CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

Off-the-shelf CAR-T cell therapies for relapsed or refractory B-cell malignancies: latest update from ASH 2023 annual meeting.

Currently, many off-the-shelf chimeric antigen receptor (CAR)-T cell products are under investigation for the treatment of relapsed or refractory (R/R) B-cell neoplasms. Compared with autologous CAR-T cell therapy, off-the-shelf universal CAR-T cell therapies have many potential benefits, such as immediate accessibility for patients, stable quality due to industrialized manufacturing and additional infusions of CAR-T cells with different targets. However, critical challenges, including graft-versus-host disease and CAR-T cell elimination by the host immune system, still require extensive research. The most common technological approaches involve modifying healthy donor T cells via gene editing technology and altering different types of T cells. This article summarizes some of the latest data from preclinical and clinical studies of off-the-shelf CAR-T cell therapies in the treatment of R/R B-cell malignancies from the 2023 ASH Annual Meeting (ASH 2023).

Bispecific antibodies targeting CD20xCD3 in immunotherapy for adult B-cell lymphoma: insights from the 65th American Society of Hematology 2023 annual meeting.

INTRODUCTION: At the 65th American Society of Hematology (ASH) 2023 Annual Meeting, the latest advancements in CD20×CD3 BsAbs for B-cell lymphoma (BCL) were highlighted, particularly in relapsed/refractory (R/R) follicular lymphoma (FL) and R/R diffuse large B-cell lymphoma (DLBCL). AREAS COVERED: This summary highlights some of the major studies on CD20×CD3 BsAbs for BCL. EXPERT OPINION/COMMENTARY: CD20×CD3 is the most widely studied BsAb, with promising results in patients with R/R DLBCL and R/R FL ≥ two prior lines of systemic therapy. Trials with the first line of B-cell lymphoma also revealed promising results. Hopefully, BsAb monotherapy or BsAb-containing regimens may become the standard therapy in patients with FL and DLBCL.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

[T-cell recruiting immunotherapies in B-cell lymphoma - the future backbone for all therapy lines?] / T-Zell-rekrutierende Immuntherapien des B-Zell-Lymphoms ­ bald in allen Therapielinien?

The introduction of immunologically targeted therapies has represented a significant advancement in the treatment of B-cell lymphomas, particularly aggressive B-cell lymphoma. CD19 CAR-T cells such as Axicabtagen-Ciloleucel (Axi-cel) and Lisocabtagen Maraleucel (Liso-cel) have been approved since 2022 and 2023, respectively, for second-line therapy of Diffuse Large B-Cell Lymphomas (DLBCL), when there is primary refractory disease or relapse within 12 months after the end of first-line therapy. These therapies result in a significant improvement in progression-free survival compared to the previous standard therapy (salvage chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation). Especially in elderly patients or patients with underlying medical conditions, CAR-T cell therapies like Axi-cel and Liso-cel demonstrate acceptable tolerability and high efficacy.Furthermore, bispecific T-cell-engaging antibodies ("bispecifics") such as Glofitamab, Epcoritamab, and Mosunetuzumab also represent promising treatment options for patients with relapsed disease after failure of second- or later line therapy and show efficacy even in a subset of patients relapsing after CD19 CAR-T cells. However, randomized study results for these substances are not yet available. They are expected to be used in earlier lines of therapy in the future, especially in combination with standard chemotherapy regimens. Common side effects of bispecific antibody therapies are cytokine release syndrome (CRS) and immune-mediated cytopenias, whereas immune-cell associated neurotoxicity syndrome (ICANS) is relatively rare compared to CD19 CAR T cells. In summary, bispecifics represent a novel, highly effective immunotherapy for the treatment of lymphomas with a very favourable toxicity profile.

Population pharmacokinetics and CD20 binding dynamics for mosunetuzumab in relapsed/refractory B-cell non-Hodgkin lymphoma.

Mosunetuzumab (Mosun) is a CD20xCD3 T-cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. The approved step-up dose regimen of 1/2/60/30 mg IV is designed to mitigate cytokine release syndrome (CRS) and maximize efficacy in early cycles. A population pharmacokinetic (popPK) model was developed from 439 patients with relapsed/refractory B-Cell Non-Hodgkin lymphoma receiving Mosun IV monotherapy, including fixed dosing (0.05-2.8 mg IV every 3 weeks (q3w)) and Cycle 1 step-up dosing groups (0.4/1/2.8-1/2/60/30 mg IV q3w). Prior to Mosun treatment, ~50% of patients had residual levels of anti-CD20 drugs (e.g., rituximab or obinutuzumab) from prior treatment. CD20 receptor binding dynamics and rituximab/obinutuzumab PK were incorporated into the model to calculate the Mosun CD20 receptor occupancy percentage (RO%) over time. A two-compartment model with time-dependent clearance (CL) best described the data. The typical patient had an initial CL of 1.08 L/day, transitioning to a steady-state CL of 0.584 L/day. Statistically relevant covariates on PK parameters included body weight, albumin, sex, tumor burden, and baseline anti-CD20 drug concentration; no covariate was found to have a clinically relevant impact on exposure at the approved dose. Mosun CD20 RO% was highly variable, attributed to the large variability in residual baseline anti-CD20 drug concentration (median = 10 µg/mL). The 60 mg loading doses increased Mosun CD20 RO% in Cycle 1, providing efficacious exposures in the presence of the competing anti-CD20 drugs. PopPK model simulations, investigating Mosun dose delays, informed treatment resumption protocols to ensure CRS mitigation.

CD70-specific CAR NK cells expressing IL-15 for the treatment of CD19-negative B-cell malignancy.

ABSTRACT: Chimeric antigen receptor (CAR) natural killer (NK) cells can eliminate tumors not only through the ability of the CAR molecule to recognize antigen-expressed cancer cells but also through NK-cell receptors themselves. This overcomes some of the limitations of CAR T cells, paving the way for CAR NK cells for safer and more effective off-the-shelf cellular therapy. In this study, CD70-specific (a pan-target of lymphoma) fourth-generation CAR with 4-1BB costimulatory domain and interleukin-15 (IL-15) was constructed and transduced into cord blood-derived NK cells by Baboon envelope pseudotyped lentiviral vector. CD70-CAR NK cells displayed superior cytotoxic activity in vitro and in vivo against CD19-negative B-cell lymphoma when compared with nontransduced NK cells and CD19-specific CAR NK cells. Importantly, mice that received 2 doses of CD70-CAR NK cells showed effective eradication of tumors, accompanied by increased concentration of plasma IL-15 and enhanced CAR NK cell proliferation and persistence. Our study suggests that repetitive administration-based CAR NK-cell therapy has clinical advantage compared with a single dose of CAR NK cells for the treatment of B-cell lymphoma.

High yield killing of lymphoma cells by anti-CD22 CAR-NK cell therapy.

Chimeric antigen receptors (CARs) offer a promising new approach for targeting B cell malignancies through the immune system. Despite the proven effectiveness of CAR T cells targeting CD19 and CD22 in hematological malignancies, it is imperative to note that their production remains a highly complex process. Unlike T cells, NK cells eliminate targets in a non-antigen-specific manner while avoiding graft vs. host disease (GvHD). CAR-NK cells are considered safer than CAR-T cells because they have a shorter lifespan and produce less toxic cytokines. Due to their unlimited ability to proliferate in vitro, NK-92 cells can be used as a source for CAR-engineered NK cells. We found that CARs created from the m971 anti-CD22 mAb, which specifically targets a proximal CD22 epitope, were more effective at anti-leukemic activity compared to those made with other binding domains. To further enhance the anti-leukemic capacity of NK cells, we used lentiviral transduction to generate the m971-CD28-CD3ζ NK-92. CD22 is highly expressed in B cell lymphoma. To evaluate the potential of targeting CD22, Raji cells were selected as CD22-positive cells. Our study aimed to investigate CD22 as a potential target for CAR-NK-92 therapy in the treatment of B cell lymphoma. We first generated m971-CD28-CD3ζ NK-92 that expressed a CAR for binding CD22 in vitro. Flow cytometric analysis was used to evaluate the expression of CAR. The 7AAD determined the cytotoxicity of the m971-CD28-CD3ζ NK-92 towards target lymphoma cell lines by flow cytometry assay. The ELISA assay evaluated cytokine production in CAR NK-92 cells in response to target cells. The m971-CD28-CD3ζ NK-92 cells have successfully expressed the CD22-specific CAR. m971-CD28-CD3ζ NK-92 cells efficiently lysed CD22-expressing lymphoma cell lines and produced large amounts of cytokines such as IFN-γ and GM-CSF but a lower level of IL-6 after coculturing with target cells. Based on our results, it is evident that transferring m971-CD28-CD3ζ NK-92 cells could be a promising immunotherapy for B cell lymphoma.

EBV-associated lymphoproliferative disease post-CAR-T cell therapy.

Epstein-Barr virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs) are common complications that occur after solid organ transplantation or allogeneic hematopoietic stem-cell transplantation (HSCT). However, their occurrence and treatment post-chimeric antigen receptor-modified T (CAR-T) cell therapy has not been reported. Two patients had been diagnosed with EBV-positive aggressive B-cell lymphoma and experienced relapses after multiple lines of treatment. After receiving CAR-T cell therapy in tandem with autologous HSCT, the patients achieved complete remission. However, with a median time of 38.5 months after CAR-T cell therapy, B-cell-derived EBV-LPDs were diagnosed, and they were relieved through the administration of immune checkpoint inhibitor or B-cell-depleting agents. Collectively, our report suggests that EBV-LPDs may represent a long-term adverse event after CAR-T cell therapy, especially in patients who previously had EBV-positive disorders, and they can be resolved by immune normalization strategy or B-cell depleting therapy.

The role of chimeric antigen receptor T cells targeting more than one antigen in the treatment of B-cell malignancies.

Several products containing chimeric antigen receptor T cells targeting CD19 (CART19) have been approved for the treatment of patients with relapsed/refractory non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukaemia (ALL). Despite very impressive response rates, a significant percentage of patients experience disease relapse and die of progressive disease. A major cause of CART19 failure is loss or downregulation of CD19 expression in tumour cells, which has prompted a myriad of novel strategies aimed at targeting more than one antigen (e.g. CD19 and CD20 or CD22). Dual targeting can the accomplished through co-administration of two separate products, co-transduction with two different vectors, bicistronic cassettes or tandem receptors. In this manuscript, we review the pros and cons of each strategy and the clinical results obtained so far.

Engineering CD20 CARs with a Twist.

CD20 is highly expressed in several types of B-cell lymphoma and is an intuitive target for chimeric antigen receptor (CAR) T-cell therapy. However, with conventional approaches, it has been challenging to provide CD20 CAR designs that confer efficacy in preclinical models and in clinical trials. In this issue, Chen and colleagues report several improved CD20 CARs, developed with minimal deviations from conventional design principles, that confer curative anti-lymphoma efficacy in preclinical models. These novel CD20 CARs enrich the pipeline for clinical development and provide an example of rational CAR design that is informed by insights into the structural biology of CAR domains. See related article by Chen et al., p. 150 (3).

Exosomal lncRNA HOTAIR induces PDL1+ B cells to impede anti-tumor immunity in colorectal cancer.

Regulatory B cells (Bregs) contribute to tumor immunosuppression. However, how B cells acquire their regulatory features in tumors remain unclear. Exosomes are important messengers that transmit tumor information to remodel tumor immunity. Here we revealed that tumor-derived exosomes drive Bregs to suppress anti-tumor immunity by delivering long non-coding RNAs (lncRNAs). HOTAIR was screened by lncRNA profiling in both colorectal cancer (CRC)-derived exosomes and infiltrating B cells. Tumor-derived HOTAIR polarized B cells toward a regulatory feature marked by programmed cell death-ligand 1 (PDL1) in CRC, and induced PDL1+ B cells to suppress CD8+ T cell activity. Exosomal HOTAIR bound to and protected pyruvate kinase M2 (PKM2) against ubiquitination degradation, resulting in STAT3 activation and PDL1 expression. Results from CRC patients showed a positive correlation between exosomal HOTAIR and tumor-infiltrating PDL1+ B cells. These findings reveal how B cells acquire PDL1-dominant regulatory feature in CRC, implying the clinical significance of exosomal therapy targeting HOTAIR.

Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Adaptable Leukemia Cells Resisting CAR T-cell Attack via B-cell Activation.

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable milestones in the treatment of B-cell malignancies. However, cancer cells frequently survive CAR T-cell killing in a large cohort of patients. Relapse oftentimes is associated with antigen loss. In this issue, Im and colleagues report a new mechanism of leukemic-cell resistance to anti-CD19 CAR T cells: Leukemic cells can enable a B-cell activation and germinal center reaction signature, which causes CD19 transcriptional downregulation and survival from CAR exposure. See related article by Im et al., p. 1055 (5).

Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma.

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy.

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.

Limited increase in antibody titers following mRNA SARS-CoV-2 vaccination for more than 3 years after final dose of anti-CD20 antibody.

We investigated the efficacy of BNT162b2 mRNA COVID-19 vaccine in patients with B-cell malignancies treated with anti-CD20 antibody. Although T-cell-mediated immune responses were detected even in patients receiving R-CHOP treatment, the S1 antibody titer following BNT162b2 vaccination remained only marginally increased for more than 3 years after the final dose of anti-CD20 antibody. We found no relationship between the percent of B-cells and S1 antibody titer. The duration of this suppression was much longer than we anticipated. Further protection and treatment strategies against COVID-19 for these patients are warranted.

Impact of TP53 Genomic Alterations in Large B-Cell Lymphoma Treated With CD19-Chimeric Antigen Receptor T-Cell Therapy.

PURPOSE: Tumor-intrinsic features may render large B-cell lymphoma (LBCL) insensitive to CD19-directed chimeric antigen receptor T cells (CAR-T). We hypothesized that TP53 genomic alterations are detrimental to response outcomes in LBCL treated with CD19-CAR-T. MATERIALS AND METHODS: Patients with LBCL treated with CD19-CAR-T were included. Targeted next-generation sequencing was performed on pre-CAR-T tumor samples in a subset of patients. Response and survival rates by histologic, cytogenetic, and molecular features were assessed. Within a cohort of newly diagnosed LBCL with genomic and transcriptomic profiling, we studied interactions between cellular pathways and TP53 status. RESULTS: We included 153 adults with relapsed or refractory LBCL treated with CD19-CAR-T (axicabtagene ciloleucel [50%], tisagenlecleucel [32%], and lisocabtagene maraleucel [18%]). Outcomes echoed pivotal trials: complete response (CR) rate 54%, median overall survival (OS) 21.1 months (95% CI, 14.8 to not reached), and progression-free survival 6 months (3.4 to 9.7). Histologic and cytogenetic LBCL features were not predictive of CR. In a subset of 82 patients with next-generation sequencing profiling, CR and OS rates were comparable with the unsequenced cohort. TP53 alterations (mutations and/or copy number alterations) were common (37%) and associated with inferior CR and OS rates in univariable and multivariable regression models; the 1-year OS in TP53-altered LBCL was 44% (95% CI, 29 to 67) versus 76% (65 to 89) in wild-type (P = .012). Transcriptomic profiling from a separate cohort of patients with newly diagnosed lymphoma (n = 562) demonstrated that TP53 alterations are associated with dysregulation of pathways related to CAR-T-cell cytotoxicity, including interferon and death receptor signaling pathway and reduced CD8 T-cell tumor infiltration. CONCLUSION: TP53 is a potent tumor-intrinsic biomarker that can inform risk stratification and clinical trial design in patients with LBCL treated with CD19-CAR-T. The role of TP53 should be further validated in independent cohorts.

Combined strategies for effective cancer immunotherapy with a novel anti-CD47 monoclonal antibody.

CD47 is a widely expressed cell-surface protein that regulates phagocytosis mediated by cells of the innate immune system, such as macrophages and dendritic cells. CD47 serves as the ligand for a receptor on these innate immune cells, signal regulatory protein (SIRP)-α, which in turn inhibits phagocytosis. Several targeted CD47 therapeutic antibodies have been investigated clinically; however, how to improve its therapeutic efficacy remains unclear. Herein, we developed a CD47 blocking antibody, named IBI188, that could specifically block the CD47-SIRP-α axis, which transduces the "don't eat me" signal to macrophages. In vitro phagocytosis assays demonstrated the pro-phagocytosis ability of IBI188. Furthermore, several in vivo models were chosen to evaluate the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment.