Treatment Of B-cell non-Hodgkin Lymphoma Research Articles

Efficacy and safety of lenalidomide in the treatment of B-cell non-Hodgkin lymphoma

BackgroundThe combination of rituximab and chemotherapy is a first-line treatment for patients with B-cell non-Hodgkin lymphoma. Lenalidomide is an immunomodulatory drug that has shown promising properties and activity in a variety of hematological malignancies. This study evaluated the efficacy and safety of lenalidomide-based regimens in the treatment of B-cell non-Hodgkin lymphoma.MethodsThe PubMed, Science Direct, ClinicalTrials.gov, and Web of Science databases were searched for relevant studies published up to May 2022. Studies with patients diagnosed with non-Hodgkin B-cell lymphoma, who were randomly assigned to a lenalidomide treatment group or a non-lenalidomide control group were considered for inclusion in this review and meta-analysis. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) of the time-to-event outcomes and risk ratios (RRs) with 95% CIs of dichotomous data were estimated.ResultsA total of 3593 patients from 10 studies were evaluated. The results of the pooled analysis indicated that the lenalidomide-based regimen was associated with prolonged overall survival (HR, 0.85; 95% CI 0.74–0.97; P = 0.02) and progression-free survival (HR, 0.70; 95% CI 0.57–0.88; P = 0.002). Significant differences were found in the overall response rate (RR, 1.18; 95% CI 1.04–1.33; P = 0.01) and complete response rate (RR, 1.18; 95% CI 1.00–1.39; P = 0.05) between the treatment and control groups.ConclusionsLenalidomide appears to be a promising therapeutic agent that offers the possibility of a novel combination of chemotherapy free regimen for patients with B-cell non-Hodgkin lymphoma.

Preclinical Study of DZD8586, a Non-Covalent LYN/BTK Dual Inhibitor with Excellent BBB Penetration, for the Treatment of B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Bruton's Tyrosine Kinase (BTK) inhibitors have been approved for the treatment of B-NHL, such as CLL and MCL. However, resistance to BTK inhibitors still inevitably occurred, and different types of mutations were identified post covalent and non-covalent BTK inhibitor treatment. Although BTK inhibitors showed some activities in the treatment of relapsed or refractory non-germinal center B-cell (non-GCB) DLBCL, no drugs have been approved due to unsustainable tumor response. It is hypothesized that blockage of BTK pathway alone is not sufficient to achieve optimal anti-tumor efficacy in DLBCL. Due to limited blood-brain barrier (BBB) penetration of the current therapies, treatment of central nervous system lymphoma (CNSL) remains a clinical challenge. DZD8586 is a rationally designed, oral, non-covalent, LYN and BTK dual inhibitor with excellent BBB penetration as a potential treatment option for B-NHL. Here we report the preclinical activity of DZD8586 in lymphoma cell lines and animal models which supports its clinical development to address the above unmet medical needs. Methods The enzymatic activity of DZD8586 against multiple kinases was assessed at Eurofins panel with purified enzymes. TMD-8-IbruR cells carrying the C481S mutation were generated by stepwise induction of increasing concentrations of Ibrutinib. Cells carrying pirtobrutinib resistance mutations were obtained by transfection of different BTK mutations into RI-1 cells using lentivirus. Cell growth inhibition was determined by CellTiter-Glo. A pBTK enzyme-linked immunosorbent assay (ELISA) was used to evaluate the pharmacodynamic profile of DZD8586. Western blot was used to test the modulations of signaling pathways with DZD8586 treatment. Animal models were established by subcutaneous and intracranial inoculation of tumor cells, respectively, and animals were treated with DZD8586 or other BTK inhibitors. The anti-tumor efficacy was assessed by comparing the change in mean tumor volume between the different treatment groups and vehicle controls. Results Kinase panel profiling showed that DZD8586 potently inhibited LYN and BTK, with good selectivity against other kinases. In cell lines expressing C481X and pirtobrutinib resistance mutations, DZD8586 demonstrated concentration dependent anti-proliferative effects, with similar GI 50s among cells carrying different BTK mutations. In a panel of DLBCL cell lines, DZD8586 exhibited potent cell growth inhibition and induced cell death in a variety of cell lines, and its effect correlated with modulations of both LYN and BTK pathways. In both subcutaneous and CNS tumor models, DZD8586 exhibited profound tumor growth inhibition in a dose-dependent manner, and induced tumor regression at the doses of 50 mg/kg and above. There was a good correlation between plasma concentrations of DZD8586 and modulations of pBTK in whole blood and tumor tissue. DZD8586 has good cell permeability and is not significantly transported by P-gp and BCRP expressed at the BBB. The K puu,CSF of DZD8586 in rat and monkey were 1.2 and 1.3, respectively, suggesting its excellent BBB penetration in humans. Conclusion DZD8586 is a novel non-covalent LYN/BTK dual inhibitor, which could overcome resistance mutations to the approved covalent and non-covalent BTK inhibitors, and derive clinical benefit to patients with DLBCL and CNSL by blocking both BTK-dependent and BTK-independent signaling pathways with excellent BBB penetration.

Addition of Inotuzumab Ozogamicin to the Conditioning Regimen of Allogeneic Stem Cell Transplantation (allo-SCT) for Relapsed CD22 (+) Lymphoid Malignancies: Long-Term Survival Results

Background: Inotuzumab ozogamicin (InO) is a humanized antibody-drug conjugate that targets CD22+ B-cells. InO demonstrated antitumor activity and manageable toxicity in phase 1/2 trials for the treatment of B-cell non-Hodgkin lymphoma (NHL) as a single agent and in combination with rituximab. In order to improve outcomes in patients with relapsed CD22 (+) NHL, or chronic lymphocytic leukemia (CLL) who failed targeted therapies and were candidates for allo-SCT, we prospectively studied the addition of InO to our standard chemo-conditioning of BFR (bendamustine, fludarabine and rituximab-Khouri Blood 2014). Herein we report long-term survival outcomes. Methods: InO was infused intravenously (iv) on day -13 outpatient, with a dose cohort of 0.6, 1.2 or 1.8 mg/m2. Bendamustine 130 mg/m 2 iv daily on days -5 to -3 together with 30 mg/m 2 iv of fludarabine on days -5 to -3 were given prior to transplantation. Rituximab was given at a dose of 375 mg/m 2 iv on days -6, +1, and +8. Tacrolimus and mini-methotrexate were used for graft versus host disease (GVHD) prophylaxis. Results: The study included 26 patients. Median age was 59 (range, 26-70) years. Disease types: CLL [n=11 (27%) ; 64% with TP53 mutations (either alone, or with other mutations such as BTK, CDH2, ZMYM3); 22% with complex cytogenetics; 75% with unmutated IGHV; mantle cell lymphoma ( MCL) [n=8 (31%); 83% had Ki 67 >30%; 25% TP53 mutation; and 25% blastoid histology]; Follicularlymphoma (n=5, 19%), and diffuse large b cell ( DLBCL[n= 2; (8%)]. Median # prior treatments was 2.5 (range, 1-6). Patients with CLL/MCL were previously treated with ibritunib (n=10), venetoclax (n=5), idelalisib (n=2), nivolumab (n=1) and CAR T (n=1). At study entry, 18 (69%) patients were in CR, 7 (27%) in PR, and 1 (4%) had SD. Eleven (42%) received their transplants from matched sibling donors (MSDs) and 15 (58%) from matched unrelated donors (MUDs). The number of patients who received the 0.6, 1.2 or 1.8 mg/m2 of InO were 4, 2 and 20 patients, respectively. No DLT was observed. Forty-two percent of patients never experienced severe neutropenia and 77% never experienced platelet counts < 20K x 10 9/L. Only 1 patient developed veno-occlusive disease, confounded by the simultaneous manisfestation of hyper-acute GVHD related to prior nivolumab pre-alloSCT. Treatment-related mortality (TRM) at 2-years was 12%. With a median follow-up of 48.7 months (range, 3.6-82.8), the 5-year overall survival (OS) and progression-free survival rates (PFS) were 84% and 80%, respectively. Seven of 8 (87.5%) patients with PR/SD at study entry converted to CR after allo-SCT. There was no significant difference in OS or PFS by histology subtype. Patients who received a transplant from MSDs had OS and PFS rates of 100% v 79% ( P = .060) and 64% ( P = .032) for those who received MUDs, respectively (Figure 1). We compared results of this trial to a group of patients (n=56) with relapsed lymphoid malignancies who received allo-SCT at our center in a preceding prospective trial using BFR conditioning without InO and the same GVHD prophylaxis (clinicaltrials.gov #NCT00880815) which was previously published. There was no statistically significant difference in patients, disease (including histology, disease status pre-transplant) or transplant characteristics between the 2 groups. We found no statistically significant differences in engraftment times, incidence and grades of liver toxicity, TRM, risk of acute grade II-IV or III-IV GVHD. However, the study group containing InO had a higher incidence of extensive chronic GVHD (mainly de novo) than the control group (50% vs 25%, P = .019), respectively. There was a trend in patients with NHL to have a better 5-year OS (93% vs 68%) and 5-year PFS (93% vs 58%, Figure 2) in the study group vs the control groups. We did not observe such a trend in patients with CLL (5-year PFS 62% vs 59%): this could be related to small #patients, level of expression of CD22, more adverse mutations in the study group. Conclusions: Our results show that InO is safe when combined with an allo-SCT conditioning regimen and may improve survival outcomes in patients with CD22 (+) NHL. This needs to be validated in a larger number of patients. An ongoing trial at our center involves fractionating InO dose pre-and post-allo-SCT in patients with lymphoma or acute lymphoblastic leukemia receiving a reduced-intensity conditioning, and adding post-transplant cyclophosphamide to decrease the risk of GVHD.

Incidence of Infections Associated with the Use of Bispecific Antibodies in B-Cell Non-Hodgkin Lymphomas

Background: Despite recent advances in the treatment of B-cell non-Hodgkin lymphomas (NHL), most patients with relapsed/refractory (R/R) disease are at high risk of dying from their disease. Bispecific antibodies (BsAbs) are novel therapies with favorable response rates in R/R B-cell NHL even after cellular therapies. Mosunetuzumab, epcoritamab, glofitamab, and odronextamab are CD20 targeting BsAbs that have proven efficacy for R/R B-cell NHL. These treatments are generally well tolerated, but the infectious complications are not well described in the literature. Our study aims to quantify the relative incidence of infections associated with B-cell NHL targeted BsAbs. Methods: We conducted a preliminary evaluation, of an ongoing pooled analysis encompassing single agent BsAbs used in B-cell NHL with no prior use of alternate BsAbs. Exclusion criteria included studies reporting the use of BsAbs as part of combination therapy and trispecific antibodies. Results: A total of 546 B-cell NHL patients (number of trials, n=4) were treated with BsAbs in the studied period. Pooled median follow up was 11.7 . Relevant adverse events included all grade neutropenia in 26.9% (n=147/546), grade III/IV neutropenia in 20% (n=109/546), all grade infections in 40.1% (n=219/546), grade III/IV infections in 16.8% (n=92/546), all grade pneumonia in 7.2% (n=17/235), and coronavirus 2019 (COVID-19) in 1.3% (n=3/235). Incidence of grade III/IV and all grade infections of the BsAbs in each study are shown in figure 1. In addition, all grade cytokine release syndrome was seen in 56.4% (n=308/546) and death was reported in 33 patients of which 4 (0.7%) were reported to be secondary to infections. Conclusion: Data from this ongoing analysis better describes the various infectious risks of patients with B-cell NHL receiving BsAbs. Providers should remain vigilant regarding these risks and intervene promptly in such situations. The finalized pooled analysis with a larger sample size may corroborate these findings.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

Combination of CDK4/6 Inhibitor Palbociclib and PI3K Inhibitor Idelalisib Synergistically Induces an Anti-Tumor Effect in B-Cell Lymphoma and Overcomes Ibrutinib Resistance

Introduction: Aberrant activity of cyclin-dependent kinases 4/6 (CDK4/6) is frequently detected in B-cell non-Hodgkin's lymphoma (B-NHL), indicating the therapeutic implications of CDK4/6 inhibitors for B-cell malignancies. What's more, the introduction of agents targeting B cell receptor signaling, especially phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, is rapidly changing how B cell malignancies are treated. In this study, we aim to investigate whether the combination of CDK4/6 inhibitor palbociclib (PAL) and PI3Kδ inhibitor idelalisib (IDE) has synergistic anti-tumor effects in B cell lymphoma. Besides, we also evaluated the effect of co-administration PAL and IDE in Bruton's tyrosine kinase inhibitor (BTKi) resistant model. Methods: In vitro, cell viability was analyzed using Cell Titer-Glo ®️ Luminescent Cell Viability Assay. Flow cytometry was used to detect apoptosis and cell cycle arrest induction. Western Blotting analysis was used to detect the changes in essential proteins in related signaling pathways. We also conducted RNA-seq to evaluate the whole transcriptome changes brought by co-treatment with PAL and IDE. The synergistic anti-tumor effects of PAL and IDE were also evaluated in vivo. BTKi secondary resistant cell lines were generated by long-time exposure to ibrutinib and BTK C481S mutation cell line was conducted by lentivirus infection and puromycin selection. Results: Firstly, we analyzed diffuse large cell lymphoma (DLBCL) gene expression profile results from GEO database, and there were more than 50% patients carried cell cycle-related genes alterations, especially for CDKN2A-CDK4/6/CCND1-Rb machinery. Then, it was noteworthy that the high level of CDK4/6 mRNA and protein expression correlated with worse survival of DLBCL patients. Next, we illustrated that pharmacological inhibition of CDK4/6 kinase activity induced inhibition of cell proliferation, tumor cell apoptosis, and cell senescence. Meanwhile, PAL treatment upregulated the expression of PI3Kδ, p-AKT (S473), and p-mTOR (S2448), suggesting compensatory activation of the PI3K-AKT-mTOR signaling pathway. Thus, combination treatment of PAL and PI3K inhibitor IDE was assessed, and this combination synergistically induced a more potent anti-proliferative effect in several B-NHL cell lines, including mantle cell lymphoma cell lines (Z-138 and Jeko-1), DLBCL cell lines (U2932 and OCI-Ly8) and Burkitt lymphoma cell line (Raji). Meanwhile, co-administration treatment could also trigger synergistic anti-tumor activity in BTK inhibitor ibrutinib-resistant HBL-1 BTK C481S and HBL-1 IR cells (long time exposure to ibrutinib). Next, the flow cytometry results indicated that the B-NHL cells became apoptotic after the PAL and IDE treatment. The activation of apoptosis-related proteins (cleaved-caspase3 and cleaved-PARP) and decrease of anti-apoptosis proteins expression (BCL-2, BCL-xL, XIAP, and MCL-1) were induced by combination of CDK4/6 and PI3K inhibitor treatment. The combination of PAL and IDE also substantially increased the cell population of G0/G1 phase and elevated expression of CDK4, CDK6, and CyclinD1. Furthermore, the gene expression profile analysis demonstrated that the mRNA expression level of PLK1 was significantly decreased by co-administration treatment, rather than in PAL or IDE treatment alone. Finally, the combination treatment also showed synergistic anti-tumor activity in multiple tumor-bearing mice models. Conclusion: The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

Changing Trends in B-Cell Non-Hodgkin Lymphoma Treatment: The Role of Novel Monoclonal Antibodies in Clinical Practice.

We are currently witnessing a dramatic shift in our approach to the treatment of B-cell non-Hodgkin lymphoma (B-NHL). In the evolving clinical landscape, novel treatments for this clinically heterogeneous disease span a wide range of interventions, encompassing targeted agents, cell therapy approaches, and novel monoclonal antibodies (NMABs). Among these, the latter are likely to exert the most profound impact due to their distinctive high efficacy and versatile applicability. NMABs represent a heterogeneous group of agents, including naked antibodies, immunotoxins, and T-cell-engaging molecules. In recent times, several NMABs have either gained regulatory approval or are on the verge of introduction into clinical practice, addressing multiple therapeutic indications and treatment regimens. Their anticipated impact is expected to be broad, initially in the context of relapsed/refractory (R/R) disease and subsequently extending to early treatment lines. The scope of this review is to provide a comprehensive overview of the biological characteristics, clinical properties, efficacy, and toxicity profiles of NMABs that have recently been introduced or are nearing integration into clinical practice.

T-cell redirecting therapies for B-cell non-Hodgkin lymphoma: recent progress and future directions.

Several key advances in the treatment of B-cell non-Hodgkin lymphoma (B-NHL) over the past two decades have strategically exploited B-cell lineage markers suitable for targeting by immunotherapies. First, the addition of the anti-CD20 monoclonal antibody (mAb) rituximab to a range of standard therapies conferred remarkable outcomes improvements in diverse settings, perhaps most prominently an overall survival advantage in newly diagnosed diffuse large B-cell lymphoma (DLBCL). Subsequently, multiple chimeric antigen receptor (CAR) T-cell therapies targeting CD19 have revolutionized the treatment of relapsed/refractory (rel/ref) DLBCL and are active in other B-NHL subtypes as well. Most recently, the longstanding aspiration to exploit patients' endogenous T-cells to combat lymphoma has been achieved via T-cell redirecting therapies such as bispecific antibodies (BsAbs) that incorporate dual targeting of a T-cell antigen such as CD3 plus a B-cell antigen such as CD19 or CD20 expressed by the tumor. These novel agents have demonstrated impressive activity as monotherapies in patients with heavily pre-treated, rel/ref B-NHL of a variety of subtypes. Now, myriad clinical trials are exploring combinations of T-cell redirectors with targeted therapies, antibody-drug conjugates, conventional chemotherapy, and even new immunotherapies. Here, we highlight key landmarks in the development of T-cell redirecting therapies for the treatment of B-NHL, emerging evidence and lessons from recent clinical trials, and exciting new directions in this arena.

Glofitamab: First Approval.

Glofitamab (Columvi®) is a CD20 × CD3 T-cell-engaging bispecific monoclonal antibody being developed by Roche for the treatment of B-cell non-Hodgkin lymphomas, including diffuse large B-cell lymphoma (DLBCL). Glofitamab received its first approval (with conditions) on 25 March 2023, in Canada, for the treatment of adult patients with relapsed or refractory DLBCL not otherwise specified, DLBCL arising from follicular lymphoma, or primary mediastinal B-cell lymphoma, who have received two or more lines of systemic therapy and are ineligible to receive or cannot receive CAR T-cell therapy or have previously received CAR T-cell therapy. Glofitamab is also under regulatory review for relapsed or refractory DLBCL in the EU and USA and in April 2023 received a positive opinion recommending the granting of a conditional marketing authorization in the EU. Clinical development of glofitamab, as a monotherapy and in combination with other agents for the treatment of non-Hodgkin lymphomas, is continuing worldwide. This article summarizes the milestones in the development of glofitamab leading to this first approval for relapsed or refractory DLBCL.

2022 Chinese expert consensus and guidelines on clinical management of toxicity in anti-CD19 chimeric antigen receptor T-cell therapy for B-cell non-Hodgkin lymphoma.

Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells has emerged as a novel modality for treating relapsed and/or refractory B-cell non-Hodgkin lymphoma (B-NHL). With increasing approval of CAR T-cell products and advances in CAR T cell therapy, CAR T cells are expected to be used in a growing number of cases. However, CAR T-cell-associated toxicities can be severe or even fatal, thus compromising the survival benefit from this therapy. Standardizing and studying the clinical management of these toxicities are imperative. In contrast to other hematological malignancies, such as acute lymphoblastic leukemia and multiple myeloma, anti-CD19 CAR T-cell-associated toxicities in B-NHL have several distinctive features, most notably local cytokine-release syndrome (CRS). However, previously published guidelines have provided few specific recommendations for the grading and management of toxicities associated with CAR T-cell treatment for B-NHL. Consequently, we developed this consensus for the prevention, recognition, and management of these toxicities, on the basis of published literature regarding the management of anti-CD19 CAR T-cell-associated toxicities and the clinical experience of multiple Chinese institutions. This consensus refines a grading system and classification of CRS in B-NHL and corresponding measures for CRS management, and delineates comprehensive principles and exploratory recommendations for managing anti-CD19 CAR T-cell-associated toxicities in addition to CRS.

CD20/TNFR1 dual-targeting antibody enhances lysosome rupture-mediated cell death in B cell lymphoma.

Obinutuzumab is a therapeutic antibody for B cell non-Hodgkin's Lymphoma (BNHL), which is a glyco-engineered anti-CD20 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and causes binding-induced direct cell death (DCD) through lysosome membrane permeabilization (LMP). Tumour necrosis factor receptor 1 (TNFR1), a pro-inflammatory death receptor, also evokes cell death, partly through lysosomal rupture. As both obinutuzumab- and TNFR1-induced cell deaths are mediated by LMP and combining TNFR1 and obinutuzumab can amplify LMP-mediated cell death, we made dual-targeting antibody for CD20 and TNFR1 to enhance DCD of obinutuzumab.Obinutuzumab treatment-induced CD20 and TNFR1 colocalisation, and TNFR1-overexpressing cells showed increased obinutuzumab-induced DCD. Two targeting modes, anti-CD20/TNFR1 bispecific antibodies (bsAbs), and obinutuzumab-TNFα fusion proteins (OBI-TNFαWT and OBI-TNFαMUT), were designed to cluster CD20 and TNFR1 on the plasma membrane. OBI-TNFαWT and OBI-TNFαMUT showed significantly enhanced LMP, DCD, and ADCC compared with that induced by obinutuzumab. TNFR1 expression is upregulated in many BNHL subtypes compared to that in normal B cells; OBI-TNFαMUT specifically increased DCD and ADCC in a B cell lymphoma cell line overexpressing TNFR1. Further, OBI-TNFαMUT blocked NF-κB activation in the presence of TNF-α, implying that it can antagonise the proliferative role of TNF-α in cancers.Our study suggests that dual targeting of CD20 and TNFR1 can be a new therapeutic strategy for improving BNHL treatment. The OBI-TNFαMUT fusion protein enhances DCD and ADCC and prevents the proliferating effect of TNFα signalling; therefore, it may provide precision treatment for patients with BNHL, especially those with upregulated TNFR1 expression.

Characterization of JNJ-80948543, a Novel CD79bxCD20xCD3 Trispecific T-Cell Redirecting Antibody for the Treatment of B-Cell Non-Hodgkin Lymphoma

Characterization of JNJ-80948543, a Novel CD79bxCD20xCD3 Trispecific T-Cell Redirecting Antibody for the Treatment of B-Cell Non-Hodgkin Lymphoma

Evaluation of Epcoritamab and Rituximab Combination in Preclinical Models of B-Cell Non-Hodgkin's Lymphoma (NHL)

Background: Epcoritamab (Epco) is a CD3 bispecific antibody that induces selective, potent T-cell-mediated killing of CD20-positive malignant B-cells. In the phase 1/2 monotherapy study, subcutaneous administered Epco showed a favorable safety profile and promising efficacy, including complete responses in heavily pretreated patients with relapsed / refractory non-Hodgkin's lymphoma (Hutchings et al, Lancet 2021; Thieblemont et al, EHA 2022, LB2364; EPCORE NHL-1). Ongoing clinical studies combining Epco with standard-of-care therapies including combinations with Rituximab show promising preliminary anti-tumor activity in diffuse large B-cell lymphoma and follicular lymphoma (Falchi et al, ASCO 2022 abstracts 7523 and 7524; EPCORE NHL-2 arm 1 and 2; NCT04663347). Here we evaluated the competition for CD20 binding and functional interactions between these agents and modeled its impact on Epco, T-cell, and tumor cell trimer formation that drives Epco clinical activity. Aims: Investigate the preclinical binding and functional interaction between Epco and Rituximab to establish the potential for the combination of these agents in the treatment of B-cell NHL. Methods: Competition between Epco and Rituximab for binding to CD20 cells was assessed by Flow Cytometry after incubating the single labeled agent in the presence of increasing concentrations of the competitor and vice-versa (across 0.1 - 300 ug/ml evaluated for each agent), on a range of CD20 expressing lymphoma cell lines. Epco-induced-T cell activation and redirected T cell cytotoxicity was determined by co-culturing T cells with lymphoma cells with varying expression of CD20. Rituximab antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by incubating fresh peripheral blood mononuclear cells with lymphoma cells in the presence of a CD3 control Epco antibody (DuoBody-ctrlxCD20). Data from binding and cytotoxicity was used to quantify the degree of binding curve shifts at different Rituximab:Epco concentrations as well as the impact on predicted trimer formation and cytotoxicity, based on modeling. Results: In binding assays, the addition of Rituximab resulted in a dose-dependent reduction of Epco binding to CD20 expressing lymphoma cell lines, with a significant reduction of Epco binding observed at clinically relevant Epco concentrations in cells with high CD20 expression levels (Figure 1). Similar binding interference interactions were observed for binding of Epco to cell lines with low and intermediate CD20 expression levels. Binding of Rituximab to cell lines expressing high CD20 levels was not significantly reduced by clinically relevant concentrations of Epco. In Epco functional studies, the addition of increasing concentrations of Rituximab did not affect Epco cytotoxic activity at clinically relevant range of Rituximab doses tested (Figure 2). Similarly, no effect of Rituximab on Epco-mediated T cell activation was observed at clinically relevant concentrations of Rituximab and Epco. ADCC activity at clinically relevant concentrations of Rituximab (10-250 ug/ml) was not impacted by incubation with the CD3 control Epco antibody (DuoBody-ctrlxCD20), even at concentrations exceeding the Epco clinical concentrations. Modeling and simulation of in vitro data showed that at clinically relevant concentration of Epco, the presence of clinically relevant concentration of Rituximab does not meaningfully impact Epco trimer formation, and the predicted trimer formation remains above EC90 relative to that needed for 50% cell kill. Conclusions: Our studies showed that while Rituximab exerts significant interference on Epco binding to CD20-expressing cells, tumor-cell cytotoxicity by Epco is minimally affected by clinically relevant concentrations of Rituximab. This conclusion is supported by preclinical modeling and simulations showing that Epco activity driven by trimer formation is not meaningfully impacted by clinical concentrations of Rituximab. Furthermore, we found a limited effect of Epco on Rituximab binding to CD20 and no impact on ADCC activity by Rituximab, thereby supporting potential synergy in the combination between these agents. Results from this study confirm the ability to combine Epco with CD20 antibodies, specifically with Rituximab, and the potential for synergistic activity of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 2811: CT120, a novel fully human anti-CD19 x anti-CD22 dual targeted chimeric antigen receptor T cell product for the treatment of B-NHL and B-ALL

Abstract Background: Anti-CD19 chimeric antigen receptor T cell therapies (CAR-T) have shown impressive clinical outcomes in relapsed/refractory (R/R) CD19+ B-cell non-Hodgkin lymphomas (B-NHL) and acute lymphoblastic leukemias (B-ALL). However, 30%-60% of patients with B-ALL who initially responded to anti-CD19 CAR-T therapies relapsed either due to poor CAR-T persistence or CD19 antigen escape, and preliminary data also indicated CD19 antigen loss in patients with B-NHL relapsed after anti-CD19 CAR-T therapy. In addition, the immunogenicity of murine origin CAR construct is another cause of limited in vivo CAR-T persistence. Design: To overcome these problems, a novel fully human anti-CD19×anti-CD22 dual targeted CAR-T cell product - CT120, has been developed. The CT120 CAR molecule contains tandem fusion of anti-CD19 and anti-CD22 scFv, which can effectively recognize CD19 and CD22 antigen independently, to avoid antigen escape. The intracellular CAR domain contains CD3ζ and 4-1BB co-stimulatory domain, which can improve in vivo persistence. In addition, both anti-CD19 and anti-CD22 antibody were generated from proprietary fully human libraries to minimize possible immunogenicity. The CAR molecule was transferred to autologous T cells through a lentiviral backbone. Results: The anti-CD19 and anti-CD22 scFv binds to its antigen in nanomolar range, and CT120 CAR-T cells can bind to both CD19 and CD22 antigens in a noncompetitive manner. CT120 CAR-T cells showed potent in vitro cytotoxicity not only to target B-cell lymphoma/leukemia cell lines (Raji, Daudi, and NALM-6), but also to the CD19 knockout (CD19-CD22+) and CD22 knockout (CD19+CD22-) Raji cell lines. CT120 can secret multiple cytokines, such as IL-2, TNF-α and IFN-γ, and proliferate efficiently after engagement of target cells. In vivo efficacy study demonstrated that CT120 CAR-T cells expanded effectively in mice and completely inhibit Raji and NALM-6 tumor cell growth. Furthermore, CT120 showed a favorable safety profile and promising efficacy in patients with r/r B-NHL and B-ALL in an investigator-initiated trial (IIT). Certain patients who relapsed after previous murine origin CAR-T treatment also benefited from CT120 therapy. Conclusion: Collectively, CT120 is a potent and safe anti-CD19×anti-CD22 dual target CAR-T product for the treatment of B-NHL and B-ALL, and now it is in registered phase 1/2 clinical trial. Citation Format: Panpan Niu, Qianli Hu, Jialu Mo, Guangrong Meng, Xiangyin Jia, Wei Cheng, Qiaoe Wei, Zhenyu Dai, Xuefeng Wu, Guang Hu, Taochao Tan, Jianfeng Zhou, Yongkun Yang, gang Hu. CT120, a novel fully human anti-CD19 x anti-CD22 dual targeted chimeric antigen receptor T cell product for the treatment of B-NHL and B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2811.

Nanobody-based anti-CD22-chimeric antigen receptor T cell immunotherapy exhibits improved remission against B-cell acute lymphoblastic leukemia

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 can achieve impressive clinical remission rates in the treatment of B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. However, relapse after CD19-CAR T treatment remains a major issue, with CD19 antigen-negative relapse being one of the main reasons. CD22, another antigen expressed in a B-cell lineage-specific pattern, is retained following CD19 loss. Accordingly, we hypothesized that CD22 could represent an alternative target to alleviate or compensate for the ineffectiveness of CD19-CAR T therapy. To this end, we generated camelid-derived CD22 nanobodies, whose smaller size, greater stability, and lower immunogenicity offer better quality than classical antibodies, and we used them to construct third-generation CD22-CARs containing 4-1BB and ICOS co-stimulatory domains. The novel CD22-CAR T cells exhibited impressive cytotoxicity both in vitro and in vivo and significantly prolonged the overall survival of tumor-bearing NSG mice. These findings provide the basis for further translational studies employing CD22-CARs.

Incidence and Characteristics of CD20 Monoclonal Antibody-Induced Interstitial Pneumonitis

Background:CD20 monoclonal antibody therapy is the backbone of treatment for B-cell non-Hodgkin lymphoma. Several studies have described an association between the use of rituximab and non-infectious interstitial pneumonitis, with estimates ranging from 3.9% to 8.4%. 1-3 However, several novel CD20 monoclonal antibodies have been introduced in the past decade, with obinutuzumab gaining FDA approval in 2013, ofatumumab first approved in 2014, and rituximab/hyaluronidase human approved as a subcutaneous route of rituximab in 2017. While the use of these agents has rapidly increased in clinical practice, the incidence of pneumonitis with these agents is not well known. Thus, we have performed a retrospective review of patients with B cell lymphoma treated with CD20 monoclonal antibody therapy to determine the incidence and characteristics of pneumonitis with these novel agents.Methods:We evaluated all patients at the University of North Carolina at Chapel Hill with B-cell lymphoma who were treated with CD20 monoclonal antibody therapy from 2014 to 2020. We performed individual chart review to verify the presence of interstitial pneumonitis, as defined by presence of respiratory symptoms (cough, dyspnea, fever, chest pain) in addition to imaging findings (pulmonary opacity on chest X-ray and/or ground glass opacities on chest computed tomography (CT)) without alternative cause (CHF with pulmonary edema, COPD flare, infectious cause including bacterial, fungal or viral pneumonia/bronchitis). We extracted demographic data, comorbid conditions, tobacco use history, and timing of pneumonitis related to CD20 monoclonal antibody exposure. We determined rates of pneumonitis based on agent and chemotherapy backbone.Results:We identified 18 cases of chart-confirmed interstitial pneumonitis over a six-year period, out of 2,207 patients treated with CD20-monoclonal antibody therapy. Seven (39%) were current or former smokers, with a median of 30 pack-years smoked. Five (29%) had a history of hypersensitivity reaction to rituximab infusion. Pneumonitis developed after a median of 4 cycles, and 16 days from the most recent CD20 monoclonal antibody infusion. Ten (56%) had diffuse large B-cell lymphoma, 4 (22%) had mantle cell lymphoma, and 1 (6%) each had follicular lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and Waldenstrom's macroglobulinemia. Eight cases (44%) occurred among patients being treated with R-CHOP, 2 (11%) were treated with BR, 2 (11%) were receiving single agent rituximab, although several other regimens were represented. Most cases occurred in patients being treated with infusional rituximab, but the rate remained quite low (14/1654, 0.8%). Four occurred in patients receiving subcutaneous rituximab hycela out of 167 treated patients (2.4%), less than prior estimates of pneumonitis with monoclonal antibody therapy. Most cases of pneumonitis were treated with steroids (n=15, 83%), although none required infliximab or anti-TNF agents, and no cases of pneumonitis were fatal. Most notably, we did not identify any cases of pneumonitis among patients treated with ofatumumab (n=51) or obinutuzumab (n=58).Conclusion:This retrospective review reinforced the safety of novel anti-CD20 agents, with low rates of interstitial pneumonitis among patients treated with infusional or subcutaneous rituximab. Cases that did occur were effectively treated with steroids, and there were no fatalities associated with pneumonitis. Ofatumumab and obinutuzumab were not associated with any cases of interstitial pneumonitis.Bibliography1. Katsuya H, Suzumiya J, Sasaki H, et al. Addition of rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisolone therapy has a high risk of developing interstitial pneumonia in patients with non-Hodgkin lymphoma. Leuk Lymphoma. 2009;50(11):1818-1823. doi:10.3109/104281909032587802. Salmasi G, Li M, Sivabalasundaram V, et al. Incidence of pneumonitis in patients with non-Hodgkin lymphoma receiving chemoimmunotherapy with rituximab. Leuk Lymphoma. 2015;56(6):1659-1664. doi:10.3109/10428194.2014.9630753. Liu X, Hong X-N, Gu Y-J, Wang B-Y, Luo Z-G, Cao J. Interstitial pneumonitis during rituximab-containing chemotherapy for non-Hodgkin lymphoma. Leuk Lymphoma. 2008;49(9):1778-1783. doi:10.1080/10428190802270886 [Display omitted] DisclosuresGrover: Novartis: Consultancy; Genentech: Research Funding; ADC: Other: Advisory Board; Kite: Other: Advisory Board; Tessa: Consultancy. Dittus: BeiGene: Other: Advisory Board; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding.

CAR T cells for the long run in aggressive B-cell lymphoma.

Since first being reported in 2010, 1 Kochenderfer JN Wilson WH Janik JE et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 2010; 116: 4099-4102 Crossref PubMed Scopus (912) Google Scholar CD19-directed chimeric antigen receptor (CAR) T cells have led a revolution in the treatment of B-cell non-Hodgkin lymphomas. The US Food and Drug Administration have approved three CAR T-cell products for the treatment of relapsed or refractory aggressive B-cell lymphomas: tisagenlecleucel, axicabtagene ciloleucel, and lisocabtagene maraleucel. All three showed high rates of complete responses in patients with refractory disease in pivotal trials (37 [40%] of 93, 2 Schuster SJ Bishop MR Tam CS et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N Engl J Med. 2019; 380: 45-56 Crossref PubMed Scopus (1179) Google Scholar 59 [58%] of 101, 3 Locke FL Ghobadi A Jacobson CA et al. Long-term safety and efficacy of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. Lancet Oncol. 2019; 20: 31-42 Summary Full Text Full Text PDF PubMed Scopus (693) Google Scholar and 136 [53%] of 256, 4 Abramson JS Palomba ML Gordon LI et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020; 396: 839-852 Summary Full Text Full Text PDF PubMed Scopus (271) Google Scholar respectively), taking into account different patient selection and trial designs in each study. In parallel, real-world data have emerged for these products, showing similar overall responses of 61·8% (95% CI 53·6–69·6) for tisagenlecleucel and 70% (61–78) for axicabtagene ciloleucel, and complete responses in 60 (39%) of 152 patients for tisagenlecleucel and 61 (50%) of 122 patients for axicabtagene ciloleucel. 5 Pasquini MC Hu ZH Curran K et al. Real-world evidence of tisagenlecleucel for pediatric acute lymphoblastic leukemia and non-Hodgkin lymphoma. Blood Adv. 2020; 4: 5414-5424 Crossref PubMed Scopus (71) Google Scholar , 6 Jacobson CA Hunter BD Redd R et al. Axicabtagene ciloleucel in the non-trial setting: outcomes and correlates of response, resistance, and toxicity. J Clin Oncol. 2020; 38: 3095-3106 Crossref PubMed Scopus (72) Google Scholar Real-world studies have supported the efficacy and safety of CD19-directed CAR T-cell therapies, including in many patients who would not have been eligible for the pivotal clinical trials. Among patients treated with axicabtagene ciloleucel in the ZUMA-1 study 7 Jacobson C Locke FL Ghobadi A et al. Long-term survival and gradual recovery of B cells in patients with refractory large B cell lymphoma treated with axicabtagene ciloleucel (axi-cel). Blood. 2020; 136: 40-42 Google Scholar with at least 4 years of follow-up (median follow-up 51·1 months), the median overall survival was 25·8 months, and the 4-year estimated overall survival was 44%. The reported duration of follow-up in the JULIET study was relatively short until now. Long-term clinical outcomes of tisagenlecleucel in patients with relapsed or refractory aggressive B-cell lymphomas (JULIET): a multicentre, open-label, single-arm, phase 2 studyTisagenlecleucel shows durable activity and manageable safety profiles in adult patients with relapsed or refractory aggressive B-cell lymphomas. For patients with large B-cell lymphomas that are refractory to chemoimmunotherapy or relapsing after second-line therapies, tisagenlecleucel compares favourably with respect to risk–benefit relative to conventional therapeutic approaches (eg, salvage chemotherapy). Full-Text PDF

Efficacy of 90Y-Ibritumomab Tiuxetan in the Older Population with Non-Hodgkin Lymphoma Pre-Treated with Rituximab-Based Regimens: A Single-Institution Experience

Background: The use of (90)Y-Ibritumomab tiuxetan (90Y-IT) as radioimmunotherapy in the treatment of B-cell non-Hodgkin lymphoma (NHL) has been approved for consolidation and salvage treatment of follicular and indolent lymphoma. It has also been studied in the treatment of diffuse large B-cell and mantle cell lymphoma. Toxicities primarily include myelosuppression with a risk of treatment-associated myelodysplastic syndrome (t-MDS).Methods: Retrospective cohort analysis of patients with B-cell NHL who received 90Y-IT from 2010 to 2016 at Geisinger Medical Center. 90Y-IT was administered as both consolidation and salvage treatment according to recommended dosing guidelines and cytopenia was monitored at routine intervals. A random subset of patients received maintenance Rituximab (R) at 375 mg/m2 every three months for eight doses.Results: Sixty-six patients with NHL received 90Y-IT at a median age of 68 years (range 32-91), 33 (50%) patients were male, and 37 (56%) were greater than 65 years of age. Disease subtypes included: follicular center cell (n=31, 47%), diffuse large B-cell (n=22, 33%), mantle cell (n=3, 4.5%), marginal zone (n=7, 10.6%), small lymphocytic (n=1, 1.5%), and Waldenstrom macroglobulinema (n=2, 3.03%). Thirty-three patients (50.0%) had aggressive NHL and 38 (57.6%) had stage III/IV disease. Disease status prior to 90Y-IT was as follows: CR (n=33, 50%), PR (n=22, 33.3%), and relapse/refractory (n=11, 16.7%).Twenty-nine patients (43.9%) received >1 line of treatment prior to 90Y-IT therapy. The median duration of disease prior to 90Y-IT was 18 months (range 4-184). Sixty-four patients received R prior to 90Y-IT and no patient received fludarabine-based therapy. The median PFS and OS were 27 and 30 months, respectively, for all patients. There was no significant difference in PFS and OS between patients younger and older than 65 years of age, between patients in CR and not in CR at the time of 90Y-IT, or between patients with aggressive and indolent disease.The median follow-up period was 30 months (range 15-87). The overall response rate (ORR) was 92.4% (n=61) with a PR-to-CR conversion rate of 22.7%. In the salvage setting, the ORR was 84.8%. In the relapse/refractory setting, the ORR was 72.7%. Patients not in CR at the time of 90Y-IT were found to be responsive to R maintenance: PFS improved from median 21 to 56 months and OS improved from median 28 to 66.5 months (p=0.002).Fifteen patients (22.7%) received R maintenance after 90Y-IT and demonstrated improvement in PFS (mean 26.9 to 49.8 months, p=0.0004) and OS (mean 32.7 to 55.7 months, p=0.0004). In the diffuse large B-cell lymphoma population, the patients with R maintenance had demonstrated benefit in PFS (mean 21.4 to 46.0 months, p=0.0423) and OS (mean 23.7 to 46.0 months, p=0.0417). Among the indolent lymphomas, the addition of R maintenance demonstrated improvement in OS from 35.4 to 57.1 months (p=0.0197).Among patients with grade 3/4 hematologic toxicities, six (9.1%) required blood transfusion, four (6.1%) needed platelet transfusion, and 17 (25.8%) received pegfilgastrim. Two patients required hospitalization for febrile neutropenia. Ten patients (15.2%) relapsed after 90Y-IT at median 65.0 months, and one patient (1.5%) developed t-MDS (del 5q, del 20q) 54 months after treatment. There were 14 deaths (six were secondary to progressive lymphoma or chemotherapy-related neutropenic sepsis).Conclusions: This is one of the largest single-center experiences of 90Y-IT administered to a predominantly elderly population with B-cell NHL pre-treated with Rituximab-based regimens. This study demonstrates the safety and feasibility of 90Y-IT in the elderly population with manageable toxicity, with no increased risk of treatment-related myelodysplastic events. This study also demonstrated PFS and OS benefit in administering Rituximab maintenance post 90Y-IT to both indolent and aggressive B-cell NHL and should be further investigated in future studies. DisclosuresKhan:Spectrum Pharmaceuticals, Inc.: Other: Advisory Board, Speakers Bureau.

Overcoming the Hurdles of Autologous T-Cell-Based Therapies in B-Cell Non-Hodgkin Lymphoma.

Simple SummaryThe activity of novel therapies that utilize patient’s own T-cells to induce remission of B-cell non-Hodgkin lymphoma (B-NHL), including chronic lymphocytic leukemia (CLL), is still suboptimal. In this review, we summarize the clinical efficacy of T-cell-based therapies in B-NHL and provide a biologic rationale for the observed (lack of) responses. We describe and compare the acquired T-cell dysfunctions that occur in the different subtypes of B-NHL. Furthermore, we discuss new insights that could enhance the efficacy of T-cell-based therapies for B-NHL and CLL.The next frontier towards a cure for B-cell non-Hodgkin lymphomas (B-NHL) is autologous cellular immunotherapy such as immune checkpoint blockade (ICB), bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T-cells. While highly successful in various solid malignancies and in aggressive B-cell leukemia, this clinical success is often not matched in B-NHL. T-cell subset skewing, exhaustion, expansion of regulatory T-cell subsets, or other yet to be defined mechanisms may underlie the lack of efficacy of these treatment modalities. In this review, a systematic overview of results from clinical trials is given and is accompanied by reported data on T-cell dysfunction. From these results, we distill the underlying pathways that might be responsible for the observed differences in clinical responses towards autologous T-cell-based cellular immunotherapy modalities between diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). By integration of the clinical and biological findings, we postulate strategies that might enhance the efficacy of autologous-based cellular immunotherapy for the treatment of B-NHL.

Improved Safety and Efficacy of a Multi-Target Chimeric Antigen Receptor Modified T Cell Therapy (4SCAR2.0) Against Relapsed or Refractory Lymphomas

BACKGROUND: Chimeric antigen receptor-modified T cell (CART) therapy holds great promise as a novel cancer therapy approach. Although CART cell therapy has revolutionized the treatment of B-cell non-Hodgkin lymphomas (NHLs), relapsed/refractory NHLs including diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL), and follicular lymphoma (FL) have not responded well to the currently approved CART cell products. The existing limitations include antigen escape, toxicities of currently approved CART cells, and the high cost of manufacture of a product which is only used for a single patient (pt). The development of an innovative multi-CART therapy using a safety improved CAR design may overcome the above limitations. METHODS: Lymphoma pts who have exhausted all available treatments with progressive or stable disease and life expectancy >2 months were enrolled in the study. Lymphoma biopsies were immunostained for various target antigens including CD19, CD20, CD22, CD30, CD38, CD70 and PSMA. An advanced CART therapy regimen, CAR 2.0, has been developed, which involves a primary and a booster CAR-T cell infusions targeting multiple antigens identified on each pt's tumor. Autologous T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine chemotherapy conditioning 1-2 days before infusion of 1.21- 4.87x106 CART cells/kg per infusion. The quality of apheresis cells, efficiencies of gene transfer and T cell proliferation, CAR T infusion dose and blood CAR copies were quantitatively documented. RESULTS: Total 60 pts were evaluated at three months follow-up time, including 42 pts with DLBCL, 10 with PMBCL, 7 with FL and 1 with gastric mucosal associated lymphoid tissue lymphoma (MALT). The median age was 47 years (range, 2-77), including 31 males and 29 females. Pt characteristics include 16 pts with stage IV disease (27%), 2 after allo-transplantation (3.3%), 4 with bone marrow involvement (6.7%), 8 with CNS involvement (13%), and 7 received prior PD-1 antibody treatment (12%). The performance status of 52 of the 60 patients (87%) was Eastern Co-operative Oncology Group score 0 at time of infusion. The study included two treatment cohorts: 30 pts received single 4SCAR19 cells, and 30 pts received CAR2.0 targeting CD19 plus an additional target, depending on the individual pt's tumor antigen staining results. In the DLBCL cohort, 25 pts received single target 4SCAR19 cell infusion, at an average dose of 1.42x106 CART cells/kg, and resulted in 7 complete response (CR), 9 partial response (PR), 3 stable disease (SD) and 6 progressive disease (PD). The other 17 DLBCL pts received dual target CART infusions (CD19 plus CD20, CD22, CD38 or CD70) at an average dose of 2.32x106 CART cells/kg, and resulted in 7 CR, 8 PR ,1 SD and 1 PD. The 5 FL pts received dual target CD19+CD22 CART infusions, at an average dose of 4.64x106 CAR-T cells/kg, and resulted in 4 CR, and 1 PR. The 10 PMBCL pts, 3 received single target 4SCAR19 cell infusions, at an average dose of 2.85x106 CART cells/kg, and resulted in 1 PR and 2 PD; the other 7 PMBCL pts received dual target CART infusions (CD19+CD22, CD30, or CD20), at an average dose of 4.87x106 CART cells/kg, and resulted in 4 CR, 2 PR and 1 PD. The MALT pt received double CART cell infusions, 2.8x106 4SCAR19 cells/kg and 2x106 4SCAR-PSMA cells/kg, and achieved PR. None of these pts developed greater than grade 2 CRS response, except for 1 DLBCL pt, who achieved CR following a grade 3 CRS. The toxicity profile of the 4SCART is consistent with our previous experience with B cell acute lymphoblastic leukemia. In summary, the comparison between single versus double CART cell infusions clearly illustrates an increased response rate for the double CART cohorts, either in DLBCL pts or in all lymphoma pts, as illustrated in the summary pie graphs below. CONCLUSIONS: The results of the multi-target 4SCAR2.0 therapy for the treatment of highly resistant lymphomas have demonstrated increased safety and improved response rate with this novel approach. There is clear overall clinical benefit with the multi-target CART regimen as compared with the single CD19 CART treatment. Continued follow-up will verify whether the 4SCAR2.0 therapy regimen can achieve long term overall survival. Figure Disclosures No relevant conflicts of interest to declare.