Challenges and considerations in developing trispecific CAR-Ts for B-cell malignancies

A Single-Cell Atlas of Classical Mantle Cell Lymphoma Reveals Paired Tumor-Immune Malignant States Correlating with Prognosis

The coexistence of mycosis fungoides, a peripheral T-cell lymphoma, and B-cell malignancies or Hodgkin's lymphoma in the same patient is unusual. Most descriptions are isolated case reports and case series are strikingly sparse. To detect cases of mycosis fungoides associated with B-cell malignancies or Hodgkin's lymphoma and to analyse the characteristics of and the interplay between the lymphoproliferative neoplasms. Patients with mycosis fungoides who had B-cell malignancies or Hodgkin's lymphoma were selected from among 398 patients either treated or followed up in two tertiary medical centres during a 7-year period. Eleven patients with mycosis fungoides and B-cell malignancy were detected (seven of non-Hodgkin's lymphoma, three of chronic lymphocytic leukaemia, one of multiple myeloma). No case of Hodgkin's lymphoma was found. In seven patients the mycosis fungoides preceded the B-cell malignancy whereas in four it was the B-cell malignancy which occurred first. The time elapsed between onset of the two malignancies ranged from 4 to 22 years (average: 12 years). Patients who had mycosis fungoides as the first neoplasm presented with earlier stages of mycosis fungoides (four of seven: IA, three of seven: IB) than those who had mycosis fungoides as their second neoplasm (of four, one: IB, one: folliculotropic, two: IIB). Among the four patients in whom the appearance of mycosis fungoides followed the B-cell malignancy, three had been treated with multiagent chemotherapy. Two patients who presented with early-stage mycosis fungoides (IA) as the first lymphoma developed mycosis fungoides tumours after becoming immunosuppressed. In two patients infiltrates composed of both malignant T- and B-cell populations were found in a single biopsy. One showed two distinct populations of the malignant cells in the skin tumour, thus constituting a classical composite lymphoma of mycosis fungoides and chronic lymphocytic leukaemia, while in the other patient the two malignant populations of marginal B-cell lymphoma and mycosis fungoides (as evidenced by both phenotypic and genotypic findings) were intermingled. This case series indicates that while the coexistence of Hodgkin's lymphoma and mycosis fungoides is extremely rare, the association of mycosis fungoides and B-cell malignancies is not as rare as reflected in the literature, with non-Hodgkin's lymphoma constituting the most common associated B-cell malignancy. In this series as well as in the cases reported in the literature mycosis fungoides usually preceded the development of B-cell malignancies, which may be in accordance with previous reports of an increased risk of developing a second haematological neoplasm. The importance of a competent immune system for patients with mycosis fungoides is well demonstrated in these cases. It is suggested that for greater precision the criteria for diagnosis of composite lymphoma of the skin should include both phenotypic and genotypic features.

Although the development of targeted approaches has been effective for the treatment of B-cell malignancies, outcomes remain poor in the relapsed and refractory settings. To expand the portfolio of B-cell-selective drugs, we developed an interactive computational tool (lymphoblasts.org) and identified ferroptosis, a form of cell death driven by iron-dependent membrane lipid peroxidation, as a previously unrecognized selective vulnerability in B-cell malignancies. Although ferroptosis has shown potential in therapy-resistant tumors, no potent ferroptosis inducers are available clinically, and a better understanding of the factors regulating ferroptosis is required to allow its therapeutic targeting and the development of clinical grade ferroptosis inducers. Our comparative analyses of CRISPR dependency (DepMap) and drug (CTD, GDSC) screens revealed that B-cell malignancies are particularly sensitive to ferroptosis and depend on several anti-ferroptotic molecules that notably include components of the glutathione synthesis machinery (GCLC, GCLM, GSS) and selenoprotein synthesis (SEPHS2, LRP8). Strikingly, this analysis also identified pro-ferroptotic genes involved in iron (TFRC) and polyunsaturated fatty acid (PUFA; ACSL4) metabolism as selective B-cell dependencies, suggesting that addiction to these pro-ferroptotic processes makes B-cell malignancies intrinsically vulnerable to ferroptosis. Accordingly, iron chelation treatment showed a much higher toxicity in B-cell malignancies compared to solid tumors, and Cre-mediated ablation of Tfrc in murine models of B-cell acute lymphoblastic leukemia (B-ALL) and B-cell lymphoma induced rapid cell death. Cre-mediated loss of ACSL4, while making cells almost completely resistant to ferroptosis, significantly compromised murine B-ALL cell viability, suggesting a key role of ACSL4 and PUFA in B-cell malignancy survival. An important role of ACSL4 and PUFA metabolism in B-cell malignancies was also suggested by analysis of clinical data from the diffuse large B-cell lymphoma MMMLNP trial, which revealed that greater than median expression of ACSL4 is associated with significantly worse survival (p = 0.0007).To uncover new ferroptotic regulators in B-cell malignancies, we performed whole-genome CRISPR knockout screens under the selective pressure of the ferroptosis inducers RSL3, Erastin, and FINO2, which highlighted several known ferroptosis regulators and uncovered new ferroptosis-related genes and pathways including sphingolipid metabolism (KDSR), phosphatidylcholine synthesis (FLVCR1, PCYT1A), inositol metabolism (ITPK1, IPMK), and lipid membrane remodelling (ATP8B2). Taken together, our findings uncover iron and PUFA metabolism as selective B-cell dependencies that likely contribute to their exquisite sensitivity to ferroptosis and identifies novel regulators of ferroptosis in B-cell malignancies. Citation Format: Etienne Leveille, Eden Bramson, Mark Robinson, Thierry Bertomeu, Andrew Chatr-Aryamontri, Shalin Kothari, Markus Müschen. Metabolic determinants of ferroptosis in B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2819.

Summary Background The coexistence of mycosis fungoides, a peripheral T-cell lymphoma, and B-cell malignancies or Hodgkin's lymphoma in the same patient is unusual. Most descriptions are isolated case reports and case series are strikingly sparse. Objectives To detect cases of mycosis fungoides associated with B-cell malignancies or Hodgkin's lymphoma and to analyse the characteristics of and the interplay between the lymphoproliferative neoplasms. Method Patients with mycosis fungoides who had B-cell malignancies or Hodgkin's lymphoma were selected from among 398 patients either treated or followed up in two tertiary medical centres during a 7-year period. Results Eleven patients with mycosis fungoides and B-cell malignancy were detected (seven of non-Hodgkin's lymphoma, three of chronic lymphocytic leukaemia, one of multiple myeloma). No case of Hodgkin's lymphoma was found. In seven patients the mycosis fungoides preceded the B-cell malignancy whereas in four it was the B-cell malignancy which occurred first. The time elapsed between onset of the two malignancies ranged from 4 to 22 years (average: 12 years). Patients who had mycosis fungoides as the first neoplasm presented with earlier stages of mycosis fungoides (four of seven: IA, three of seven: IB) than those who had mycosis fungoides as their second neoplasm (of four, one: IB, one: folliculotropic, two: IIB). Among the four patients in whom the appearance of mycosis fungoides followed the B-cell malignancy, three had been treated with multiagent chemotherapy. Two patients who presented with early-stage mycosis fungoides (IA) as the first lymphoma developed mycosis fungoides tumours after becoming immunosuppressed. In two patients infiltrates composed of both malignant T- and B-cell populations were found in a single biopsy. One showed two distinct populations of the malignant cells in the skin tumour, thus constituting a classical composite lymphoma of mycosis fungoides and chronic lymphocytic leukaemia, while in the other patient the two malignant populations of marginal B-cell lymphoma and mycosis fungoides (as evidenced by both phenotypic and genotypic findings) were intermingled. Conclusions This case series indicates that while the coexistence of Hodgkin's lymphoma and mycosis fungoides is extremely rare, the association of mycosis fungoides and B-cell malignancies is not as rare as reflected in the literature, with non-Hodgkin's lymphoma constituting the most common associated B-cell malignancy. In this series as well as in the cases reported in the literature mycosis fungoides usually preceded the development of B-cell malignancies, which may be in accordance with previous reports of an increased risk of developing a second haematological neoplasm. The importance of a competent immune system for patients with mycosis fungoides is well demonstrated in these cases. It is suggested that for greater precision the criteria for diagnosis of composite lymphoma of the skin should include both phenotypic and genotypic features.

CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies [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 2902.

A method is described that permits colony formation in culture by B lymphocytes from normal blood and from blood, marrow or lymph nodes of patients with myeloma or lymphoma. The method depends on: (1) exhaustively depleting cell suspensions of T lymphocytes, (2) a medium conditioned by T lymphocytes in the presence of phytohaemagglutinin (PHA-TCM), and (3) irradiated autologous or homologous T lymphocytes. Under these conditions the assay is linear. Cellular development of B lymphocytes can be followed; differentiation to plasma cells is seen in cultures of cells from normal individuals and myeloma patients, but not lymphoma patients. Malignant B lymphocytes in culture produced immunoglobulin of the class identified in the patient's blood, or in freshly obtained cells. We conclude that the assay is suitable for studying the growth, differentiation and regulation of normal and malignant B lymphocytes in culture.

Reduced Effect of Inotuzumab Ozogamicin (CMC544) on P-Glycoprotein Positive Malignant B Cells and Its Restoration by Multidrug Resistance Modifiers.

A Phase 1 Dose-Escalation Study of the Oral CDK Inhibitor Voruciclib in Patients with Relapsed/Refractory B-Cell Malignancies or Acute Myeloid Leukemia (AML): Preliminary Results of the Completed Dose Escalation Stage in AML

A First-in-Human Phase 1 Study of Abbv-319, an Antibody-Drug Conjugate Composed of a CD19 Antibody Linked to a Potent Proprietary Glucocorticoid Receptor Modulator, in Patients with Relapsed or Refractory B-Cell Malignancies

Content List ‐ Read more articles from the symposium: Targeted therapy in B‐cell malignancies.

Simple SummaryPatients with B-cell malignancies benefit from the third vaccination against SARS-CoV-2 with regards to antibody production. In this cohort, patients with B-cell malignancies experienced greater difficulties in developing neutralizing antibodies, particularly under active treatment with anti-CD19 CAR T-cells or anti-CD20 monoclonal antibodies. Nevertheless, the third dose achieved an enhancement of the serological responses and increased humoral immunity. These observations are in line with recent studies. Future studies with larger cohorts on this research topic will broaden the understanding of an effective anti-SARS-CoV-2 vaccination scheme and guide the timing for upcoming booster vaccinations in high-risk hematological patients. Additionally, future studies should also include data on circulating B- and T-cells in order to gain a more complete picture of the immune system of patients with B-cell malignancies upon different treatments and their influence on immune protection in response to vaccinations.Patients with B-cell malignancies are at a higher risk of severe SARS-CoV-2 infections. Nevertheless, extensive data on the immune responses of hematological patients and the efficacy of the third dose of the vaccine are scarce. The goal of this study was to determine standardized anti-SARS-CoV-2 S antibody levels and to evaluate differences between treatment modalities in response to the second and third vaccines among patients with B-cell malignancies treated at the University Hospital Krems and the University Hospital of Vienna. The antibody levels of a total of 80 patients were retrospectively analyzed. The results indicate a significant increase in antibody production in response to the third vaccination. The highest increases could be observed in patients in a “watchful-waiting” and “off-therapy” setting. Encouragingly, approximately one-third of patients who did not develop antibodies in response to two vaccinations achieved seroconversion after the third vaccination. “Watchful-waiting”, “off-therapy” and treatment with BTK inhibitors were indicative for increased antibody response after the third dose compared to anti-CD19 CAR T-cell and anti-CD-20 antibody treatment. In summary, the results of this study underline the pre-eminent role of the need for complete vaccination with three doses for the development of protective immunity in patients with B-cell malignancies.

Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3′-end of an exon to the 5′-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.Endogenous circular RNA (circRNA) expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations. Here, the authors provide a map of circRNA expression in B-cell malignancies based on high-throughput RNA sequencing and present an enzyme-free digital counting methodology, which has the potential to become the gold standard for circRNA quantification.

Risk of Infection with Ibrutinib in Patients with B-Cell Malignancies: A Meta-Analysis of Phase III Randomized Controlled Trials

Novel targeted therapies have substantially improved the prognosis of patients with B-cell malignancies. However, a substantial fraction of patients relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance [EMDR]). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumor cell adhesion enabling the physical displacement from protective niches. In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we antagonized stromal expressed PKC-beta, which we have previously shown to be a stroma-autonomous signaling pathway critical for the survival of malignant B cells (Lutzny et al., Cancer Cell 2013). The dependency on stroma PKC-b was uniformly found for acute (ALL) and chronic (CLL, MCL) B-cell malignancies. In particular, our data demonstrate that stroma PKC-b is of key importance for multidrug resistance of malignant B cells (Park et al., Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B-cell malignancies. We identified that stroma PKC-b drives a transcriptional program, activating TGF-b and BMP-signaling in tumor cells. Our data show that antagonizing stroma signals with TGF-b inhibitors abrogated upregulation of BCL-XL and overcomes stroma-dependent resistance to venetoclax. This activation operates in parallel to the activation of the transcription factor EB (TFEB) as a downstream target of PKC-b. Interference with these signaling pathways impairs plasma membrane integrity of MSCs by downregulation of numerous adhesion and signaling molecules (e.g., ADAM17), required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-b for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EuTCL-1 driven B-cell tumors and treated with venetoclax in combination with or without enzastaurin (PKC-b inhibitor). Combined treatment significantly prolonged survival, based on PKC-b mediated impairment of lysosome biogenesis in vivo. Similarly, concurrent treatment of PKC-b inhibitors with chemotherapy also improved survival in an ALL-PDx model. Our data demonstrate that mitigating EMDR with small-molecule inhibitors of PKC-b or TGF-b signaling enhances the effectiveness of both targeted and nontargeted chemotherapies and, moreover, has the ability to overcome venetoclax resistance in B-cell malignancies in vivo. A clinical trial to test the dual inhibition of stroma and tumor cells in lymphoma patients is in preparation. Citation Format: Eugene Park, Jingyu Chen, Andrew Moore, Maurizio Mangolini, Joseph R. Byod, Hilde Schjerven, James C. Williamson, Paul J. Lehner, Michael Leitges, Alexander Egle, Marc Schmidt-Supprian, Seth Frietze, Ingo Ringshausen. Overcoming venetoclax resistance in B-cell malignancies by antagonism of stromal TGF-beta-mediated drug resistance [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-62.

Circular RNAs (circRNAs) are covalently closed endogenous RNA molecules with tissue- and disease specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Based on high-throughput RNA sequencing (RNA-seq) data, we designed assays for analyzing 52 unique circRNAs simultaneously, using a digital, enzyme-free technology termed NanoString, in cell lines and paired fresh frozen and formalin-fixed, paraffin-embedded (FFPE) patient samples (including mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia). The data obtained using NanoString were compared to RNA-seq and reverse transcription-qPCR (RT-qPCR) data obtained on the same samples. RNA-seq profiling revealed a high expression of circRNAs in B-cell malignancies and the NanoString circRNA expression profiles were able to distinguish different B-cell malignancies. We detected circRNAs known to be deregulated in other cancers, including ciRS-7, circHIPK3 circCCDC66, circCDYL, circZKSCAN1 and circFBXW7, and identified a novel circRNA from the IKZF3 oncogene. NanoString data were more reproducible and quantitatively accurate than RNA-seq data and the technology works in particular well for low quality RNA samples. Together, we demonstrate that the NanoString technology enables specific, sensitive and accurate quantification of circRNAs in FFPE samples and provide a map of circRNA expression in B-cell malignancies. Citation Format: Lasse Sommer Kristensen, Mette Dahl, Maria S. Andersen, Iben Daugaard, Thomas B. Hansen, Kirsten Grønbæk, Jørgen Kjems. Profiling of endogenous circular RNA molecules in formalin-fixed paraffin-embedded tissues from patients with B-cell malignancies using an enzyme-free digital counting method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-394.