CD19 Chimeric Antigen Receptor T-cell Research Articles

Nonactivated and IL-7 cultured CD19-specific CAR T cells are enriched in stem cell phenotypes and functionally superior

AbstractCD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.

Methylated Whole Genome Cell-Free DNA Sequencing before Chimeric Antigen Receptor T-Cell Therapy for Large B-Cell Lymphoma Predicts Treatment Outcomes

Introduction More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes. Methods Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2. Results A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007). Discussion This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.

Development and Validation of a Clinical Prediction Model for the Efficacy of CD19 CAR T-Cell Therapy in DLBCL

Currently, Autologous CD19 chimeric antigen receptor T-cell therapy (CART) has become the most important treatment modality for relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) beyond conventional multi-line treatments. However, it is worth noting that over 60% of CD19 CAR-T recipients will eventually experience relapse. A substantial amount of clinical research has been conducted to explore the reasons for the ineffectiveness or relapse of CAR-T therapy. Based on a comprehensive analysis of clinical data, we selected various combinations of predictive factors and used the TRIPOD guidelines to construct a logistic regression model for predicting the efficacy of CD19 CAR-T therapy. This study was conducted as a retrospective cohort study, including patients with confirmed diagnosis of DLBCL who underwent CD19 CAR-T therapy and met the following criteria: (1) aged 18 years or older but not exceeding 80 years; (2) voluntarily participated in the study and signed an informed consent form. The exclusion criteria for this study were: (1) addition of other chemotherapy drugs or targeted drugs within 90 days before assessing the best response after infusion, except for those who achieved complete response (CR) or progressive disease (PD); (2) death caused by non-tumor progression factors within 90 days after infusion. After the inclusion and exclusion criteria, a total of 54 patients with DLBCL undergoing CD19 CART at Shanghai Tongji Hospital of Tongji University ultimately participated in this study from January 1, 2019, to April 31, 2023. The derivation cohort consisted of 45 patients enrolled in clinical trials (NCT02537977, NCT03154775, CTR20201986, CTR20200561). The median age of the patients in the derivation cohort was 60 years (IQR: 50-67 years). Out of the 45 participants, 25 (55.6%) were male, and 29 (64.4%) had the non-germinal center B-cell (non-GCB) subtype, 1 had grade 3-4 Cytokine Release Syndrome (CRS). The validation cohort consisted of 9 patients involved in real-world studies. The median age of the patients in the validation cohort was 55 years (IQR: 52.5-69 years). Out of the 9 participants, 5 (55.6%) were male, and 6 (66.7%) had the non-GCB subtype, 2 had grade 3-4 CRS. The demographic and clinical characteristics of the derivation cohort were similar to those of the validation cohort. Based on the best CD19 CART response at 90 days, we divided the derivation cohort into two groups, CR and non-CR. Then, we selected a series of candidate predictive factors from baseline data such as clinical features, genetic features, and laboratory features before CART (Table 1). The results of univariate and multivariate logistic regression analysis were used to select specific factors (Gender, ECOG, TP53) as model prediction indicators based on p-values (p<0.05). There was no significant correlation between these factors (p>0.1). A score nomogram (Model1) was plotted using the regression coefficients of each predictive factor as weights (Fig.1 A), C-index (Model1) =0.872 (Fig.1 C). To facilitate better clinical use, we also constructed a risk nomogram (Model2) to assess the degree of risk (Fig.1 D). Model2 considers three factors: Male, ECOG≥2, TP53 altered. If a person has not any of these risk factors, they are categorized as low risk. If there is one risk factor present, they are deemed to be at moderate risk, whereas having two or more risk factors classifies them as high risk. C-index (Model2) =0.845 (Fig.1 F). Finally, the model's clinical efficacy was validated using real-world CART-treated patients, and the C-index was calculated as Model1=0.875 (Fig.1 G), Model2=0.7 (Fig.1 H), confirming the clinical impact of the model. In conclusion, this study has successfully developed an easily implementable clinical prediction model. For the first time, we have used CART clinical studies as the derivation cohort and real-world results as the validation cohort. Our scoring system provides clinicians with accurate and objective efficacy information, helping them identify the appropriate population for CAR-T therapy in DLBCL.

Total Body Irradiation Versus Chemotherapy-Only Conditioning in Autologous Hematopoietic Stem Cell Transplantation for Large B-Cell Lymphoma

Introduction Recent data suggest that compared with high-dose chemotherapy conditioning and autologous stem cell transplant (ASCT), CD19-chimeric antigen receptor (CAR) T-cell therapy as second-line therapy improves outcomes in patients with primary refractory or early relapsed large B-cell lymphoma (LBCL). However, more than half of patients will not attain a long-term remission with CAR T-cell therapy, and may subsequently be considered for ASCT, indicating an unmet need for alternative therapies including strategies to improve ASCT outcomes. Given that LBCL is radioresponsive, we evaluated whether total body irradiation (TBI)-based conditioning may improve outcomes compared to chemotherapy-only conditioning among LBCL patients who underwent peripheral blood ASCT at our institution. Methods We reviewed the records of adults with relapsed/refractory LBCL who underwent ASCT at Fred Hutch Cancer Center between 2012-2021. LBCL was categorized as de novo DLBCL, transformed DLBCL, and double-hit lymphoma (DHL). Patients with primary CNS lymphoma were excluded. Given that TBI is generally reserved for younger patients (≤ 60 years), we excluded patients in the chemo-only group older than the oldest patient in the TBI group (58.7 years) to reduce the bias due to age differences. The hazards for failure for progression-free survival (PFS) and for overall mortality (OM) were compared between TBI-based and chemo-only groups using Cox regression with adjustment for clinically important variables including age, sex, histology, LDH at ASCT, conditioning type, PET positivity at the time of ASCT and year of ASCT. LDH at ASCT, age and year of ASCT were modeled as continuous linear variables. Results Among 225 patients, we excluded 121 patients in the chemo-only group who were older than 58.7 years, leaving 104 patients for the analysis ( Table 1). A total of 48 (46%) patients received TBI-based and 56 (54%) received chemo-only conditioning. All patients in the TBI group received 12 Gy in 6-8 fractions. Most patients (n=46, 82.14%) in the chemo-only group received BEAM conditioning. Patients receiving TBI-based conditioning had more adverse features including male sex (72.9% vs 58.9%), DHL histology (31.3% vs 7.1%), and PET-positive disease at the time of ASCT (39.6% vs 23.2%). With a median follow-up of 51.2 months (range 1.3-134.2) among survivors, point estimates of PFS at 2 and 5 years were 74.3% and 66.3%, respectively among TBI patients and 68.5% and 64.0%, respectively among the chemo-only patients ( Figure 1). Time to engraftment was similar between the groups. On multivariable analysis, compared to chemo-only conditioning, TBI was not associated with improved PFS (TBI vs chemo-only: HR 1.02, 95%CI 0.45-2.31, P=0.96) or OS (HR 0.85, 95%CI 0.37-1.98, P=0.7). Predictors of worse PFS were PET positivity at the time of ASCT (HR 3.39, 95%CI 1.61-7.17, P < 0.01) and higher LDH (HR 1.0011, 95%CI 1.0002-1.0020, P=0.02). Similarly, both PET positivity (HR 3.12, 95%CI 1.47-6.63, P<0.01) and higher LDH (HR 1.0013, 95%CI 1.0003-1.0022, P <0.01) were associated with worse OS. Age, sex, histology and year of ASCT were not significant. Conclusions Despite worse prognostic factors, TBI-based conditioning yielded similar outcomes compared to chemo-only conditioning for patients with relapsed/refractory LBCL undergoing ASCT. These results confirm prior findings from a registry-based study. Although a proportion of patients may achieve a long-term remission with ASCT, other strategies are needed to improve ASCT outcomes.

Machine Learning-Based Time-Series Clustering Identifies Archetypal Trajectories of Hematotoxicity after CAR T-Cell Therapy

BACKGROUND Severe hematotoxicity has been reported in a subset of patients (pts) after chimeric antigen receptor (CAR) T-cell therapy, leading to severe infections, transfusion dependency, and worse outcomes. Early identification of pts at risk for severe hematotoxicity could inform the consideration of an autologous stem cell boost, allogeneic HCT, and/or additional infectious prophylaxis. While distinct archetypal trajectories of blood count recovery have been proposed (e.g., quick vs. intermittent vs. aplastic), manual trajectory classification is subjective and labor-intensive. To address these limitations, we automated the identification of distinct trajectories of hematotoxicity by applying time-series clustering to longitudinal absolute neutrophil count (ANC) data from >400 pts treated with CAR T-cell therapy. Next, we sought to identify factors associated with the identified trajectories and to assess the predictive ability of the CAR-HEMATOTOX score (Rejeski, Blood 2021). STUDY DESIGN AND METHODS Adults ≥18 years of age who received their first infusion of CAR T cells for hematologic malignancies with commercial or investigational products at the Fred Hutchinson Cancer Center (2013-2023) were included. ANC trajectories were clustered using non-supervised longitudinal k-means based on Euclidean distances using the latrend and kml packages in R 4.1.3. We applied logistic regression to pre-lymphodepletion (pre-LD) variables to predict poor/very poor ANC recovery. Sensitivity and specificity were computed using a probability threshold based on the Youden criteria. RESULTS A total of 509 pts were identified; 106 were excluded due to insufficient data, with 403 pts included for the analysis. The most common disease types were aggressive NHL (n = 161; 40%), indolent NHL (n = 82; 20%), and ALL (n = 74; 18%). CAR T-cell products were axi-cel, n= 101 (25%); brexu-cel, n = 24 (6%); cilta-cel, n = 21 (5%); liso-cel, n = 46 (11%); ide-cel, n = 25 (6%); tisa-cel, 12 (3%); and investigational CD19 or CD20 CAR T-cell products, n = 174 (43%). As shown in Figure 1A, the ANC longitudinal data clustered into four distinct trajectories as follows: 1) high nadir followed by rapid recovery (“very good recovery”), n = 294 (73%); 2) low nadir followed by rapid recovery (“good recovery”), n = 87 (22%); 3) low nadir followed by intermittent recovery (“poor recovery”), n = 13 (3%); 4) aplastic phenotype (“very poor recovery”), n = 9 (2%). In univariate logistic regression, ALL (reference: aggressive NHL; OR = 5.43, 95% CI, 1.71-20.6, p = 0.006), pre-LD lower ANC (OR = 3.33 per log 10 , 95% CI, 1.82-6.25, p < 0.001), lower pre-LD platelet count (OR = 10 per log 10, 95% CI, 3.70-33.3), p < 0.001), and higher pre-LD disease burden as measured by lactate dehydrogenase (LDH) were associated with poor/very poor ANC recovery (OR = 16.6 per log 10, 95% CI, 4.79-59.3, p < 0.001). Next, we assessed the predictive ability of CAR-HEMATOTOX to identify poor/very poor ANC recovery in n = 356 pts with a calculable score. The CAR-HEMATOTOX score high/low showed modest discrimination (C-index: 0.65), which improved when used as a continuous variable (C-index: 0.85). As previously reported, these models had high sensitivity (>90%) but low specificity (high/low: 31%, continuous: 58%). To improve risk prediction of poor/very poor ANC recovery, we built a logistic regression model using restricted cubic splines - allowing for non-linear effects - including pre-LD ANC, platelet, Hb, LDH, CRP, and ferritin, which showed higher discrimination (C-index: 0.91), high sensitivity (88%), and higher specificity (79%) ( Figure 1B). CONCLUSION We introduce an automated, scalable, and disease-agnostic framework to analyze hematologic toxicity after CAR T-cell therapy. K-means clustering of longitudinal ANC data categorized pts into archetypal trajectories of hematologic recovery. A logistic regression model using pre-LD ANC, platelet, Hb, LDH, CRP, and ferritin showed improved discrimination and sensitivity compared to CAR-HEMATOTOX in our training set, but both models had low specificity (poor ability to “rule in” pts at risk of severe hematotoxicity) and thus low clinical utility at this time. We plan to further improve our predictive models by incorporating post-infusion biomarkers of systemic inflammation that we have previously shown to be associated with delayed count recovery after CAR T-cell therapy (Juluri, Blood Advances 2022).

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

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

CD19 CAR-T cell therapy for relapsed or refractory diffuse large B cell lymphoma: Why does it fail?

Chimeric antigen receptor T (CAR-T) cell therapy is an effective treatment for relapsed or refractory diffuse large B cell lymphoma (DLBCL) with 3 CD19 targeting products now FDA-approved for this indication. However, up to 60% of patients ultimately progress or relapse following CAR-T cell therapy. Mechanisms of resistance to CAR-T cell therapy in patients with DLBCL are likely multifactorial and have yet to be fully elucidated. Determining patient, tumor and therapy-related factors that may predict an individual's response to CAR-T cell therapy requires ongoing analysis of data from clinical trials and real-world experience in this population. In this review we will discuss the factors identified to-date that may contribute to failure of CAR-T cell therapy in achieving durable remissions in patients with DLBCL.

Long-term follow-up of humanized and murine CD19 CAR-T-cell therapy for B-cell acute lymphoblastic leukemia

Objective: Murine CD19 chimeric antigen receptor T-cell (CAR-T) products have been approved for the treatment of refractory/relapsed (R/R) B-cell acute lymphocytic leukemia (B-ALL) ; moreover, humanized products are also undergoing clinical trials. This study aimed to explore the differences in safety and short- and long-term follow-up efficacy between humanized and murine CD19 CAR-T-cells for treating relapsed and refractory B-ALL. Methods: Clinical data of 80 patients with R/R B-ALL treated with CD19-targeted CAR-T-cells at the Union Hospital of Tongji Medical College of Huazhong University of Science and Technology between May 2016 and March 2023 were analyzed, which included 31 patients with murine CAR-T and 49 with humanized products. Results: The proportion of patients with cytokine-release syndrome (CRS) in the murine and humanized groups was 63.1% and 65.3%, respectively. Moreover, a higher proportion of patients suffered from severe CRS in the murine group than in the humanized CAR-T group (19.4% vs 8.2%, P=0.174). Furthermore, one patient per group died of grade 5 CRS. The incidence of grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS) was 12.9% and 6.1%, respectively; severe ICANS were not observed. Among patients receiving murine CAR-T-cells, an overall response (OR) was observed in 74.2%. Conversely, the OR rate of patients receiving humanized CAR-T-cells was 87.8%. During the median follow-up time of 10.5 months, the median recurrence-free survival (RFS) of patients with murine CAR-T-cells was 12 months, which was as long as that of patients with humanized CAR-T-cells. The median overall survival (OS) were not reached in both groups. Of the 45 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a significantly improved RFS (43.25% vs 33.33%, P=0.027). Bridging transplantation was an independent factor in prolonging OS (χ(2)=8.017, P=0.005) and PFS (χ(2)=6.584, P=0.010). Common risk factors, such as age, high proportion of bone marrow blasts, and BCR-ABL fusion gene expression, had no significant effect on patients' long-term follow-up outcomes. Three patients reached complete remission after reinfusion of humanized CAR-T-cells. However, one patient relapsed one month after his second infusion of murine CAR-T-cells. Conclusions: The results indicate that humanized CAR-T therapy showed durable efficacy in patients with a higher tumor burden in the bone marrow without any influence on safety. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients who were not treated successfully with CAR-T therapies.

Efficacy and safety of fourth-generation CD19 CAR-T expressing IL7 and CCL19 along with PD-1 monoclonal antibody for relapsed or refractory large B-cell lymphoma

Objective: This study systematically explore the efficacy and safety of fourth-generation chimeric antigen receptor T-cells (CAR-T), which express interleukin 7 (IL7) and chemokine C-C motif ligand 19 (CCL19) and target CD19, in relapsed or refractory large B-cell lymphoma. Methods: Our center applied autologous 7×19 CAR-T combined with tirelizumab to treat 11 patients with relapsed or refractory large B-cell lymphoma. The efficacy and adverse effects were explored. Results: All 11 enrolled patients completed autologous 7×19 CAR-T preparation and infusion. Nine patients completed the scheduled six sessions of tirolizumab treatment, one completed four sessions, and one completed one session. Furthermore, five cases (45.5%) achieved complete remission, and three cases (27.3%) achieved partial remission with an objective remission rate of 72.7%. Two cases were evaluated for disease progression, and one died two months after reinfusion because of uncontrollable disease. The median follow-up time was 31 (2-34) months, with a median overall survival not achieved and a median progression-free survival of 28 (1-34) months. Two patients with partial remission achieved complete remission at the 9th and 12th months of follow-up. Therefore, the best complete remission rate was 63.6%. Cytokine-release syndrome and immune effector cell-associated neurotoxicity syndrome were controllable, and no immune-related adverse reactions occurred. Conclusion: Autologous 7×19 CAR-T combined with tirelizumab for treating relapsed or refractory large B-cell lymphoma achieved good efficacy with controllable adverse reactions.

Tislelizumab augment the efficacy of CD19/22 dual-targeted chimeric antigen receptor T cell in advanced stage relapsed or refractory B-cell non-Hodgkin lymphoma.

Dual-targeted chimeric antigen receptor T (CAR-T) cell is an important strategy to improve the efficacy of CD19 CAR-T cell against refractory or relapsed B cell non-Hodgkin lymphoma (R/R B-NHL). However, durable responses are not achieved in most patients, in part owing CAR-T cell exhaustion caused by PD-1/PD-L1 pathway.We conducted a prospective, single-arm study of dual-targeted CD19/22 CAR-T cell combined with anti-PD-1 antibody, tislelizumab, in R/R B-NHL (NCT04539444). Tislelizumab was administrated on +1day after patients received infusion of CD19/22 CAR-T cell. Responses, survival and safety were evaluated. From 1 August 2020 to 30 March 2023, 16 patients were enrolled. The median follow-up time is 16.0 (range: 5.0-32.0months) months. Overall response was achieved in 14 of 16 (87.5%) patients, and the complete response (CR) was achieved in 11 of 16 (68.8%) patients. The 1-year progression-free survival and overall survival rates were 68.8% and 81.3%, respectively. Of the 14 patients responded, 9 patients maintained their response until the end of follow-up. Among the 15 out of 16 (93.8%) patients who had extranodal involvement, 14 (93.3%) patients achieved overall response rate with 11 (73.3%) patients achieving CR. Eight (50%) patients experienced cytokine release syndrome. No neurologic adverse events were reported. Gene Ontology-Biological Process enrichment analysis showed that immune response-related signaling pathways were enriched in CR patients. Our results suggest that CD19/22 CAR-T cell combined with tislelizumab elicit a safe and durable response in R/R B-NHL and may improve the prognosis of those patients.

Optimizing CAR-T cell therapy in adults with B-cell acute lymphoblastic leukemia.

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated unprecedented success in the treatment of various hematologic malignancies including relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). Currently, there are two FDA-approved CD19-directed CAR-T cell products for the treatment of adults with R/R B-ALL. Despite high remission rates following CD19 CAR-T cell therapy in R/R B-ALL, remission durability remains limited in most adult patients, with relapse observed frequently in the absence of additional consolidation therapy. Furthermore, the burden of CAR-T cell toxicity remains significant in adults with R/R B-ALL and further limits the wide utilization of this effective therapy. In this review, we discuss patient and disease factors that are linked to CAR-T cell therapy outcomes in R/R B-ALL and strategies to optimize durability of response to reduce relapse and mitigate toxicity in the adult population. We additionally discuss future approaches being explored to maximize the benefit of CAR-T in adults with B-ALL.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from theimmunekilling of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

The shifting roles and toxicities of cellular therapies in B-cell malignancies.

Cellular therapies provide a curative-intent option for patients with relapsedand refractory lymphomas. Current options including high dose chemotherapyfollowed by autologous or allogeneic hematopoietic stem cell transplantation or CD19 chimericantigen receptor T-cell (CART) therapy. The indication varies according to lymphoma sub-type and line oftherapy. The sequencing of these therapies and their use in second-line orlater settings to manage these diseases is undergoing significant changes, withCD19 CAR T becoming a preferred option for relapsed aggressive B-cell lymphoma.The mechanism of both therapies causes significant yet distinctlymphodepletion, infectious, and inflammatory toxicities. The resulting patternand timing of immune reconstitution helps guide risk-mitigating strategies,revaccination, and infectious prophylaxis. In this review, we discuss theindication, efficacy, toxicity and immune reconstitution of autologoushematopoietic stem cell transplantation and CAR T therapy for use in thetreatment of lymphoma.

Effectiveness and safety of CD22 and CD19 dual-targeting chimeric antigen receptor T-cell therapy in patients with relapsed or refractory B-cell malignancies: A meta-analysis.

The efficacy of CD22 or CD19 chimeric antigen receptor T (CAR-T) cells in the management of acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) was observed. Because antigen loss and lack of CAR-T-cell persistence are the leading causes of progressive disease following single-antigen targeting, we evaluated CD22/CD19 dual-targeting CAR-T-cell therapy efficacy and safety in relapsed/refractory B-cell malignancies. The Web of Science, PubMed, Cochrane, and Embase databases were searched until July 2022. Patients confirmed with any relapsed/refractory B-cell hematological malignancies were included regardless of age, gender, or ethnicity, receiving CD22 and CD19-dual-targeting CAR-T-cell therapy. The studies conducted on patients with coexisting other cancer were excluded. We used random-effect models to explore the outcome, and heterogeneity was investigated by subgroup analysis. Fourteen studies (405 patients) were included. The pooled overall response (OR) and complete remission (CR) were 97% and 93%, respectively, for ALL patients. The 1-year proportions of overall survival (OS) and progression-free survival (PFS) were 70% and 49%, respectively. For NHL, OR occurred in 85% of patients, and 57% experienced CR. The results illustrated that the 1-year OS and 1-year PFS were 77% and 65%, respectively. The subgroup analysis showed that the dual-targeting modality achieved higher CR in the following cases: coadministration of CD22/CD19-CAR-T cells and third-generation CAR-T cells combined with ASCT and BEAM pretreatment. The ALL and NHL groups seemed similar in treatment-related toxicity: all grade cytokine release syndrome (CRS), severe CRS, and neurotoxicity occurred in 86%, 7%, and 12% of patients, respectively. Our meta-analysis demonstrated that the CD22/CD19 dual-targeting CAR-T-cell strategy has high efficiency with tolerable adverse effects in B-cell malignancies.

Anti-CD19 CAR T-cell consolidation therapy combined with CD19+ feeding T cells and TKI for Ph+ acute lymphoblastic leukemia.

We conducted a single-arm, open-label, single-center phase 1 study to assess the safety and efficacy of multicycle-sequential anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in combination with autologous CD19+ feeding T cells (FTCs) and tyrosine kinase inhibitor (TKI) as consolidation therapy in patients under the age of 65 years with de novo Ph-positive CD19+ B-cell acute lymphoblastic leukemia. Participants were given induction chemotherapy as well as systemic chemotherapy with TKI. Afterward, they received a single cycle of CD19 CAR T-cell infusion and another 3 cycles of CD19 CAR T-cell and CD19+ FTC infusions, followed by TKI as consolidation therapy. CD19+ FTCs were given at 3 different doses. The phase 1 results of the first 15 patients, including 2 withdrawals, are presented. The most common adverse events were cytopenia (13/13) and hypogammaglobinemia (12/13). There was no incidence of cytokine release syndrome above grade 2 or immune effector cell-associated neurotoxicity syndrome or grade 4 nonhematological toxicities. All 13 patients achieved complete remission, including 12 patients with a complete molecular response (CMR) at the data cutoff. The relapse-free survival was 84%, and the overall survival was 83% with a median follow-up of 27months. The total number of CD19-expressing cells decreased with an increasing CMR rate. CD19 CAR T cells survived for up to 40months, whereas CD19+ FTCs vanished in 8 patients 3months after the last infusion. These findings could form the basis for the development of an allo-HSCT-free consolidation paradigm. This trial was registered at www.clinicaltrials.gov as #NCT03984968.

ICOS and OX40 tandem co-stimulation enhances CAR T-cell cytotoxicity and promotes T-cell persistence phenotype.

Chimeric Antigen Receptor (CAR) T-cell therapies have emerged as an effective and potentially curative immunotherapy for patients with relapsed or refractory malignancies. Treatment with CD19 CAR T-cells has shown unprecedented results in hematological malignancies, including heavily refractory leukemia, lymphoma, and myeloma cases. Despite these encouraging results, CAR T-cell therapy faces limitations, including the lack of long-term responses in nearly 50-70% of the treated patients and low efficacy in solid tumors. Among other reasons, these restrictions are related to the lack of targetable tumor-associated antigens, limitations on the CAR design and interactions with the tumor microenvironment (TME), as well as short-term CAR T-cell persistence. Because of these reasons, we developed and tested a chimeric antigen receptor (CAR) construct with an anti-ROR1 single-chain variable-fragment cassette connected to CD3ζ by second and third-generation intracellular signaling domains including 4-1BB, CD28/4-1BB, ICOS/4-1BB or ICOS/OX40. We observed that after several successive tumor-cell in vitro challenges, ROR1.ICOS.OX40ζ continued to proliferate, produce pro-inflammatory cytokines, and induce cytotoxicity against ROR1+ cell lines in vitro with enhanced potency. Additionally, in vivo ROR1.ICOS.OX40ζ T-cells showed anti-lymphoma activity, a long-lasting central memory phenotype, improved overall survival, and evidence of long-term CAR T-cell persistence. We conclude that anti-ROR1 CAR T-cells that are activated by ICOS.OX40 tandem co-stimulation show in vitro and in vivo enhanced targeted cytotoxicity associated with a phenotype that promotes T-cell persistence.

CD19 chimeric antigen receptor T cell therapy in leukemia xenograft mouse: Anti-leukemic efficacy, kinetics, and 4-week single-dose toxicity

CD19 Chimeric antigen receptor T (CAR-T) cell therapy has shown a promising response rate for relapsed/refractory B-cell malignancies. However, serious side effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome arose in early case reports. Though several preclinical and clinical studies of CAR-T cell therapy have been reported, there is a lack of toxicological assessments. This study was carried out as a preclinical assessment of CD19 CAR-T cell therapy, including the anti-leukemic efficacy, kinetics in peripheral blood, and 4-week single-dose toxicity evaluation in leukemia xenograft mice. Leukemia xenograft mice model was established by injecting 1.0 × 105 cells/mouse of luciferase-labeled human B cell acute lymphoblastic leukemia (B-ALL) cell line via the tail vein, and after 3 days, 2.0 or 4.0 × 106 cells/mouse of CD19 CAR-T cells were injected intravenously. CD19 CAR-T cells showed significant anti-leukemic efficacy, showing inhibition of tumor progression in the bioluminescence-based in-vivo imaging system. In the kinetics study using qPCR, CAR-T cells peaked in peripheral blood on day 60 in males and day 30 in females. In a 4-week single-dose toxicity study, CD19 CAR-T cell injected groups showed no mortality and toxicological signs, or changes in body weight, food/water consumption, hematology, clinical chemistry, organ weights, and histopathology compared to control groups. These results suggested that 4.0 × 106 cells/mouse of CD19 CAR-T cells were effective in B-ALL xenograft mice without serious side effects, so the no-observed adverse effect level (NOAEL) was estimated to be higher than 4.0 × 106 cells/mouse, under the condition examined in the current study.

Low toxicity and excellent outcomes in patients with DLBCL without residual lymphoma at the time of CD19 CAR T-cell therapy.

CD19 chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough for patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), inducing sustained remissions in these patients. However, CAR T cells can result in significant toxicities. Preinfusion disease burden is associated with toxicities and outcomes after CAR T-cell therapy. We identified 33 patients with R/R DLBCL treated at 8 academic centers who had no detectable disease at the time of CAR T-cell therapy. The median time from leukapheresis to CAR T-cell infusion was 48 (19-193) days. Nine patients received axicabtagene ciloleucel, and 24 received tisagenlecleucel. There was no severe (grade ≥3) cytokine release syndrome, and only 1 patient developed severe neurotoxicity (grade 4). After a median follow-up of 16 months, 13 patients relapsed (39.4%) and 6 died (18.1%). One-year event-free survival and overall survival were 59.6% and 81.3%, respectively. Our findings suggest that, in patients with R/R DLBCL who have an indication for CAR T-cell therapy, treating patients in complete remission at the time of infusion is feasible, safe, and associated with favorable disease control. Further exploration in a larger clinical trial setting is warranted.

Outcomes among adult recipients of CD19 CAR T-cell therapy for Burkitt lymphoma.

7571 Background: Burkitt lymphoma (BL) is a rare, but highly aggressive B-cell lymphoma associated with especially poor outcomes in patients with relapsed/refractory disease. Multiple CD19 chimeric antigen receptor (CAR) T-cell products have been FDA approved to treat relapsed/refractory B-cell non-Hodgkin lymphoma in the second-line setting, however little is known about CAR T-cell responses in patients with BL. This multicenter study aims to assess real-world outcomes among patients with BL treated with CAR T-cell therapy across the United States. Methods: This multicenter, retrospective chart review was conducted by abstracting data from the medical charts of patients with BL who received CAR T-cell therapy. Demographic characteristics were assessed along with pre-CAR treatments, bridging therapies, infusion-related complications, CAR-T responses, and the use of post-CAR systemic therapy and/or hematopoietic stem cell transplant. Results: A total of 13 patients with relapsed/refractory BL received CAR T-cell therapy after a median of 3 prior therapies (range 1-6). Most patients received axi-cel (n = 8), although tisa-cel (n = 3) and liso-cel (n = 2) were also represented. The median age of these patients was 38 (range 24-68). A total of 6 (46.2%) had evidence of central nervous system involvement at diagnosis and 5 (38.5%) had a prior history of autologous stem cell transplant. The overall response rate 1 month after infusion was 69.2% with a complete response rate of 53.8%. All patients experienced grade 1-2 cytokine release syndrome (CRS). No one experienced grade ≥3 CRS, but 2 patients developed grade ≥3 immune effector cell-associated neurotoxicity syndrome (ICANS). Twenty-eight-day mortality was 23%, including one patient who died from grade 4 ICANS. Regarding long term data, only 4 (30.8%) patients sustained a complete response for more than 6 months. Median progression-free survival was 4.2 months (95% CI 28.2-257.6), and the 1-year overall survival rate was 61.5% (95% CI 0.56-0.67). Two (15.4%) patients received consolidative allogeneic stem cell transplants after CAR T-cell therapy, but both ultimately developed progressive disease. Conclusions: Relapsed/refractory BL is a rare and aggressive disease that remains challenging to treat despite the advent of CAR T-cell therapy. Further investigation is needed to determine the most effective management of these patients. We expect to include additional patients in this analysis over the coming months.

Developing CAR‐T‐sparing Radiotherapy ‐ early dosimetric results

Background/objective: Radiotherapy (RT) is an effective treatment for residual lymphoma following CD19 chimeric antigen receptor T-cell (CAR-T) therapy. However, there are concerns regarding the potential effect of RT on CAR-T, particularly early after infusion. We initiated a prospective protocol to explore the effect of different RT planning parameters and techniques on dose to the blood, and thus CAR-T cells, in order to develop a “CAR-T sparing RT” for early RT after CAR-T therapy. Methods: We analysed 33 RT treatment plans for 11 lesions in 7 patients. Each lesion was planned 3 times; (1) a conventional plan using volumetric modulated arc therapy (VMAT) technique, (2) a plan with optimisation for blood vessels in the region as well as blood-rich organs and bone marrow, and (3) same as plan 2 but delivered with Flattening Filter-Free (FFF) beams to increase dose rate. Plans were compared with regards to planning target volume (PTV) coverage, dose homogeneity, organs at risk (OAR) doses including blood vessels, dose rate and beam-on time. Results: Using CAR-T sparing techniques, the mean dose to major blood vessels was reduced by a mean of 12% compared to the conventional VMAT plan. The beam-on time was reduced by a mean of 60% due to the increase in dose rate. The blood-rich organ doses were not significantly affected. There was no detrimental effect on the overall quality of plans in terms of PTV coverage and dose homogeneity. The effect of different planning measures employed varied in different cases depending on the PTV volume, the anatomical location, and the proximity of blood vessels. Examples of clinical cases will be presented in the meeting. Conclusion: Reduction of radiation dose to the blood is possible using a variety of technical adjustments as well as reduction of the beam-on time. Further work continues to understand the relative contribution of the individual parameters and optimise RT in the post-CAR-T setting. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Radiation Therapy Conflicts of interests pertinent to the abstract. N. G. Mikhaeel Honoraria: Gilead, Educational meeting honoraria, Oct 2022