile following var-cel infusion shall help prognosticate safety outcomes.

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.

Safety and Efficacy of GPRC5D CAR T Cell Therapy in Relapsed/Refractory Multiple Myeloma Patients

B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapies have shown breakthrough therapeutic effects in treating relapsed/refractory (R/R) multiple myeloma patients. However, relapses, including some that are BCMA negative, remain a challenge. Here we have developed a second-generation GPRC5D CAR-T, composed of a GPRC5D-binding scFv (derived from a humanized rabbit monoclonal antibody), a 4-1BB costimulatory domain, and a CD3ζ signaling domain. Following pre-clinical studies, we initiated a phase 1 dose-escalation clinical trial. Lymphodepletion with fludarabine and cyclophosphamide was performed before administering 3E6, 6E6, or 1E7/kg anti-GPRC5D CAR T cells. To date, we have enrolled a total of 7 R/R MM patients, including 3 patients with prior BCMA CAR-T treatment. All patients received autologous CAR T-cell infusions and were clinically evaluated between February 7th, 2023, and June 26th, 2023. No dose-limiting toxicities (DLT) were observed, even at the highest dose of 1E7/kg. Grade 3 or higher adverse events (AEs) were mainly hematological toxicities, such as neutropenia (6 [85.7%]), anemia (3 [43%]), and thrombocytopenia (3 [43%]). Cytokine release syndrome occurred in 6 (85.7%) of 7 patients, with all cases classified as grade 1 or 2. Notably, no neuro-toxicities were observed in any of the seven patients. The overall response rate was 85.7% (6/7 patients), comprising 3 complete responses or better, 3 partial responses, and one no-response (NR). The NR patient had previously shown no response to BCMA CAR-T therapy, with FACS analysis confirming the absence of both BCMA and GPRC5D on their tumor cells. Interestingly, the other two patients previously treated with BCMA CAR T-cell therapy, one reached complete response (CR), and the other reached partial response (PR). Remarkably, one patient diagnosed as COVID-19 positive two days before CAR-T infusion still received the CAR-T cell product, which led to a dramatic in vivo expansion of CAR-T cells, resulting in CR within one month after cell infusion. In conclusion, GPRC5D CAR T-cell therapy demonstrated promising clinical efficacy and tolerable safety in patients with R/R MM. For those who relapsed after anti-BCMA CAR T-cell therapy, GPRC5D CAR T-cell therapy offered an effective option (NCT05739188).“