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Introduction Lymphoid malignancy (LM) is a heterogenous neoplasm with respect to biology, aggressiveness, and treatment with over 100 subtypes, including Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM). Genome-wide association studies have identified common variants associated with risk of specific LM subtypes, but less is known about the contribution of rare inherited pathogenic variants (PV) in the genetic architecture of LM risk. Rare PV have been shown to contribute to the genetic architecture of solid tumor cancer risk, many of which have been found in genes that are important to the DNA-damage repair (DDR) pathway (e.g., BRCA1, BRCA1, ATM). Here, we investigated the prevalence of PV in cancer predisposition genes regularly included on multi-gene panel testing based on NCCN guidelines and correlated with risk of LM overall and by common subtypes. Methods In this association study, cases were newly diagnosed lymphomas (excluding chronic lymphocytic leukemia) from Mayo Clinic who were enrolled between 2002 and 2015 in the Lymphoma SPORE Molecular Epidemiology Resource (MER) and MM from Mayo Clinic enrolled primarily between 1998 and 2022 in a clinical registry. Controls were from the Mayo Clinic Biobank who were enrolled between 2009 and 2016, from which we excluded individuals with a history of hematologic malignancy. All participants provided a blood sample for whole exome sequencing, performed by Regeneron on an Illumina NovaSeq panel (mean coverage of 48X). Variants were called using GATK v4 and variant annotation was performed using BioR. All loss-of-function variants (i.e., nonsense, frameshift, and consensus splice sites) and pathogenic missense in 19 cancer predisposition genes with a minor allele frequency < 0.5% were included in the analyses. Logistic regression was used to estimate odds ratio (OR) and 95% confidence intervals (CI) for the association between mutation status by gene (burden test) and risk of LM overall, and by subtype that had more than 5 mutation carriers. All analyses were adjusted for age (at diagnosis for cases and at enrollment for controls) and sex. Results A total of 6990 cases and 42432 controls were included. Median age was 63 years for both cases and controls (case range 18-94 years, control range 18-99 years). Males accounted for 58.5% (n=4090) of the cases and 41.7% (n=17762) of the controls. MM accounted for 31% (n=2138) of the cases, followed by diffuse large B-cell lymphoma (DLBCL, n=1146) and follicular lymphoma (FL, n=1132) each accounting for 16% of cases. CHEK2 (1.0%), ATM (0.4%), BRCA2 (0.4%), and BRCA1 (0.3%) had the most PV carriers in controls; these frequencies are in the range of published control populations. A total of 1816 (3.7%) individuals had a PV across the 19 genes, with 4.7% of cases and 3.5% of controls carrying any PV (OR=1.34, 95% CI: 1.18-1.51). ATM (OR=1.86, 95% CI: 1.36-2.49), CHEK2 (OR=1.74, 95% CI: 1.42-2.13) and TP53 (OR=9.07, 95% CI: 4.51-18.87) were all significantly associated with increased risk of LM overall (Figure 1). When investigating subtypes (Table 1), CHEK2 (OR=1.93, 95% CI: 1.23-2.89) and TP53(OR=10.97, 95% CI: 3.06-31.43) were significantly associated with DLBCL. ATM (OR=2.22, 95% CI: 1.13-3.90) was significantly associated with FL. ATM (OR=1.97, 95% CI: 1.16-3.12), CHEK2 (OR=1.75, 95% CI: 1.24-2.41) and TP53 (OR=7.67, 95% CI: 2.43-20.72) were significantly associated with MM and ATM (OR=8.62, 95% CI: 4.47-15.12), NBN (OR=6.94, 95% CI: 2.41-15.76) and TP53 (OR=38.30, 95% CI: 10.27-116.69) were significantly associated with mantle cell lymphoma (MCL). When subset to LM cases overall, there was no evidence of enrichment for young onset (age <60 years), male sex, or family history of hematologic malignancy between PV carriers and non-PV carriers. However, FL cases with an ATM PV tended to be less than 60 years (OR=3.21, P=0.09) compared to non- ATM PV carriers. Similarly, MM cases with a TP53 PV tended to be less than 60 years of age (OR=7.39, P=0.07) compared non-carriers. Discussion In this large case-control study, PV in established cancer predisposition genes were associated with increased risk of LM overall, demonstrating that rare inherited variants may play an important etiologic role. This study also suggests that PV in cancer predisposition genes may be subtype specific. These results will help inform LM risk for individuals who undergo genetic testing of cancer predisposition genes.

Background The class of CD20×CD3 bispecific antibodies (BsAbs) has emerged as an important anti-lymphoma modality for patients (pts) with R/R B-cell non-Hodgkin lymphoma. Odronextamab, a novel, off-the-shelf, CD20×CD3 BsAb, has previously demonstrated compelling efficacy in both R/R diffuse large B-cell lymphoma and R/R FL. In pts with R/R FL, odronextamab monotherapy achieved 75% complete response (CR) rates and encouraging durability of responses, and showed generally manageable safety, in the Phase 2 ELM-2 study (NCT03888105; Kim TM, et al. ASH. 2022). Here, we report the results of a second, pre-specified interim analysis of ELM-2. Methods Intravenous odronextamab was administered weekly in 21-day cycles during Cycles (C) 1-4. Optimization of the step-up regimen to further mitigate cytokine release syndrome (CRS) was reported previously (Kim TM, et al. ASH. 2022). Odronextamab was administered with steroid prophylaxis and step-up doses of 0.7/4/20 mg during C1, followed by 80 mg on Days 1, 8, and 15 of C2-4. After C4, odronextamab maintenance treatment continued at 160 mg every 2 weeks until disease progression or unacceptable toxicity. Pts who achieved a durable CR for ≥9 months transitioned to dosing every 4 weeks. A pre-specified interim analysis was performed when 80 pts had completed ≥12 months follow-up. The primary endpoint was objective response rate (ORR), assessed by independent central review (ICR) according to the Lugano classification. Key secondary endpoints included CR rate, duration of response (DoR), progression-free survival (PFS), overall survival (OS), and changes in scores of patient-reported outcomes. Minimal residual disease (MRD) was included as an exploratory endpoint. Results At data cut off (Jan 31, 2023), the global study was fully enrolled and included 140 safety-evaluable pts with FL; 128 pts were efficacy-evaluable and 85 pts had ≥12 months follow-up. The median duration of study follow-up for efficacy was 26.6 months. In the safety-evaluable FL population, median age was 60.5 years (range 22-84), 53% were male, and pts had received a median of 3 (range 2-13) prior lines of therapy. 73% of pts were refractory to their last therapy, 41% were double refractory, and 74% were refractory to an anti-CD20 antibody. 50% of pts had progression of disease within 2 years (POD24) and 29% had a prior autologous hematopoietic stem cell transplant. ORR and CR rate by ICR were 80% (102/128) and 72% (92/128), respectively, and were consistent across pts with high-risk features. Responses were durable, with both median DoR and duration of CR of 21.7 months; the probability of maintaining CR for 24 months was 48%. Median PFS was 20.7 months (95% CI: 16.7-26.5) and median OS was not reached; the probability of survival at 3 years was 63%. Patient-reported overall quality of life (QoL) scores were maintained during the course of treatment. MRD status at 12 weeks was highly predictive of PFS. Safety was generally consistent with previous reports, and no new treatment-related Grade (Gr) 5 events were recorded since the first interim analysis. The most common treatment-emergent AEs (>30% all grades) were CRS (55%), anemia (34%), neutropenia (34%), and pyrexia (33%). With the optimized 0.7/4/20 mg step-up regimen (n=72), 98% of CRS events were Gr 1/2, and only one pt had Gr 3 CRS. All CRS events resolved with supportive measures; 13 pts received tocilizumab for CRS management with the optimized regimen. Only one low-grade ICANS event was reported with the optimized regimen. Gr ≥3 infections occurred in 50 (36%) pts (Gr 5, n=14 [10%]); COVID-19 infections were reported in 46 (35%) pts (Gr 5, n=8 [6%]). Conclusions The results of this pre-specified analysis of the Phase 2 ELM-2 study continue to demonstrate deep and durable responses with odronextamab in heavily pretreated, refractory pts with R/R FL. 90% of responders were complete responders, with a 48% probability of maintaining CR for 2 years. High CR rates appear to translate to favorable, long-term survival outcomes in the setting of incurable disease. The incidence of AEs was consistent with earlier reports, with no new treatment-related Gr 5 events. Importantly, continued treatment with odronextamab had no detrimental effects on overall QoL. These data underscore the promise of odronextamab as a potentially effective agent for the management of R/R FL. Updated data will be presented.

Background: There are a lack of clinical prediction tools in early-stage Hodgkin lymphoma (E-HL). We developed and validated a modern-day model, known as E-HIPI, to predict progression-free survival (PFS) within the first 5 years (y) incorporating detailed individual patient (pt) data from international clinical trials and prospective registry data that were standardized, normalized & harmonized as part of the HoLISTIC Consortium (www.hodgkinconsortium.org). Methods: Model development utilized Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) guidelines (Moons, Ann Intern Med 2015) on a cohort of 3,080 newly-diagnosed E-HL pts treated on 4 seminal phase 3 clinical trials (NCIC: Meyer, NEJM 2012; RAPID: Radford, NEJM 2015; EORTC-GELA H9U: Ferme, E ur J Cancer 2017; EORTC/LYSA/FIL H10: Andre, JCO 2017). External validation was done in an independent cohort of 462 E-HL pts from a large registry (Princess Margaret, Toronto). Pts with classic HL, stage I or II disease, and ages 18-65 y were included. The primary outcome was PFS. Cox models were utilized with follow-up truncated at 5 years. Baseline predictors were: sex, stage, B symptoms, histology, number & location of nodal sites, and continuous data values of age, maximum tumor diameter (MTD), white blood cell count (WBC), lymphocyte count, hemoglobin, albumin & erythrocyte sedimentation rate (ESR). Multiple imputation was used for missing data. To consider possible non-linear relationships for continuous variables we examined plots & cubic splines. We used backward elimination ( P<0.1) to develop the models & internal validation with 200 bootstrap samples was conducted to estimate optimism & correct for overfitting. The final prediction equations applied optimism corrections to beta coefficients & hazard ratios (HR). Sensitivity analyses included model results stratified by PET staging (vs CT) and each HL trial. The models were evaluated in the external validation cohort & compared using c-statistics. Model scores were also grouped by tertiles for visualization in Kaplan-Meier (KM) curves. Results: Median age in the development cohort was 35y; 51% were female; 81% had nodular sclerosis & 77% stage II disease; 26% had B symptoms; 32% had mediastinal bulk (≥10cm or >1/3 diameter) and the mean MTD was 6.4 cm (interquartile range (IQR), 3.9-8.2). Median follow-up was 60 months (IQR 45-75). KM estimates at 5y was 90.7% (264 events) for PFS, and 97.1% (78 events) for overall survival (OS). After backward elimination, significant variables retained were age, sex, nodal location (cervical region), MTD & albumin. Stage, ESR, or the number of nodal sites were not significant. Age was modeled with piecewise linear splines due to its non-linear relationship. See the Table for the final prediction model and optimism-corrected HRs. Optimism-corrected c-statistics in the development model was 0.63. Sensitivity analyses of stratification by type of imaging at staging or by each clinical trial demonstrated no change in model parameters or c-statistics. Most baseline characteristics and outcomes of the external validation cohort were similar besides median follow-up time (74 months, IQR 37-129). C-statistics for the E-HIPI model in external validation was 0.63. In addition, observed 5y outcomes stratified by tertile of predicted risk in the external cohort separated pts into a low & higher-risk group, which comprised 1/3 & 2/3 of pts, respectively ( Figure). Finally, model diagnostics revealed the potential for time dependency in the retained covariates. Further assessment of differential prediction between early (<2y) vs. later (2-5y) PFS events is ongoing. Conclusion: Following TRIPOD methodology, we rigorously developed and externally validated the E-HIPI among >3,500 E-HL pts.Discrete risk groups were delineated, and we identified several novel factors, including nodal location, sex & key continuous variables that predict 5y PFS. Additional model refinement is underway, including formal calibration & external validation via other HL registries (Stanford, Mayo/Iowa SPORE). A web-based calculator to simplify application of the E-HIPI will also be presented at ASH. Future planned modeling includes integration of differing treatments & response-adapted imaging via multistate modeling to enrich individualized survival estimates and simulation modeling to predict late consequences.

Background: Maintenance therapy with brentuximab vedotin (BV) after autologous stem cell transplantation (ASCT) improved progression-free survival (PFS) among high-risk patients (pts) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the phase III AETHERA trial. However, cHL treatment has changed significantly since that trial with frequent incorporation of BV and PD-1 inhibitors into earlier lines of therapy. The efficacy of BV maintenance may be different among pts who receive novel agents before ASCT. Methods: Pts with a diagnosis of R/R cHL who underwent ASCT between 2010 and 2022 were identified at 5 US transplant centers. Pts receiving no systemic post-ASCT maintenance or BV maintenance were included, while pts receiving investigational maintenance treatments were excluded. Medical records were reviewed to identify clinical variables. Results: 921 pts were identified. Median age was 32 yrs (IQR 24-44). 641 pts (70%) had primary refractory disease or relapsed within 12 months of frontline therapy, 326 (35%) had extranodal disease at relapse, and 173 (19%) had B symptoms at relapse. Pts received a median of 2 (1-9) lines of therapy before ASCT, including 295 (32%) who received ≥3 lines. 425 (46%) pts received BV before ASCT (including 24 pts as part of frontline treatment). 70 pts (8%) were BV-refractory (defined as failure to achieve an objective response to any BV-based treatment). 169 (18%) received a PD-1 agent with salvage treatment. 638 pts (69%) had a complete response on pre-ASCT positron emission tomography (PET). BEAM (71%) or CBV (21%) conditioning were used for most pts and 236 pts (26%) received peri-ASCT radiation. 224 pts (24%) received post-ASCT BV maintenance (including 37% of pts undergoing ASCT from 2015-2022). Compared to pts receiving no maintenance, pts receiving BV maintenance were more likely to have primary refractory/early relapsed disease (79% vs 67%, p<0.001), have received only 1 salvage regimen (79% vs 65%, p=0.001), and received BEAM conditioning (83% vs 67%, p<0.001). BV maintenance pts were less likely to receive peri-ASCT radiation (20% vs 28%, p=0.022). The median number of BV maintenance cycles was 10 (1-18) and the most common reason for discontinuation was neuropathy. Median post-ASCT follow-up was 4.9 yrs. 5-yr PFS and OS after ASCT were 70% (95% CI 67-74%) and 85% (83-88%), respectively. Because BV-refractory pts were much less likely to receive BV maintenance (p=0.0044) and had inferior PFS (HR 2.2, p<0.001) (and therefore would serve as a confounder), we excluded BV-refractory pts from additional analyses. Use of BV maintenance was associated with improved PFS (5-yr 81% vs 67%, HR 0.47 [0.33-0.69], p<0.001) and OS (5-yr 92% vs 83%, HR 0.38 [0.20-0.73], p=0.004). The benefit of BV maintenance depended upon pre-ASCT therapy. BV maintenance was associated with a significant improvement in PFS for pts who received no novel agents before ASCT (HR 0.41 [0.25-0.65], p<0.001), but not for BV-treated (HR 0.69 [0.38-1.25], p=0.22) or PD-1-treated pts (HR 0.63 [0.13-3.03], p=0.56) ( Figure). Among pts with 0-1 modified AETHERA risk factors, BV maintenance was not associated with a significant PFS benefit in any treatment subgroup (no novel agents, HR 0.50, p=0.087; BV-treated, HR 1.28, p=0.67; PD-1-treated, HR 2.97, p=0.44). For pts with 2+ modified AETHERA risk factors, BV maintenance significantly improved PFS in the no novel agent group (HR 0.35, p<0.001) but not in the BV-treated (HR 0.55, p=0.099) or PD-1 treated groups (HR 0.24, p=0.19). In multivariable analyses that included key variables (age, year of ASCT, conditioning regimen, peri-ASCT radiation, early relapse/primary refractory disease, B symptoms, extranodal sites, pre-ASCT PET, lines of therapy), BV maintenance was associated with a significant PFS benefit among pts who received no novel agents before ASCT (HR 0.27 [0.12-0.65], p<0.001), but not for pts treated with BV (HR 0.56 [0.28-1.10], p=0.091) or PD-1 blockade (HR 1.10 [0.22-5.56], p=0.91) before ASCT. Conclusions: In this large cohort, BV maintenance was associated with the clearest benefit among pts who received only chemotherapy before ASCT. We were unable to identify a significant improvement in PFS for pts receiving novel agents before ASCT (which could be due to insufficient power to detect a small benefit in this population), suggesting that if these pts benefit from BV maintenance, its impact is likely more limited.

Introduction: Immune-mediated graft failure (also termed graft rejection) is a feared complication of hematopoietic stem cell transplant (HSCT) and there is no effective intervention. Multiple pre-transplant risk factors for graft failure are known and recent studies from our groups identified interferon gamma (IFNγ) as a mechanistically important and pharmacologically targetable cytokine involved in graft failure pathophysiology. CXCL9, a downstream marker of IFNγ production, was also identified as a biomarker. We describe an international multicenter retrospective pooled analysis of emapalumab (EMA), an IFNγ neutralizing agent, for the prevention/treatment of immune-mediated graft failure after HSCT. Methods: The safety and preliminary efficacy of EMA were studied in predominantly pediatric patients who received EMA using two different strategies: 1) graft failure treatment and 2) graft failure prophylaxis. Patients at US centers were treated with EMA if they developed clinical and laboratory signs of graft failure after HSCT. All treatment patients met 2 or more of the following criteria: 1) HLA mismatched donor and/or prior graft failure, 2) unexplained fever >39°C (or >38°C if prior history of graft failure) after HSCT, 3) absolute neutrophil count decline after initial engraftment, 4) delayed neutrophil engraftment after HSCT, 5) real-time CXCL9 > 2.5x upper limit of normal (ULN). Temperature and CXCL9 cutoffs were based on a prior graft failure biomarker publication (Sabulski et al, Blood Advances 2021). Patients treated at European centers received prophylactic EMA to prevent graft failure if they were deemed at high-risk prior to transplant. High-risk patients had a prior history of graft failure after HSCT or had two or more of the following established risk factors: a) affected by a disease with high risk of graft failure, including acquired severe aplastic anemia, thalassemia, primary HLH; b) ex-vivo T cell-depleted stem cell product; c) transplant from unrelated cord blood unit or mismatched related/unrelated donor. Results: Thirty-six HSCTs used EMA for graft failure treatment or prophylaxis and were included in this study.EMA was used for graft failure treatment in 25 HSCTs wherein patients developed clinical and laboratory signs of graft failure (Table 1). Most HSCTs in the treatment cohort were performed for bone marrow failure syndromes (n=16). All patients tolerated EMA without complications and no side effects or infections were attributed to EMA. Fifty six percent (14/25) of patients treated with EMA for impending graft failure engrafted and had resolution of graft failure concerns, while the remaining 44% suffered graft failure. Responders had a median ANC of 0.29 x10 3 cells/μL (range 0.05-0.79) prior to EMA, which increased to a median of 0.6 x10 3cells/μL (range, 0-5.1) and 2.97 x10 3 cells/μL (range, 0.85-8.7) on days 3 and 7 after EMA dosing. Eighty percent (20/25) of CXCL9 levels were elevated in the treatment cohort prior to EMA dosing. The median CXCL9 level prior to EMA dosing was elevated 2.4 x ULN (range, 0.3-157.8). The median pre-EMA CXCL9 level in patients who ultimately suffered graft failure in the treatment cohort was 3.3x ULN (range, 0.7-157.8) compared to a median of 2.2x ULN (range, 0.3-30.4) in subjects who had signs of graft failure but did not suffer graft failure (p=0.24). Eleven patients received prophylactic EMA based on a prior history of graft failure or other pre-transplant risk factors. EMA was well tolerated in these patients and 91% (10/11) of patients engrafted while one patient with prior history of graft failure suffered recurrent graft failure. The pre-HSCT (day -2) CXCL9 level in the lone prophylactic patient who suffered graft failure was 4x the ULN. Conclusions: This study reports the largest cohort of HSCTs that used EMA therapy for the novel purpose of treating or preventing graft failure. EMA was well tolerated in all patients. Efficacy conclusions are preliminary and limited by the study design, however our treatment study successfully identified a cohort of patients with a markedly high graft failure rate (44%) and we show preliminary evidence to suggest emaplaumab prevented graft failure in patients who had signs of impending graft failure at the time of treatment. The safety and preliminary efficacy observations in this study support a larger prospective study of IFNγ blockade for graft failure prevention after HSCT.

Objective: This research delineates the temporal alterations of cell-free circulating tumor DNA (ctDNA) in the plasma and cerebrospinal fluid (CSF) of patients harboring primary and secondary central nervous system (CNS) lymphoma. The aim is to establish that CSF serves as a superior reservoir of ctDNA compared to plasma, thereby aiding in diagnostic processes and monitoring therapeutic efficacy. Concurrently, this study underscores the enhanced response and survival rates in patients administered Bruton's tyrosine kinase (BTK) inhibitors over an extended period exceeding three months. Methods: Data including cranial-enhanced magnetic resonance imaging (MRI) scans, leukocyte and protein concentrations in the CSF, exfoliated CSF cytology, flow cytometry, interleukin-10 to interleukin-6 ratios, ctDNA in plasma and CSF, were gathered from patients presenting primary and secondary CNS lymphoma（7 PCNSL,9 SCNSL）. The temporal ctDNA modifications in plasma and CSF during the patient's disease progression-initial onset, remission, and relapse-were scrutinized. Additionally, the implications of BTK inhibitor usage on patient response rates and survival rates were assessed. Results: Within the cohort of primary CNS lymphoma patients, ctDNA was detected in the CSF and plasma of 2 and 5 patients, respectively. Conversely, in patients with secondary CNS lymphoma, CSF and plasma ctDNA were discovered in 8 and 4 cases, respectively. The detection rate was contingent upon the central lesion's magnitude and position and the patient's surgical history related to the brain. Four patients, including two from each group, underwent cranial-enhanced MRI to appraise tumor burden at various disease stages, simultaneously with the monitoring of ctDNA in plasma and CSF. It was revealed that ctDNA levels in the CSF were in synchrony with the tumor burden, whereas plasma ctDNA levels remained relatively static. This indicates that CSF ctDNA may serve as a viable tool to assess therapeutic responses in CNS lymphoma patients. In two patients where surgical biopsy was unfeasible and both CSF exfoliation cytology and flow cytometry returned negative results, CSF ctDNA was positive, thereby underscoring its auxiliary role in diagnosing CNS lymphoma. Furthermore, the study indicated improved response and survival rates in patients subjected to BTK inhibitors for over three months. Conclusion: CSF ctDNA demonstrates potential in detecting CNS lymphoma and in the dynamic monitoring of therapeutic responses. Compared to cranial-enhanced MRI, exfoliated CSF cytology, and flow cytometry, CSF ctDNA testing offers superior sensitivity. For patients for whom brain surgical biopsy is not an option, CSF ctDNA testing emerges as a valuable supplementary diagnostic tool. CSF ctDNA allows for dynamic monitoring of therapeutic responses in CNS lymphoma patients, enabling the detection of residual lesions, and thereby guiding the formulation and duration of the treatment regimen. Patients administered oral BTK inhibitors for more than three months demonstrate higher treatment response and survival rates.

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.

Introduction: Chronic lymphocytic leukaemia (CLL) is the most common leukaemia type. In 2020, there were an estimated 207,463 people living with CLL, with an annual incidence of approximately 4.9 per 100,000 in the United States (US). Zanubrutinib is a Bruton tyrosine kinase inhibitor that is FDA-approved and an NCCN preferred and recommended treatment of CLL. In the phase 3 ALPINE trial (NCT03734016), zanubrutinib elicited a significantly higher overall response rate and significantly longer progression-free survival (PFS) than ibrutinib. This study aimed to compare zanubrutinib versus ibrutinib in Relapsed/Refractory (R/R) CLL by calculating the number needed to treat (NNT) to avoid one progression or death and associated incremental costs. Methods:A health-economic model was developed to evaluate the number of R/R CLL patients needed to be treated to avoid a progression or death from the US payer perspective. Payer blend was assumed with 40% Commercial and 60% Medicare payer mix. Clinical efficacy data were extracted from the ALPINE trial. Final analysis result of 24-month PFS (79.5% for zanubrutinib and 67.3% for ibrutinib) was used for the base-case analysis in the model. Treatment, adverse event management, other medical resource use, and subsequent treatment costs were considered in the model. The NNT, incremental cost per treated patient, and incremental cost per additional patient with progression or death were captured. Deterministic sensitivity analyses were conducted to assess parameter uncertainties and explore key model drivers. Scenario analyses were conducted to test the impact of different PFS estimates. Results: The base-case results fromthe NNT model showed that for every 8 patients treated with zanubrutinib, 1 event of progression or death would be avoided compared to using ibrutinib. The total costs per patient treated with zanubrutinib and ibrutinib are $370,558 and $430,150, respectively, with a cost savings of $59,593 associated with using zanubrutinib (Table). Drug costs and PFS have the greatest impact on the incremental cost per patient. Varying the PFS scenarios, including adjustment with drug interruption, COVID death, or treatment discontinuation, change the NNT from 8 to 12, and are associated with cost savings of $58,179 to $67,153 per zanubrutinib-treated patient in a 24-month time frame. Applying the model result to a hypothetical scenario of a clinical practice of 100 patients treated with zanubrutinib versus ibrutinib suggest that approximately 13 patients will avoid disease progression events or death. Conclusions: The NNT model suggests thatusing zanubrutinib to treat R/R CLL patients, compared to ibrutinib, will result in more favorable clinical and economic outcomes in the US.

Ciltacabtagene autoleucel (cilta-cel) consists of autologous T cells genetically modified to express a two-binding domain chimeric antigen receptor (CAR). The target antigen of the CAR is B-cell maturation antigen (BCMA), which is highly expressed on malignant plasma cells (PC) from multiple myeloma subjects. In the Phase 1b/2 CARTITUDE-1 study, cilta-cel led to early, deep, and durable responses and was approved for the treatment of patients with relapsed or refractory multiple myeloma (RRMM) after four or more prior lines of therapy by FDA. Despite cilta-cel's outstanding overall response rate (97.9%) and duration of response (DOR) in RRMM patients, relapse still occurs. Biomarkers associated with this durable response and acquired resistance remain to be elucidated. We present updated correlative data from the CARTITUDE-1 study. At the ~3 year follow-up (October 2022 data cutoff), 97 patients had received a single infusion of cilta-cel (median 0.71 x10 6 cells/kg [range 0.52 x10 6-0.94 x10 6]); the median follow up was 33.4 months (range 1.5-45.2), median DOR was 33.9 months (95% CI, 25.5-not estimable [NE]) and median PFS was 34.9 months (95% CI, 25.2-NE) (Lin Y et al, ASCO 2023). Drug product (DP), baseline and on-treatment whole blood and bone marrow samples from enrolled patients were analyzed by methods including flow cytometry, MSD immunoassays, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and TCR sequencing. In CARTITUDE-1, the DP contained a mixture of transduced and non-transduced T cells; the median transduction efficiency was 16% (range 5-32%), with a balanced distribution of CAR+CD4+ and CAR+CD8+ cells, median frequency 12% (range 2-28%) and 6% (range 2-20%), respectively. Further, the DP T cell subset composition was variable but balanced between central (Tcm) and effector memory (Tem) phenotypes. CAR+ T cells expanded reaching a median peak concentration (C max of 730 cells/µL; range 3-13805 cells/µL) in blood between days 12-14 post-infusion and persisted in circulation for a median of 100 days (range 20-912 days). High efficacy (best response) and DOR were achieved despite variable CAR-T cell expansion and lack of detectable CAR-T cell persistence over time. At C max, the CD4:CD8 ratio (based on % of CAR+ cells) was 0.29, demonstrating a CAR+CD8+ T cell preferential expansion, and both CAR+CD4+ and CAR+CD8+ T cells were predominantly of a Tcm phenotype, median 95% (range 62-99.5%) and 96% (range 33-99.7%), respectively. Higher frequency of early-memory T cell phenotypes has been associated with clinical responses to CAR-T therapies in MM (Dancy et al., Blood 2018). CITE-seq analyses showed that DP characteristics such as a high CD8+ stem-like phenotype and a low CD4+ Treg-like phenotype in the CAR+ T cell compartment associated with longer DOR. Some of the most abundant clones in the DP single cell TCR data were CAR+CD8+ T cells expressing granzyme B, suggesting high cytotoxic potential. Additional bulk TCR sequencing data suggest both diversity and individual clonal expansion longitudinally after cilta-cel infusion. We also evaluated tumor and patient intrinsic characteristics at baseline. BCMA expression on bone marrow PC was prevalent and highly variable among RRMM patients; however, expression did not associate with best response or DOR. Further, high ORR was observed in patients with high-risk cytogenetics, high tumor burden (>= 60% bone marrow PC), or baseline plasmacytomas, although with a non-statistically significant shorter PFS. Sustained MRD negativity strongly associated with longer DOR. Conversely, patient-intrinsic characteristics associated with inflammation (CRP, ferritin, neutrophils, pro-inflammatory cytokines) correlated with shorter DOR. Treatment benefit of the magnitude seen with cilta-cel had not been reported with any other approved therapy in this setting. Correlative analyses suggest an emerging profile to help understand such outstanding clinical activity: a balanced CD4/CD8 ratio and Tcm/Tem phenotype of the manufactured DP and preferential expansion of CAR+CD8+ Tcm cells. Further, certain baseline patient-intrinsic and tumor characteristics may explain the longer DOR in the RRMM setting. These investigations help identify markers of response to cilta-cel and may lead to CAR-T cell design or manufacturing strategies that enhance drug product characteristics and thus clinical efficacy.

Introduction Relapsed or refractory (r/r) acute myeloid leukemia (AML) has a dismal prognosis. Treatment options are limited and include fludarabine (Flu) and cytarabine (Ara-C), +/- anthracyclines (e.g., FLAG-Ida). Recent progress has been limited to targeted therapies, which benefit only a subset of the population; efforts to develop cellular therapies have not been met with success. NKX101 is an NK cell therapy derived from healthy donors and engineered to express an NKG2D ligand-directed chimeric antigen receptor (CAR) to enhance killing of malignant cells, as well as a membrane bound form of interleukin (IL)-15 to promote persistence and activity. Because Ara-C is known to upregulate NKG2D ligand expression, use of Flu/Ara-C as an alternative to standard Flu/cyclophosphamide (Flu/Cy) for lymphodepletion (LD) was tested for NKX101. Methods Six patients with r/r AML were enrolled in this cohort of NKX101-101, a Phase 1 safety study. All patients had received at least one prior line of therapy, as well as approved targeted therapies for those with underlying genetic mutations. Patients received NKX101 at a dose of 1.5 billion (B) viable CAR+ cells/dose on Days 0, 7, and 14 after LD comprising Flu (30 mg/m2) and Ara-C (2 g/m2) each once daily for five days and 2 days of rest. Efficacy assessments were performed on Day 27 and included bone marrow aspiration and biopsy. Additional treatment cycles of LD and NKX101 were given to some patients to deepen or consolidate responses. Pharmacokinetic (PK) and cytokine sampling were performed throughout the treatment cycle(s). Results At baseline, five out of six patients had poor-risk genetic features, including TP53 mutation; the median bone marrow blast count was 35% (range: 20 - 86%). Patients had received a median of two prior lines of therapy, with all patients having previous venetoclax exposure (Table 1). All patients received at least three doses of NKX101 at 1.5 B cells/dose. There were no cases of cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease of any grade in any patients (Table 2). Myelosuppression and infection were the most common higher-grade toxicities. Four out of six patients had CR/CRi, with three out of six achieving CR. Three of these patients had no detectable MRD by flow, and one had 0.18% RUNX1-RUNX1T1 translocation via polymerase chain reaction testing on bone marrow. One subject was taken to consolidative hematopoietic cell transplant (HCT) and remains in CR. Another subject received a consolidative cycle of therapy and remains in CR. A third subject had three cycles of treatment with successive decrease in disease burden and remains in CRi. The fourth subject had CR after one cycle of treatment. PK profiling showed that NKX101 was consistently detected and correlated with infusion days (Figure 1). Cytokine data showed minimal post-infusion elevations above baseline; no association was observed between clinical response and elevation of serum cytokines (including IL-15, IL-6, IFNγ, IL-10, and IL-8). Data on the expression of NKG2D ligands (MICA, MICB, ULBP1, and ULBP3) in subject bone marrow samples were obtained via multiplex immunohistochemistry. This was evaluated as a composite H-score. Responders were split between those with relatively low ligand expression and those with higher ligand expression, suggesting that further factors may contribute to NK cell resistance. Conclusions Alternative LD with Flu/Ara-C followed by NKX101 shows promising early responses including MRD negativity in r/r AML. The toxicity profile of this regimen is manageable and was consistent with underlying AML and exposure to LD, without any events of CRS or ICANS. Expansion of enrollment and longer follow-up are needed. Despite being an allogeneic, non-HLA-matched cell product, NKX101 persisted for up to three weeks in PK testing. Directly comparing the data from patients treated with Flu/Cy with NKX101 versus Flu/Ara-C with NKX101 suggests that Flu/Ara-C can replace the Flu/Cy regimen common to CAR T cell therapy as LD without compromising overall NKX101 exposure.