CD19-directed Chimeric Antigen Receptor Research Articles

2721. Respiratory Virus Infections Following CAR T-cell Therapy: Risk Factors and Outcomes

Abstract Background Chimeric antigen receptor (CAR) T-cell therapy is a novel cancer therapy which functions by engineering patients’ own cells to target a malignancy. The preparative lymphodepleting chemotherapy and cell versus tumor effect create a novel, dynamic state of immunocompromise. Respiratory viruses have been found to be the predominant late infectious complication, but little is known about specific risk factors and outcomes of these infections. Methods We retrospectively identified adult patients who received CD19-directed CAR T-cell therapy at our center and followed them up to two years post-infusion (or until disease progression, death, or February 2022 — whichever came first). We undertook structured chart review using REDCap. Descriptive statistics were used where indicated. An adapted ordinal scale was used to assess the severity of respiratory virus infections (RVIs) (PMCID: PMC9278199). Results We found 73 patients who met search criteria. Of those, 23 patients (32%) developed an RVI, and their baseline characteristics are listed in Table 1. There were 38 episodes of RVI with an average of 1.65 episodes per affected patient. The median time from infusion to RVI was 180 days. The etiologic agent for the majority of RVIs was either unknown or rhino/enterovirus (Figure 1). The median ordinal score for RVI severity was 1, corresponding to ambulatory management for most (Figure 2). However, 24% of patients required a higher level of care. In patients where serial NAATs were performed, the duration of viral shedding was a median of 33 days (IQR 32 – 39). Immediately prior to infection the median IgG was 370 mg/dl (IQR 292 – 566), ANC was 1690 cells/μL (IQR 873 – 3165), and ALC was 480 cells/μL (IQR 285 – 703). 10 (43%) patients were treated with IVIG and 4 (17%) patients received antivirals. Many patients did not seek care for their RVIs or sought care outside of the Hematology/Oncology clinic. The RVI diagnosis relied on the impression of the treating physician at the time the patient presented with symptoms, or when the patient described past symptoms for which they did not present to the Hematology/Oncology clinic for care. Additionally, sometimes a full panel of respiratory viral nucleic acid amplification testing was done and negative (possibly false negative due to a collection issue or non-tested virus). Other times only COVID/FLU/RSV testing was done. Other times no respiratory virus panel was done. “Unknown” is a clinical syndrome of respiratory viral infection encompassing patients either without nucleic acid amplification testing being performed or with negative testing. The Ordinal Scale for EHR Use* was used to rank the severity of each instance of RVI. Possible rankings included 1, 3, 5, 7, 9, or 11. None of the RVIs met the criteria for 9 (organ support) or 11 (death). *Khodaverdi M, Price BS, Porterfield JZ, Bunnell HT, Vest MT, Anzalone AJ, Harper J, Kimble WD, Moradi H, Hendricks B, Santangelo SL, Hodder SL; N3C Consortium Collaborators. An ordinal severity scale for COVID-19 retrospective studies using Electronic Health Record data. JAMIA Open. 2022 Jul 9;5(3):ooac066. doi: 10.1093/jamiaopen/ooac066. PMID: 35911666; PMCID: PMC9278199. Conclusion There is a high burden of delayed RVI in patients who receive CAR T-cell therapy, and these infections can lead to severe illness requiring hospitalization. Clearance of virus was delayed, with duration of shedding lasting at least one month for most. Many CAR T-cell therapy patients had lower immunoglobulin G and lymphocyte counts prior to RVI. Further research is needed to understand the best treatment and prevention strategies for RVIs in this population. Disclosures All Authors: No reported disclosures

IMMU-26. MRNA CAR T CELL TRAFFICKING FOLLOWING INTRA-VENTRICULAR ADMINISTRATION IN PRECLINICAL MODELS OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND GD2-directed chimeric antigen receptor (CAR) T cells have shown promise as a potential therapeutic for diffuse midline glioma. We have previously shown intra-tumoral infusions of mRNA CAR T cells result in tumor regression with improved safety and toxicity profile in murine models. For human trials, the optimal route of CAR T cell administration into the central nervous system should maximize in vivo potency while minimizing procedural morbidity. Prior mRNA CAR T literature has reported decreased solid tumor infiltration with systemic delivery, so we sought to determine mRNA CAR T cell trafficking into pontine tumors from the cerebral spinal fluid to inform clinical translation of this therapy. METHODS Mice engrafted with SU-DIPG13P* tumor cells in the pons were treated with 5x106 mRNA CAR T cells into the lateral ventricle (LV) and brains were harvested 24 hours later. Tumor and T cells were analyzed using confocal microscopy to determine the migration of CAR T cells. RESULTS LV injection of GD2-directed CAR T cells resulted in T cell trafficking to the subarachnoid spaces, tumor site, and surrounding parenchyma. The highest concentration of T cells was in the subarachnoid space directly adjacent to the tumor (p< 0.0001) and decreased on a gradient to subcortical tumor. Even at 24 hours after injection, T cells were identified within the center of the tumor mass. CD19-directed control CAR T cells showed similar distribution within the subarachnoid with significantly decreased T cells at the tumor site. CONCLUSION Ventricular infusions of mRNA CAR T cells showed effective migration to pontine tumors within 24 hours. While CAR T cells did not fully penetrate the parenchyma within that timeframe, GD2-directed mRNA CAR T cells were detected within the center of the tumor mass, indicating ventricular administration may be sufficient for clinical delivery. Additional routes are being explored in ongoing work.

A Novel Model Combining Circulating Absolute Monocyte Counts and a Four-Gene Monocyte Signature in Leukapheresis Identifies Lymphoma Patients at Very High Risk of Progression after Tisagenlecleucel or Axicabtagene Ciloleucel Therapy

Introduction CD19-directed chimeric antigen receptor (CAR) T can induce durable remissions in a fraction of relapsed/refractory large B-cell lymphomas (R/R LBCL), but 60% of patients (pts) still relapse. Biological mechanisms explaining lack of responses are emerging but they are largely unsuccessful in predicting disease response at the patient level. Aim To address the potential role of lymphocyte features before CAR T manufacturing, we performed transcriptomic and functional evaluations of leukapheresis products (LK) in 95 R/R LBCL pts enrolled in a prospective observational study, to identify correlates of response and survival to tisagenlecleucel (Tisa-cel) and axicabtagene ciloleucel (Axi-cel). Methods RNA of sorted CD3+ cells from LK was digitally quantified using the nCounter CAR T Characterization Panel (NanoString). Flow cytometry (FCM) was used for CAR T enumeration and monocyte analysis. Disease response at day 90 was assessed by computed tomography (CT) and positron emission tomography-CT (PET/CT) according to Lugano criteria. Statistical analyses were performed by GraphPad Prism v.9.00 and R v.4.1.2. Results We identified a 4-gene transcriptional signature (including MS4A4A, CD86, CD163 and SIGLEC5) able to divide LBCL pts into two groups, [namely Expressors (EXP) and poor-Expressors (poor-EXP)], characterized by significantly different survival probabilities. EXP have shorter PFS (P<0.0001: median PFS for EXP was 61 days and not reached for poor-EXP) (Fig. 1) and are less likely to respond (P<0.01). As the identified genes are highly expressed in monocytes but not in T cells, we sought to define whether monocytes contained in LK have a role in determining post CAR T outcome. In line with the fact that EXP had higher frequencies of monocytes in LK by FCM, we confirmed elevated absolute monocyte counts (AMC) both in LK (P<0.01) and in peripheral blood (PB) (P<0.05) of EXP. We then checked if monocyte levels could influence response and survival. When compared to responders, non-responders (NR) had higher LK AMC (P<0.01), higher percentages of monocytes (P<0.01) but also decreased percentages of lymphocytes (P<0.001), a parameter that was also associated with reduced PFS [HR: 0.97 (CI 0.96 - 0.99) P=0.01]. Consistent with the fact that circulating monocytes significantly correlated with those in LK (r= 0.6621, P<0.0001), NR also had significantly higher AMC (P<0.001) and AMC levels in PB negatively correlated with PFS [HR: 1.8 (CI 1.2 - 2.9) P=0.01]. Additionally, pts with serum C-reactive protein (CRP) and ferritin levels higher than the upper normal limit, displayed significantly higher AMC (P<0.001 for CRP; P<0.05 for ferritin). Accordingly, univariate analysis indicated that high ferritin, CRP and LDH levels at the time of LK were associated with the lack of response, but only high serum ferritin levels correlated with shorter PFS [HR: 2.86 (CI 1.25-6.55) P=0.013]. We next investigated if the combination of the monocyte signature and high ferritin levels and/or high circulating monocytes could better identify a group of pts at high risk of progression, compared to the expression of the 4 genes alone. Remarkably, albeit high ferritin levels and monocyte counts are overall associated with significantly reduced survival, the shortest PFS was achieved in EXP with monocyte counts above the median (P=0.0234: median PFS for EXP with high monocytes was 32.5 days and for EXP with low monocytes 209 days) (Fig. 2), thus supporting the hypothesis that CAR T treatment outcome is influenced by the presence of elevated levels of monocytes expressing the 4 genes. Concatenated monocyte FCM data from 30 pts analyzed with FlowSOM, followed by a consensus clustering algorithm combined with t-SNE (t-stochastic neighbor embedding), indicated that EXP had higher frequencies of CD14hi monocytes co-expressing MS4A4A, CD86, CD163 and SIGLEC5 at the protein level than poor-EXP (P<0.05), and accordingly, the presence of this population was also associated with lack of response (P<0.05). Conclusions Our data suggest that the poor outcome of some LBCL pts receiving CAR T is due to the presence of high monocytes levels expressing a four-gene signature in an inflammatory microenvironment. Additionally, the pre-manufacturing combined analysis of the gene signature and of PB AMC, could be used to plan trials evaluating CAR T cells versus other newly approved therapies such as bispecific antibodies.

Preclinical Efficacy of a Novel CD8-Targeted Fusosome for In Vivo CAR T Cell Therapy

SG299 is a CD8-targeted fusosome; a self-inactivating (SIN) lentiviral vector (LVV) pseudotyped with a CD8-targeted fusogen, which carries a transgene encoding a CD19-directed chimeric antigen receptor (CAR). We evaluated the preclinical efficacy of a representative CD8/CD19CAR fusosome comparable to SG299 for its ability to control tumor growth in vivo in an NSG mouse model engrafted with human CD19 positive tumor cells and human peripheral blood mononuclear cells (PBMC). We compared direct intravenous (IV) administration and ex vivo exposure of fusosome to resting T cells followed by IV injection of vector-cell mixtures, as has been proposed for extracorporeal delivery (ECD) methods. We have also evaluated the performance of multiple lots of fusosomes to assess consistency in manufacturing and have evaluated donor-to-donor variability in five PBMC donors in this model. To measure anti-tumor efficacy, immunodeficient NSG mice were challenged IV with NALM6 tumors expressing firefly luciferase (5E5 cells/mouse) on day 4. For direct IV, PBMC (1E7 cells/mouse) were injected IV on day -1 followed by CD8/CD19CAR fusosome (1E7 IU/mouse) on day 0. For ECD, PBMC were incubated with CD8/CD19CAR fusosome for 1 hr and the cell and fusosome mix were injected IV on day 1. Tumor growth was monitored by bioluminescence imaging over a 28-day period. In addition, we have investigated both pharmacokinetics of fusosome induced CAR T cells and NALM6 tumors by flow cytometry. CD8/CD19CAR fusosome demonstrated statistically significant control of NALM6 tumors by both direct IV and ECD routes of delivery. Kinetic analysis of peripheral blood demonstrated tumor control and in vivo CART cell generation were dose dependent. The peak CAR T cell response was at D14. CARs detected at all timepoints were CD8+ specific; no CD4 + CAR T cells were observed. In addition, all mice that received fusosome demonstrated protection from morbidity and mortality as measured by reduced weight loss and improved survival compared to control or tumor only mice. Lot-to-lot performance variability of fusosomes was minimal; three lots of fusosome that had similar physical and functional attributes showed efficient and equivalent tumor control with multiple PBMC donors. Finally, an assessment of donor-to-donor variability across five PBMC donors using a single lot of fusosome showed donor-specific variations in tumor control as measured by BLI but demonstrated a consistent protection from morbidity and mortality. Collectively, these studies demonstrate preclinical efficacy of our CD8/CD19CAR candidate, lot consistency and verify our ability to generate CD19-directed CAR T cells in vivo using a specifically targeted platform.

A CD19/CD20-Directed Bispecific CAR-T Cell Therapy in Relapsed or Refractory Aggressive B-Cell Non-Hodgkin Lymphoma (NHL)

Three CD19-directed chimeric antigen receptor (CAR) T cell products are approved in the US for patients with relapsed or refractory (R/R) aggressive B cell lymphoma. Most patients treated in the third or later line will experience disease progression. Resistance to CD19-directed CAR T cell therapy may be due to tumor, T cell, or microenvironmental factors. Tumor-intrinsic causes of relapse include CD19 antigen loss, which is observed in approximately one third of relapsed cases(Spiegel, Dahiya, et al., 2021). Lower pretreatment CD19 antigen density has also been associated with treatment failure(Spiegel, Patel, et al., 2021). Conversely, enrichment of memory CD8+ T cells within the infusion product has been associated with achievement of complete remission (CR)(Deng et al., 2020). IMPT-314 is an autologous, naïve/memory enriched T cell product transduced by a lentivirus to express a tandem, OR-gate CD19/CD20-directed CAR with a 4-1BB co-stimulatory domain(Zah et al., 2016). IMPT-314 incorporates the same CAR and a highly similar manufacturing process as CART19/20, which was evaluated in a single-institution, Phase 1 study (NCT04007029)(Larson et al., 2023; Puliafito et al., 2023). Eleven patients with R/R B cell NHL have been treated at two dose levels (50 x 10 6 ± 30%, n=8; 200 x 10 6 ± 30%, n=3). Ten patients were evaluable for safety, and eleven patients were evaluable for response. Cytokine release syndrome (CRS) occurred in 60% of patients, however there were no CRS events above Grade 1. No patients experienced immune effector cell associated neurotoxicity syndrome (ICANS) of any grade. The overall response rate (ORR) and CR rate were 91% and 73%, respectively. As of March 6, 2023, with a median follow-up of 20.7 months, median progression-free survival (PFS) was 18.2 months (2.6-not estimable), and the median overall survival (OS) has not been reached (5.7-not estimable). This study of CART19/20 demonstrates the potential of simultaneous targeting of CD19 and CD20 with a bispecific CAR. Study Design The MPCT-012L study (NCT05826535) is a Phase 1/2 multicenter, open label study to evaluate the safety and efficacy of IMPT-314 in participants with relapsed/refractory aggressive B-cell NHL, defined as diffuse large B cell lymphoma (DLBCL), transformed follicular lymphoma (tFL), primary mediastinal large B cell lymphoma (PMBCL), and high-grade B-cell lymphoma (HGBL), after 2 or more lines of systemic therapy. Participants who have progressed after prior CD19 CAR-T cell therapy are eligible and are included in a separate cohort. All participants must have received prior anti-CD20 monoclonal antibody and an anthracycline. Patients with tFL must have received at least one line of therapy after transformation to DLBCL. Subjects with active (symptomatic or untreated) CNS involvement are excluded. There is no requirement for demonstration of CD19 or CD20 tumor expression at baseline. Phase 1 will be conducted using a modified 3+3 design with a starting dose of 100 x 10 6 CAR-T cells. Dose limiting toxicities (DLTs) will be evaluated for 28 days post-dosing. If a dose level is determined to be safe based on the DLT incidence, a total of up to 15 total subjects may be backfilled at that dose, per cohort. The primary objectives for Phase 1 are to evaluate the safety of IMPT-314 and to determine the recommended Phase 2 dose. The primary objective of Phase 2 is to estimate the efficacy of IMPT-314 as measured by CR rate. Secondary endpoints include evaluation of pharmacokinetics and time to event outcomes.

Incidence of Invasive Fungal Infections in CAR T-Cell Therapy Recipients: A Single-Center Retrospective Analysis

Background: CAR T-cell therapy has transformed the treatment of hematologic malignancies, but challenges persist due to toxicities like cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and infections. Rates of invasive fungal infections (IFI) post CAR T-cell therapy are estimated to range from 1% to 7% (Haidar et al. Clin Infect Dis, 2020). Some centers use positive fungal biomarkers, such as serum (1à3)-β-D-glucan (B-DG) and galactomannan (GM), alongside surveillance radiographic imaging to guide preemptive fungal therapy in this population. The objective of the study was to assess the diagnostic performance of surveillance B-DG and GM for the diagnosis of IFI in recipients of BCMA and CD19-directed CAR T for multiple myeloma (MM) and non-Hodgkin lymphoma (NHL), respectively. Methods: We conducted a retrospective analysis on adult patients who received BCMA-directed CAR T therapy for MM or CD19-directed CAR T for NHL at our institution between 2018-2023. IFI was defined using the Revised European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) criteria (Donnelly et al. Clin Infect Dis, 2020). Only patients with proven or probable infection were included in the analysis. The choice of antifungal prophylaxis was determined based on the treating physician's discretion. In the most cases, B-DG and GM monitoring was done prior to apheresis, before CAR T infusion, and subsequently every 14 days for a duration of 6 months. All patients were followed until 7/12/2023, or until their data were censored at the last follow-up. The cumulative incidence was calculated using Kaplan-Meier method, considering the time to IFI, with IFI-free death and disease relapse as competing risks. Statistical analysis was performed using IBM SPSS (version 29.0). Result: Among the 148 subjects in this study, 51 patients (34.5%) received BCMA CAR T, and 97 (65.5%) received CD19 CAR T therapy. The median age at CAR T infusion was 66 years (range 36-78) in the BCMA cohort and 65 years (range 27-83) in the CD19 cohort (p=0.64). The BCMA cohort had a higher number of prior therapies, with a median of 6 (range 2-15) compared to the CD19 cohort, which had a median of 4 (range 2-12) (p<0.001). The median follow-up from CAR T infusion was 9.5 months (range 0-63) in both cohorts, with 12 months (range 0-63) in the BCMA cohort and 8 months (range 0-49) in the CD19 cohort. All patients received prophylactic antifungal agents, including anti-yeast (fluconazole, n=95), and anti-mold (micafungin n=5, isavuconazole n=1, or Posaconazole n=52). Among the BCMA cohort, 92% of the patients received anti-yeast prophylaxis, while 8% were given anti-mold prophylaxis. In the CD19 cohort, 49.5% received anti-yeast prophylaxis, while 50.5% received anti-mold prophylaxis. Out of the 148 patients, 21 individuals (14%) exhibited elevated B-DG levels, while only 2 patients (1%) showed elevated GM levels. However, 20 out of 21 (95%) cases with positive B-DG, and all cases with positive GM were deemed to be false positives. Among these false positives, 60% were attributed to IVIG administration, while the cause for the remaining cases remained unknown (Table 1). Only one patient in this study developed IFI. The patient received anti-yeast prophylaxis (Fluconazole) at CAR-T and had a disseminated Rhizopus infection on day 13 post CD19 CAR T, which ultimately resulted in the patient's death. The 1-year cumulative incidence of IFI, considering competing risks, was 0.7% (95% confidence interval [CI], 0- 2.1). The median progression-free survival (PFS) in the overall cohort was 11.7 months (95% CI, 6.9-16.6), with 8.6 months (95% CI, 3.2-13.97) in the BCMA cohort and 14.6 months (95% CI, NR-NR) in the CD19 cohort (p=0.324). The median overall survival (OS) of the study cohort was 32.4 months (95% CI, 16.3-48.4), with 41.4 months (95% CI, 16.74-66.08) in the BCMA cohort and 23.98 months (95% CI, NR-NR) in the CD19 cohort (p=0.260). Conclusion: The rate of IFI in our cohort was low (0.7%) post-CAR T therapy. The diagnostic performance of serial monitoring of B-DG and GM for the diagnosis of IFI is poor in recipients of BCMA-directed CAR T therapy for MM or CD19-directed CAR T for NHL.

TRANSCEND FL: Phase 2 Study Primary Analysis of Lisocabtagene Maraleucel as Second-Line Therapy in Patients with High-Risk Relapsed or Refractory Follicular Lymphoma

Background: Results with CD19-directed CAR T cell therapy in patients (pts) with second-line (2L) R/R follicular lymphoma (FL) and high-risk features, such as progression of disease within 24 months (POD24) from diagnosis or double refractory to anti-CD20 antibody plus alkylator, have not been previously reported. TRANSCEND FL (NCT04245839), a global, phase 2, open-label, single-arm, multicohort, pivotal study, assessed efficacy and safety of the anti-CD19 CAR T cell therapy lisocabtagene maraleucel (liso-cel) in pts with second line or later (2L+) R/R indolent NHL. Some data from the primary analysis were previously reported, including safety in 2L+ R/R FL, and focused on efficacy in third line or later R/R FL (Morschhauser F, et al. Hematol Oncol 2023;41[S2]:877‒880). Here, we report primary analysis results in the cohort of pts with 2L high-risk R/R FL. Methods: Eligible pts in the 2L R/R FL cohort had biopsy-confirmed FL before enrollment and must have had POD24 with treatment ≤ 6 months from original FL diagnosis and/or must have had high tumor burden as defined by modified Groupe d'Etude des Lymphomes Folliculaires (mGELF) criteria. All pts received 1 prior combination systemic therapy with an anti-CD20 antibody and alkylator. Eligible pts received liso-cel (100 × 10 6 CAR + T cells) after lymphodepleting chemotherapy (LDC). Bridging therapy was allowed with reconfirmation of PET-positive disease before LDC. The primary endpoint was ORR per independent review committee (IRC) by PET/CT using Lugano 2014 criteria. Secondary endpoints included CR rate, duration of response (DOR), PFS, OS, safety, and cellular kinetics. Pharmacodynamic endpoints were exploratory. Results: At data cutoff (January 27, 2023), 23 of 25 leukapheresed pts received liso-cel and were evaluable for safety and efficacy per IRC; 1 received nonconforming product and 1 reached CR after bridging therapy and no longer met eligibility criteria. Median (range) age was 53 y (34-69), 74% had stage III/IV disease, and 35% were high-risk per FL International Prognostic Index (FLIPI). Sixty-five percent of pts had POD24 from initiation of first-line combination chemoimmunotherapy (52% had POD24 from diagnosis), 70% met mGELF criteria (mGELF only, 48%; mGELF and POD24 from diagnosis, 22%), and 48% were double refractory to anti-CD20 antibody plus alkylator. Median (range) on-study follow-up was 18.1 months (1.0-26.8). In efficacy-evaluable pts, the ORR and CR rate were both 95.7% (95% CI, 78.1-99.9; 1-sided P < 0.0001; Table). With a median follow-up of 16.8 months and 17.8 months, respectively, median DOR and PFS were not reached; 12-month DOR and PFS were 89.8% and 91.3%, respectively. The most common grade (gr) ≥ 3 treatment-emergent AEs (TEAE) were cytopenias; neutropenia was most frequent (52%). Cytokine release syndrome (CRS) occurred in 12 (52%) pts (no gr ≥ 3). Median (range) time to onset and resolution of CRS was 6 days (2-9) and 3 days (2-7), respectively. Neurological events (NE) occurred in 4 (17%) pts, with 1 (4%) gr 3 and no gr 4-5 (Table). Median (range) time to onset and resolution of NEs was 8.5 days (6-11) and 2.5 days (1-4), respectively. Three (13%) pts received tocilizumab/steroids for CRS/NEs. Prolonged cytopenia (gr ≥ 3 laboratory values at Day 29) occurred in 3 (13%) pts; all recovered to gr ≤ 2 by Day 90. No gr ≥ 3 infections were reported. One TEAE death occurred in the context of IRC-assessed disease progression due to gr 5 macrophage activation syndrome (MAS). Liso-cel showed rapid expansion with median (range) time to maximum transgene levels of 10 days (7-11). Persistence of liso-cel transgene was detected up to Month 12 in 5 of 18 (28%) pts. B-cell aplasia (< 3% CD19 + B cells in peripheral blood lymphocytes) after liso-cel infusion was rapid and maintained in ≥ 95% of pts through Month 2. Conclusions: This is the first report of outcomes in 2L high-risk R/R FL with CD19-directed CAR T cell therapy. In this population, liso-cel achieved very high CR rates (22 of 23 pts); deep and durable remissions, with follow-up ongoing; and a favorable safety profile with low rates of severe (gr ≥ 3) CRS, NEs, and prolonged cytopenia, and no severe infections. These data support liso-cel as a potential new treatment option in pts with 2L R/R FL at high-risk for treatment failure.

Precision Targeting of the Malignant Clone in Diffuse Large B Cell Lymphoma Using Chimeric Antigen Receptor T Cells Against the Clonotypic IGHV4-34 B Cell Receptor

Introduction: CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has revolutionized the treatment of B cell lymphoma. However, most CART19-treated patients either fail to respond or show disease progression after an initial response. In fact, up to 30% of lymphoma relapses following CART19 show loss of CD19 expression. In addition, several patients who respond well to CART19 display complete loss or severe reduction of their normal B cell repertoire, leaving them exposed to recurrent infections, limited response to vaccines, and requiring prophylactic measures such as intravenous immunoglobulins. A significant portion of mature B cell malignancies express B cell receptors (BCR) that use the same immunoglobulin heavy variable gene: IGHV4-34. In particular, ~30% of activated B cell (ABC) diffuse large B cell lymphomas (DLBCL), ~35% of primary central nervous system lymphomas, ~65% of vitreoretinal lymphomas, and ~35% of hairy cell leukemia variant express IGHV4-34. This suggests that the IGHV4-34 heavy chain is critical for driving the disease by delivering cell survival and proliferation signals, and multiple studies have shown that BCR signaling is required for ABC-DLBCL survival. However, while highly enriched in several types of B cell lymphomas, IGHV4-34 expressing B cells compose only ~5% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV4-34 CAR T cells would be highly effective and safe against B cell malignancies, as they would: (i) efficiently recognize IGHV4-34+ lymphoma cells while sparing normal B cells; and (ii) target a tumor driver that is essential for lymphoma cell survival ( Fig 1A). Methods and Results:We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV4-34+ BCR (CART4-34) using a single-chain variable fragment (scFv) derived from the 9G4 rat monoclonal antibody. We used tumor B cells that endogenously (HBL1) or exogenously (Jeko1, Bonna12, Mec1) express the IGHV4-34 BCR. Using cytotoxicity, cytokine secretion and proliferation assays in vitro we showed that CART4-34 specifically target all IGHV4-34+ tumor B cells tested while sparing IGHV4-34- tumor B cells and, most importantly, healthy B cells. However, while the initial CART4-34 showed strong cytotoxicity towards IGHV4-34+ cells in short-term in vitro assays, they were significantly inferior to CART19 in in vivo xenograft models. We hypothesized that the poor performance of CART4-34 compared to CART19 was due to the unique challenge of targeting the membrane-distal portion of the BCR, 18 nm from the plasma membrane. In contrast, CART19 targets an epitope of CD19 located just 5 nm from the membrane ( Fig 1B). Therefore, to improve immune synapse (IS) formation, we designed new CAR constructs with smaller extracellular domains by replacing the original CD8 hinge (44 amino acids (aa)) with either: (i) a short version of the IgG4 hinge (12aa); or (ii) a G4S linker (5aa). We found that both CART4-34 (IgG4) and CART4-34 (G4S) exhibited significantly improved cytotoxicity towards IGHV4-34+ tumor cells in short-term and long-term in vitro assays in all IGHV4-34+ cell lines tested, while maintaining specificity. Importantly, in in vivo human xenograft models of IGHV4-34+ lymphoma (HBL1), CART4-34 with short hinges showed significantly improved tumor control comparable to, or better than, CART19 ( Fig 1B). We also found that while CART19-resistant lymphoma cells had complete loss of CD19 surface expression, CART4-34-resistant tumor cells retained high expression of IgM, suggesting a reduced likelihood of antigen-negative escape owing to the critical role of the BCR for lymphoma B cell survival. Mechanistically, we found that while the original CART4-34 (CD8 hinge) formed IS with striking morphological differences compared to IS formed by CART19, the IS formed by CART4-34 with short hinges highly resembled those formed by CART19, providing a rationale for the improved anti-tumor potency of short-hinge CART4-34. Conclusion:We implemented a novel paradigm for the treatment of B cell malignancies: the specific targeting of the malignant clone, while sparing the remainder of the healthy B cell repertoire. We designed and optimized a best-in-class anti-IGHV4-34 CAR T cell product with potent anti-tumor effects and minimal toxicity towards IGHV4-34-negative B cells. The activity of CART4-34 will be investigated in a Phase I clinical trial.

CAR19 Therapy Drives Expansion of Clonal Hematopoiesis and Associated Cytopenias

Background: For patients with relapsed/refractory large B-cell lymphomas (rrLBCL), CD19-directed chimeric antigen receptor T cells (CAR19) improve survival compared to autologous hematopoietic cell transplantation (HCT). However, major toxicities of CAR19 therapy include prolonged cytopenias and associated infections. To better understand the impact of CAR19 on such toxicities, we studied a cohort of LBCL patients achieving durable remissions to assess immune recovery after CAR19 treatment. We (1) compared immune reconstitution in HCT vs CAR19 treated patients, (2) performed molecular analysis of post-CAR19 cytopenic marrow specimens, and (3) profiled emergent mutations in cell free DNA (cfDNA) before and after CAR19. Methods & Results: We first propensity matched rrLBCL patients treated at Stanford with either CAR19 (100% axi-cel) or HCT who achieved durable remissions. Patients were matched based on age, sex, prior systemic lines of therapy, and time from infusion. From 471 eligible cases, propensity score matching was successful in 138 (n=69 per arm). When compared to HCT-treated counterparts, CAR19-treated patients had reduced recovery of absolute lymphocyte counts (ALC) and absolute neutrophil counts (ANC; longitudinal regression day 28-800 ANOVA p<0.005), but similar recovery of hemoglobin and platelets. CAR19-treated patients experienced significantly more Grade ≥3 infections in competing risk analyses (HR 2.61, CI 1.26-5.42), and higher non-relapse mortality (log-rank p=0.027). Next, to characterize mechanisms underlying cytopenias after CAR19, we profiled marrow aspirates from cases requiring biopsy due to prolonged cytopenia or clinical concern for myeloid neoplasms. No patient had evidence of lymphoma relapse at the time of biopsy, and all were >60d from CAR19 infusion (median 377d, range 86-1561d). Among 21 patients profiled by DNA sequencing, 18 (86%) had evidence of ≥1 somatic mutation in genes canonically associated with clonal hematopoiesis (CH) in the marrow ( Fig. 1A), with the most recurrently mutated genes being DNMT3A (38%), TP53 (33%), PPM1D (24%), ASXL1 (14%), and TET2 (14%). Among 6 patients diagnosed with and requiring therapy for a myeloid malignancy, all (100%) had a canonical myeloid karyotypic aberration detected by FISH, as compared with 0 in the 14 other evaluable cases ( Fig. 1A). CAR19 cells were proportionately enriched in the marrow relative to paired peripheral blood by flow cytometry (n = 12, median 2.5-fold enrichment, Wilcoxon p<0.001). Using 10x scRNA-Seq, viable CAR19 was detectable in 7 of 8 marrows (88%) and were predominantly CD4+. We hypothesized that CH clones detectable after CAR19 would also be present in pre-CAR19 blood samples and detectable in cfDNA. To test this, we profiled 786 longitudinal samples from 141 patients using CAPP-seq and serially monitored CH clones in cfDNA. Among 141 eligible cases, 40 (28%) were evaluable for CH, in having both pre- and post-CAR19 infusion specimens, while in durable remission (median follow up 967d). Of 51 CH clones detected in post-infusion cfDNA, 46 (90.1%) were also detectable prior to CAR19, typically expanding post-infusion (median = 1.8-fold, Fig. 1B). We next tested whether expanding CH clones showed evidence for selection after CAR19. Strikingly, among 48 recurrently mutated genes tested, DNMT3A and PPM1D showed significant evidence for clonal selection and expansion after CAR19 (Q-value < 0.05). When compared to patients without emerging CH clones, patients with ≥5-fold clonal expansions of canonical CH genes (23% of cases) had reduced ANC, but not ALC, recovery (median ANC=1.43 vs 2.43 at 6-mo, Wilcoxon p<0.001). This association remained significant in multivariate longitudinal regression that included age, sex, prior lines of therapy, and pre-infusion ANC (ANOVA p = 0.007). Conclusion: CAR19 treated patients experience reduced immune reconstitution and higher infection rates than HCT treated counterparts. Bone marrow findings reveal prolonged CAR19 cytopenia to commonly be associated with clonal cytopenia of undetermined significance (CCUS). Despite durable lymphoma remissions, canonical myeloid clonal expansions are common post-CAR19, and associated with myelosuppression. Non-invasive genotyping could aid in early identification of patients at risk for prolonged cytopenias after CAR19 and inform interventions to decrease associated complications.

A Phase 1 Study of PIT565, a First-in-Class, Anti-CD3, Anti-CD19, Anti-CD2 Trispecific Antibody in Patients with Relapsed and/or Refractory B-Cell Malignancies

Background and Significance: There is an unmet therapeutic need for patients with relapsed and/or refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL) and R/R B-cell acute lymphoblastic leukemia (B-ALL) who have failed multiple lines of therapy. PIT565 is an investigational, first-in-class, immunoglobulin G-like, trispecific antibody that targets CD19 on malignant B cells while engaging CD3 and CD2 (a costimulatory receptor) on T cells, leading to redirected T-cell cytotoxicity toward CD19-positive malignant B cells. CD2 signaling prevents transcriptional changes characteristic of exhaustion during persistent T-cell receptor stimulation of CD8 T cells. Furthermore, the loss of CD2 ligand, CD58, on tumor cells has been shown to mediate resistance to the antitumor activity by T cell-redirecting therapies, confirming the importance of CD2 activation in T cells. Findings from preclinical studies suggest that PIT565 mediates more potent and sustained antitumor T-cell responses compared with CD3 bispecifics (Lu H, ASH 2022). Study Design and Methods: This is an open-label, phase 1, multicenter study of PIT565 in patients with R/R B-NHL and R/R CD19-positive B-ALL who have relapsed/failed to respond to ≥2 lines of prior therapy (including an anti-CD20 monoclonal antibody-containing chemotherapy combination regimen for patients with R/R B-NHL) (NCT05397496). The study is ongoing with plans to enroll approximately 140 patients in Belgium, France, Israel, Italy, Japan, Singapore, Spain, and the USA. Eligible patients are ≥18 years with an Eastern Cooperative Oncology Group performance status ≤2 and have at least one bi-dimensionally measurable nodal lesion or one bi-dimensionally measurable extranodal lesion as measured via positron emission tomography-computed tomography for R/R B-NHL, and/ or morphologic disease in the bone marrow (³5% blasts) for R/R B-ALL. Patients with contraindication to tocilizumab; history of other malignant disease; active central nervous system involvement; autoimmune disease (other than patients with vitiligo, hypothyroidism only requiring hormone replacement, or psoriasis not requiring systemic treatment or conditions not expected to recur); or receiving systemic treatment with any immunosuppressive medication are not eligible. During the dose escalation part of the study, PIT565 is administered to patients with R/R B-NHL (Group A) and patients with R/R B-ALL (Group B). During the dose-expansion part, PIT565 is administered to patients with R/R large B-cell lymphoma (LBCL) who have received CD-19-directed chimeric antigen receptor (CAR)-T therapy (Group A1); patients with R/R LBCL who have not received CAR-T therapy (Group A2); and patients with R/R B-ALL (Group B1). PIT565 is administered either once weekly (Q1W) or once every 2 weeks (Q2W) with and without a priming dosing. Routes of administration (intravenous [IV] or subcutaneous [SC]) are explored in the dose-escalation phase. Patients initially receive PIT565 IV Q1W over a 28-day cycle. Q2W treatment in a 28-day cycle is explored if supported by preliminary pharmacokinetics (PK), pharmacodynamics, efficacy, and safety findings. The primary objective is to assess safety (including dose-limiting toxicities) and tolerability, and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD), schedule, and route of administration for each indication. Secondary objectives include evaluation of preliminary antitumor activity (overall response rate, complete response rate, best overall response, duration of response, overall survival, and progression-free survival for B-NHL or event-free survival for B-ALL), assessment of PK and immunogenicity of PIT565 in R/R B-NHL and R/R B-ALL. The dose escalation phase is guided by an adaptive Bayesian logistic regression model following the escalation with overdose control principle. The MTD and/or RD are further explored during the dose-expansion phase.

Durable Efficacy and Manageable Safety in Patients Age ≥ 75 Years with Relapsed/Refractory Large B-Cell Lymphoma Treated with Tisagenlecleucel in the Real-World Setting

Background: Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR)-T cell therapy approved for the treatment of patients (pts) with relapsed/refractory (r/r) large B-cell lymphoma (LBCL) who have received ≥2 lines of prior therapy. Previous analysis of the Center for International Blood & Marrow Transplant Research (CIBMTR) registry showed high rates of durable response with progression-free survival (PFS), duration of response, and overall survival (OS) rates at 24 mo of 28%, 53%, and 44%, respectively, as well as similar efficacy outcomes among pts age ≥65 y as compared with pts age <65 y (Landsburg et al, ASH 2022). Although it has been shown that real-world pts treated with CAR-T cell therapy are older than those included in pivotal trials, limited data on CAR-T outcomes among pts age ≥75 y are available. Here, we report the first analysis of clinical outcomes in pts age ≥75 y with r/r LBCL treated with tisagenlecleucel in the real-world setting. Methods: Data were collected as a part of a noninterventional, prospective, longitudinal study using the CIBMTR cellular therapy registry. All pts were treated in the United States, Canada, or Israel. Efficacy outcomes analyzed included overall response rate (ORR), complete response rate (CRR), PFS, and OS. Key safety outcomes included the incidence and severity of adverse events, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). Nominal P values are reported without adjusting for multiplicity. Results: As of May 4, 2023, 1375 pts had received tisagenlecleucel (infused set); 278 pts were age ≥75 y. Pts age ≥75 y had similar baseline characteristics compared with pts age <75 y (Eastern Cooperative Oncology Group 0-1, 85% in both populations; elevated lactate dehydrogenase [LDH], 47% in both populations), received similar lymphodepleting regimens, and received tisagenlecleucel in a similar number of days post relapse (81 d vs 85 d, respectively). Pts age ≥75 y had a lower frequency of prior autologous hematopoietic cell transplantation than pts age <75 y (12% vs 26%, respectively) (Figure). One hundred twenty-one pts age ≥75 y had comorbidities (121/278, 44%), including pulmonary disease (65/278, 23%), cardiac disease (60/278, 22%), and hepatic disease (21/278, 8%). Among 247 pts age ≥75 y who were evaluated for efficacy, the ORR (CR + partial response) was 64% (95% CI, 58.1-70.3) and CRR was 47% (95% CI, 41.0-53.8). ORR was similar in pts age <75 y (n=984; 60% [95% CI, 56.9-63.1]), as was CRR (46% [95% CI, 43.2-49.5]). With a median length of follow-up (infusion to data cutoff) of 30 mo, 24-mo PFS in pts age ≥75 y was 23% (95% CI, 16.7-30.1) compared with 28% in pts age <75 y (95% CI, 25.1-31.7). Twenty-four-mo OS in pts age ≥75 y was 39% (95% CI, 31.2-47.2) compared with 44% (95% CI, 40.1-47.8) in pts age <75 y (Figure). AE frequency in pts age ≥75 y (n=250) was comparable with pts age <75 y (n=1004), including the incidence of all-grade CRS (57% vs 60%, respectively; P=0.31) and grade 3/4 CRS (6% of each subgroup). Grade 5 CRS occurred in 2 pts age ≥75 y and 6 pts age <75 y. Tocilizumab was used to manage CRS in 66% of pts age ≥75 y compared with 56% of pts age <75. All-grade ICANS frequency in pts age ≥75 y was comparable with pts age <75 y (27% vs 22%, respectively; P=0.06). Grade 3/4 ICANS was experienced by 7% of each subgroup. Four pts age ≥75 y experienced grade 5 ICANS compared with 1 pt age <75 y. Median time to ICANS onset was similar in pts age ≥75 y (5 d, range 2.0-6.5) and pts age <75 y (5 d, range 3.0-8.0). Corticosteroids were used with similar frequency to manage ICANS in pts age ≥75 y (68%) compared with pts age <75 y (65%). Conclusions: In the first analysis of pts age ≥75 y, tisagenlecleucel demonstrated similar efficacy and rates of CRS and ICANS in pts with r/r LBCL age ≥75 y as compared with age <75 y. As few pts with r/r LBCL age ≥75 y received CAR-T cell therapy in clinical trials, real-world evidence for this age group is invaluable for guiding treatment decisions in this clinical setting, and supports consideration of tisagenlecleucel, a therapy that results in durable disease control in approximately one quarter of patients with a low risk of severe toxicity.

Real-World Tisagenlecleucel Outcomes in Richter-Transformed Chronic Lymphocytic Leukemia: A Center for International Blood & Marrow Transplant Research (CIBMTR) Analysis

Background: Richter transformation (RT) is a rare but serious transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most commonly large B-cell lymphoma (LBCL). There is currently no standard therapy for RT lymphoma and median overall survival (OS) is estimated to be only 8-12 months. Tisagenlecleucel is an autologous CD19-directed chimeric antigen receptor (CAR)-T cell therapy approved for the treatment of r/r LBCL in patients who have received ≥2 lines of prior therapy. Here, we describe the efficacy and safety of tisagenlecleucel in patients with RT in a real-world setting with a median follow-up (infusion to data cutoff) of 31 months. Methods: Data were collected as part of a noninterventional, prospective, longitudinal study using the CIBMTR registry. All patients were treated in the United States, Canada, or Israel. Efficacy outcomes analyzed included overall response rate (ORR), progression-free survival (PFS), and OS. Safety outcomes included the incidence and severity of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Results: As of May 4, 2023, 48 patients with RT received a single tisagenlecleucel infusion. The median age at infusion was 68 years (range, 39-83). Fifty-eight percent of patients were ≥65 years old. The median number of all prior lines of therapy was 3 (range, 1-8); 42% of patients received ibrutinib as a prior therapy. No information confirming clonal relation of LBCL to CLL in evaluated patients was available. Fifty percent of patients had primary refractory disease. Prior to infusion, 46% of patients were reported to have elevated lactate dehydrogenase (LDH). Two patients who received tisagenlecleucel infusion, and were alive at data cutoff, had not yet reached 100 days post-infusion and were therefore not evaluable for efficacy and safety. Among the 46 patients evaluated for efficacy and safety, the ORR (complete response [CR] + partial response) was 63% (95% CI, 47.5-76.8) and CR rate was 50% (n=23). In total, 23 patients (23/46, 50%) experienced disease relapse, 17 within 6 months of infusion (Figure). Twelve- and 24-month PFS were 43% and 39%, respectively; 12- and 24-month OS rates were 51% and 44%, respectively. Among 23 patients with a best overall response of CR, 57% remained in remission at data cutoff. The most common adverse events of any grade were CRS (78%), hypogammaglobinemia (46%), and ICANS (35%). Grade 3/4 CRS was reported in 11% of patients. No patient experienced grade 5 CRS. CRS was managed with tocilizumab in 81% of cases. Grade 3 ICANS was reported in 9% of patients. No grade 4/5 ICANS events were observed. Two patients went on to receive a stem cell transplant following tisagenlecleucel infusion. In total, 25 deaths were reported. The most common cause of death was progressive disease (13/48, 27%). Twelve patients (12/48, 25%) died in the absence of disease progression. Causes of non-relapse-related mortality included infection (including COVID-19, n=5), respiratory failure (n=2), malignant neoplasm (n=2), prior malignancy (n=1), multiorgan dysfunction (n=1), and severe veno-occlusive disease (n=1). Conclusions: In the largest cohort of patients with RT receiving treatment in a real-world setting, tisagenlecleucel resulted in a high overall response rate and durable survival. The incidences of CRS and ICANS were similar to other real-world reports of tisagenlecleucel for patients with LBCL. Overall, these findings demonstrate that tisagenlecleucel could be considered a potential treatment option for patients with RT who have a high unmet medical need.

Intravenous IL-1 Receptor Antagonist for Prevention of Severe Immune Effector Cell-Associated Neurotoxicity Syndrome

Background and Significance: The management of toxicities from autologous CD19-directed chimeric antigen receptor (CAR) T cell therapy have evolved and the rates of severe cytokine release syndrome (CRS) have declined from nearly 50% in early trials to 8% (CRS grade 3 or higher) from real-world retrospective data with axicabtagene ciloleucel (axi-cel) (Jacobson et al. TCT 2022). However, the rate of severe immune effector cell-associated neurotoxicity (ICANS) has only marginally improved since CAR-T cell therapy has become commercially available. With expanding indications for CAR-T cell therapy, the prevention and management of ICANS remain an unmet need. We previously conducted a pilot, investigator-sponsored phase II study using Anakinra, an IL-1 receptor antagonist, administered subcutaneously at the onset of grade 2 CRS or any grade ICANS (Oliai et al. ASCO 2021) which demonstrated that anakinra is safe and feasible. There are several prospective studies that are currently open to accrual that incorporate subcutaneous Anakinra for prevention and treatment of severe ICANS (NCT04359784, NCT04148430, NCT04150913, and NCT04432506). Anakinra crosses the blood brain barrier and has been shown to be safe and efficacious in rheumatologic conditions. Relatively high doses of IV Anakinra have been shown to be safe in severe sepsis and in subarachnoid hemorrhage. We hypothesize that IV Anakinra is safe and effective in preventing severe ICANS in patients undergoing standard of care axi-cel therapy for large B cell lymphoma. Study Design and Methods: This investigator-sponsored trial (NCT4205838) is a phase Ib/II study of adults eligible for standard of care axi-cel for large B cell lymphoma. Anakinra will be administered intravenously using a standard 3+3 design with cohort 1 receiving 200mg IV loading dose, followed by 100mg IV q6h maintenance dose and cohort 2 receiving 400mg IV loading dose, followed by 200mg IV q6h. The trigger to initiate Anakinra is any grade ICANS or grade ≥2 CRS in the absence of ICANS, based on ASTCT 2019 criteria. In addition to Anakinra, participants will receive standard of care interventions for CRS and ICANS. Subjects with secondary CNS involvement are included. Key exclusion criteria are primary CNS lymphoma, Burkitt's lymphoma, transformed DLBCL from CLL, prior allogeneic stem cell transplantation, < 30 days of exposure to immune check point inhibitor therapy. The study is open to accrual at University of California, Los Angeles (UCLA) with 2 additional sites within the University of California Hematologic Malignancies Consortium (UCHMC). Objectives: The primary objective is to evaluate the safety and tolerability of Anakinra given intravenously in patients undergoing CAR-T cell therapy and to estimate the efficacy of Anakinra in prevention of severe ICANS (grade ≥3). Secondary objectives include the impact of Anakinra on the efficacy of CAR-T cell therapy for large B cell lymphoma, the duration of ICANS, and the pharmacokinetics of Anakinra. Exploratory objectives focus on changes in inflammatory cytokines during ICANS in the peripheral blood. Statistics and Current Status: Using historical control from the real-world experience using axi-cel for relapsed/refractory large B-cell lymphoma (38%, Jacobson et al. ASH 2018), a sample size of 36 patients achieves 80% power to detect a 50% reduction in the rate of severe ICANS when comparing to the historical control using a one-sample exact binomial test at the two-sided type I error rate of 0.10. This study has been open at UCLA since February 2023. Two of 4 enrolled participants met criteria to receive IV Anakinra. Conclusions: The safety and tolerability of IV Anakinra is being evaluated in patients with large B cell lymphoma undergoing treatment with axi-cel in order to prevent severe ICANS.

Hypoimmune, Allogeneic CD22-Directed CAR T Cells That Evade Innate and Adaptive Immune Rejection for the Treatment of Large B Cell Lymphoma Patients That Are Relapsed/Refractory to CD19-Directed CAR T Cell Therapy

Despite the success of CD19CAR therapy against large B-cell lymphoma, up to 60% of CD19-directed CAR T cell treated patients relapse after, or are refractory to, treatment. Of the relapsed/refractory population a significant subset of patients, around 30%, have CD19-negative tumor and a short median survival prognosis - necessitating the implementation of rapid intervention strategies (Atilla, Transl Oncol 2022; Spiegel, Blood 2021). Sana is developing “off-the shelf” allogeneic CD22-directed CAR T cells that may enable rapid intervention and induce clinically meaningful activity for these CD19 CAR T cell relapsed/refractory patients. Here we describe methods to create hypoimmune (HIP), allogeneic CD22-directed CAR T cells using a clinically validated CD22CAR that: 1) support adaptive immune cell evasion via B2M and CIITA gene disruption, 2) promote persistence and innate immune cell evasion by overexpression of CD47, and 3) mitigate GvHD via TRAC gene disruption, (Hu, Nat Commun 2023). HIP, allogeneic CD22-directed CAR T cells (CD22 HIP CAR T cells) were derived from healthy donor CD8 + and CD4 + T cells that were gene-edited to disrupt the B2M, CIITA, and TRAC (TCR) genes and transduced to overexpress CD47 and express a second-generation, clinically validated CD22-directed CAR. Gene-edited Mock T cells and unedited, allogeneic CD47-CD22CAR T cells were generated as donor-matched controls. The CD22 HIP CAR T cells were evaluated for immune evasive activity using a previously described in vitro xCelligence cytotoxicity assay (Hu, Nat Commun 2023). The T cells were further evaluated in vitro for dose-dependent short-term and long-term cytolytic activity analysis via luciferase or IncuCyte assay, respectively. Correlate cytokine responses were performed by meso-scale discovery analysis and T cell expansion by IncuCyte classification. Systemic NALM and RAJI parental or CD19 antigen knockout tumor models (IV injection) were executed in a NSG or NSG-SGM3 mouse background. xCelligence analysis confirmed that CD22 HIP CAR T cells were protected from NK and macrophage recognition; whereas Mock control T cells that lack HLA expression succumbed to NK cell and macrophage killing within 5-hours of the 50-hour assay due to the absence of CD47 protection. Furthermore, CD22 HIP CAR T cells elicited cytotoxicity and robust cytokine responses (e.g. IFNγ, IL-2) in a dose-dependent manner comparable to allogeneic CD47-CD22CAR T cells when cultured with CD22 +/CD19 + or CD22 +/CD19 - NALM, RAJI, or K562 targets. The CD22 HIP CAR T cell and allogeneic CD47-CD22CAR T cell control produced low level IFNγ (<400pg/mL) and Granzyme B (<100pg/mL) when cultured alone or with CD22 - tumor targets. These basal levels of IFNγ and Granzyme B did not have long-term cytolytic effects on CD22 - tumor cells, nor was any significant level of IL-2 produced by CD22 HIP CAR T cells cultured alone, or in the presence of CD22-negative tumor targets. Indeed, CD22 HIP CAR T cell proliferation was dependent upon CD22 + tumor co-culture. NALM and RAJI CD19 knockout in vivo tumor models demonstrated that CD22 HIP CAR T cells significantly reduced flux (Day 21 AUC vs Mock: NALM and RAJI (p=0.0001)) and prolonged survival (Median vs Mock: p<0.004). Furthermore, CD22 HIP CAR T cells elicited comparable, dose-dependent antitumor activity relative to allogeneic CD47-CD22CAR T cell controls in a systemic NALM tumor model. Our data provide evidence that CD22 HIP CAR T cells elicit robust antitumor activity in an antigen and dose-dependent manner. Furthermore, these studies demonstrate that CD22 HIP CAR T cells are agnostic to CD19 expression and selectively elicit pharmacologic activity against, or in response to, CD22 + tumor. Results also demonstrate that the HIP approach to generate allogeneic CAR T cells that evade innate and adaptive immune rejection can be expanded as a platform to generate CAR T cells against target antigens alternative to CD19. Collectively, the data suggest that CD22 HIP CAR T cells display a combination of antigen-specific pharmacologic activity and immune evasion that support their progression into human clinical studies for the treatment of CD19 CAR T cell-refractory patients.

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.

Impact of Sarcopenia and Subsequent Muscle Loss on Post-CAR T-Cell Outcomes in Relapsed/Refractory Large B-Cell Lymphoma

Background: Sarcopenia is a hallmark of cachexia commonly found in cancer patients that is caused by immune and metabolic mediators of catabolism. Chimeric antigen receptor (CAR) T-cell therapy is used to treat cancer and is characterized by the release of inflammatory cytokines and other potentially catabolic mediators. Previously, we showed that baseline sarcopenia and other immuno-nutritional scores were associated with poor outcomes after CD19-directed CAR T-cell therapies in patients with R/R LBCL (Rejeski et al. Cancer Immunol. Res. 2023). Here we examined the relationship between body composition and serum metabolite levels over time after CAR T-cell infusion. Methods: This is a single-center retrospective study of N=83 patients with R/R LBCL who received CAR T-cell therapy. The median clinical follow-up was 18.4 months. Muscle and adipose tissue distribution were calculated from clinical pre-lymphodepletion, day (d) 30, and day (d) 90 CT imaging using Slice-O-Matic software (v5.0, Tomovision). Baseline sarcopenia status was determined using established criteria (SMI 52.4 cm2/m2 for Males and 38.5 cm2/m2 for Females) . Immuno-nutritional scores included: GPS Score: CRP ≤ 10 mg/L = 0; CRP > 10 mg/L and albumin > 3.5 g/dL = 1; CRP > 10 mg/L and albumin < 3.5 g/dL = 2. PNI Score: Albumin (g/L) + 5 × total lymphocyte count (10^9/L). Muscle loss at d30 or d90 after CAR T-cell therapy was defined by a decrease of > 10% in their skeletal muscle index (SMI) from baseline. Patients who had relapsed, died or had missing scans were censored during the analysis. Univariate and multivariable Cox regression models using LASSO regularization assessed the association between various factors and survival outcomes. Serum metabolites were profiled at baseline and at regular time points up to 4 weeks after CAR T cell therapy (n=55) as previously described (Fahrmann et al. Cell Rep. Medicine 2022). Results: Patients with baseline sarcopenia (n=44) had worse progression-free (PFS) and overall survival (OS) after CD19 CAR T cell therapy for R/R LBCL compared to non-sarcopenic patients (n=39) (OS: HR=2.5, 95% CI: 1.3-4.9, P=0.008; PFS: HR=1.8, 95% CI: 1.0-3.2, P=0.037). In multivariable Cox fits (MVA) for OS, male gender (HR=3.3, 95% CI: 1.3-8.8, P=0.014), and GPS score of 2 (HR=3.8, 95% CI: 1.4-10.6, P=0.011) were associated with worse OS. On MVA for PFS, a lower PNI score was mildly associated with worse outcomes (HR=0.9, 95% CI: 0.8-1.0, P=0.039). Muscle loss occurred between baseline and d30 and d90 after CAR T-cell therapy in 34% and 32% of patients, respectively. Muscle loss correlated with concurrent adipose tissue loss and overall weight loss. Patients who had baseline sarcopenia had more pronounced muscle loss at d30 and d90 compared to patients who were non-sarcopenic at baseline. Associations with d30 muscle loss included ICANS grade 2 or higher (P=0.046) and baseline LDH (P=0.033). Further, patients with ICANS grade 2 or higher had a significantly higher d30 median muscle loss compared to patients with grade 0-1 ICANS (9.1% versus 6.4%; p=0.049). However, whether landmarked at d30 or d90, muscle loss after CAR T-cell therapy did not significantly associate with the risk of subsequent progression or death. Individual serum metabolites that significantly decreased between pre-infusion and after CAR T-cell infusion included those potentially associated with gut microbiome alterations (indole-3-acetate and xanthosine). Pathway analysis revealed changes in purine and pyrimidine metabolism over time after CAR T-cell therapy. Subgroup analyses comparing metabolites over time in sarcopenia versus non-sarcopenia, as well as based on muscle loss are in progress. Conclusions: Sarcopenia is highly prevalent (53%) in R/R DLBCL patients presenting for CAR-T and approximately 1/3 of patients lose muscle mass during the course of therapy, with the greatest risk noted in those with baseline sarcopenia. High immuno-nutritional scores are associated with inferior outcomes. Muscle loss was associated with a higher disease burden and higher-grade toxicities. Further study is needed to understand the association between antibiotics use and clinical factors for microbiome-related metabolites that change over time after CAR T-cell infusion. Interventional approaches to improve functional and nutritional reserve to optimize outcomes are warranted.

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) (>4 mg/dL, 10.6% vs 35.4%, p<0.001), ferritin (≥400 ng/mL, 37.5% vs 62.4%, p<0.001), lactate dehydrogenase (LDH) (≥225 U/L, 51% vs 76.4%, p<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>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<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<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<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.

Cladribine and Cyclophosphamide Lymphodepletion Prior to Axicabtagene Ciloleucel in Relapsed or Refractory Large B-Cell Lymphoma

Introduction : Chemotherapy-based lymphodepletion (LD) with fludarabine and cyclophosphamide (Flu/Cy) is a standard preparatory step for chimeric antigen T-cell (CAR T) therapy and its importance for optimal CAR T expansion and efficacy is well-established. An international Flu shortage required our center and others to utilize alternative agents for LD or risk interruption in patient therapy. When unable to utilize Flu as part of the LD regimen for patients with relapsed/refractory large B-cell lymphoma (RR LBCL) that required CD19-directed CAR T as a standard of care therapy, our center adopted a standard operating procedure to substitute Flu with Cladribine (Clad), an alternate purine analogue with similar chemical structure, as part of Clad/Cy LD prior to axicabtagene ciloleucel (axi-cel). Here we report outcomes in these patients. Methods : We performed a retrospective, single-center analysis of consecutive RR LBCL patients who received Clad/Cy LD prior to axi-cel at Moffitt Cancer Center between 8/2022 and 1/2023, and compared outcomes to a historical cohort, consisting of 144 Flu/Cy axi-cel LBCL patients treated between 11/2017 and 2/2021. The Clad/Cy LD regimen, given as standard care, maintained Cy at 500 mg/m 2 and substituted Flu 30 mg/m 2 with Clad 5 mg/m 2/day with both agents given on Days -5, -4, and -3. For efficacy we compared best overall response (BOR) at 90 days and progression-free survival (PFS) comparing the two LD regimens. For safety we compared the rates of cytokine release syndrome (CRS) and neurotoxicity as graded by ASTCT criteria. We also performed correlative analyses exploring absolute lymphocyte count (ALC) kinetics, axi-cel expansion by quantitative PCR, and changes in the cytokine milieu as assessed by the enzyme-linked immunosorbent Ella™ system (Bio-Techne, Oxford, UK) at serial time points following infusion. Results : 23 patients received Clad/Cy LD followed by axi-cel for R/R DLBCL. Baseline characteristics of the study cohort based on LD are shown in the Table. Compared to the historical cohort, Clad/Cy patients received axi-cel after fewer lines of treatment (median 2 [range, 1-3] vs 2 [range, 1-7]; Rank-Sum test p<0.01) and had a greater proportion of relapsed (not refractory) disease (52% vs 22%, p<0.01). The 90-day BOR did not differ between the Clad/Cy and Flu/Cy groups (79% vs 84%; P=0.54) with a similar distribution of complete (22% vs 24%) and partial responders (57% vs 60%). With a median follow-up of 4.6 (95% CI, 3.2-6.2) months for Clad/Cy and 24.2 (95% CI, 21.5-25.8) months for Flu/Cy cohorts, PFS was similar (Figure 1B, Log-Rank p=0.84). Rates of CRS grade ≥ 2 were higher in the Clad/Cy cohort (70% vs 44%; p=0.02), but no CRS grade ≥ 3 was observed in the Clad/Cy compared to 13 (9%) of Flu/Cy patients, including three grade 5 events. Neurotoxicity ≥ grade 2 (44% vs 43%; p=0.77) and peak C-reactive protein levels (15 vs Flu/Cy 12; p=0.3) did not differ for the Clad/Cy and Flu/Cy cohorts. Both regimens had comparable peak axi-cel copy numbers (Clad/Cy 7.9 × 10 6 vs Flu/Cy 5.1 × 10 6 copies/µg). Clad/Cy resulted in a predictable decrease in the ALC from Day -5 to Day 0, but a less profound degree of lymphopenia on Day 0 (0.04 vs 0.02; p<0.01) with ALC 0 achieved later (Day +2 [IQR, +1 to +4] vs Day +1 [IQR, -1 to +2] for Flu/Cy). The median magnitude of change of cytokine levels (Delta = Day -5 - Day 0 concentration) differed between Clad/Cy and Flu/Cy for all tested analytes except IL-6 (3.4 vs 4.6 pg/mL for Flu/Cy; p=0.29). Clad/Cy resulted in greater increases in IL-2 (0.5 vs 0.2 for Flu/Cy; p=0.01) and GM-CSF (2.0 vs 0.9 for Flu/Cy; p=0.03), whereas Flu/Cy had higher IL-15 (31.4 vs 16.8 for Clad/Cy; p<0.01). Interestingly, IFN-γ tended to increase in Clad/Cy and to decrease in Flu/Cy (1.2 vs 0.0; p=0.01). Conclusions : Clad/Cy LD is feasible for axi-cel conditioning in the treatment of RR LBCL. Although it resulted in less profound LD on the day of CAR T infusion, a later ALC nadir, and a different cytokine milieu compared to Flu/Cy, it was associated with similar peak axi-cel expansion and, at short term follow-up, there appears to be similar initial efficacy and toxicity. Future analyses focusing on response durability and long-term toxicities such as infections and post-CAR T cytopenias are warranted.