Primary CD138 Research Articles

Combined MEK1/2 and ATR inhibition promotes myeloma cell death through a STAT3-dependent mechanism invitro and invivo.

Mechanisms underlying potentiation of the anti-myeloma (MM) activity of ataxia telangiectasia Rad3 (ATR) antagonists by MAPK (Mitogen-activated protein kinases)-related extracellular kinase 1/2 (MEK1/2) inhibitors were investigated. Co-administration of the ATR inhibitor (ATRi) BAY1895344 (BAY) and MEK1/2 inhibitors, for example, cobimetinib, synergistically increased cell death in diverse MM cell lines. Mechanistically, BAY and cobimetinib blocked STAT3 Tyr705 and Ser727 phosphorylation, respectively, and dual dephosphorylation triggered marked STAT3 inactivation and downregulation of STAT3 (Signal transducer and activator of transcription 3) downstream targets (c-Myc and BCL-XL). Similar events occurred in highly bortezomib-resistant (PS-R) cells, in the presence of patient-derived conditioned medium, and with alternative ATR (e.g. M1774) and MEK1/2 (trametinib) inhibitors. Notably, constitutively active STAT3 c-MYC or BCL-XL ectopic expression significantly protected cells from BAY/cobimetinib. In contrast, transfection of cells with a dominant-negative form of STAT3 (Y705F) sensitized cells to cobimetinib, as did ATR shRNA knockdown. Conversely, MEK1/2 knockdown markedly increased ATRi sensitivity. The BAY/cobimetinib regimen was also active against primary CD138+ MM cells, but not normal CD34+ cells. Finally, the ATR inhibitor/cobimetinib regimen significantly improved survival in MM xenografts, including bortezomib-resistant models, with minimal toxicity. Collectively, these findings suggest that combined ATR/MEK1/2 inhibition triggers dual STAT3 Tyr705 and Ser727 dephosphorylation, pronounced downregulation of cytoprotective targets and MM cell death, warranting attention as a novel therapeutic strategy in MM.

Toll-like receptor signaling in multiple myeloma cells promotes the expression of pro-survival genes B-cell lymphoma 2 and MYC and modulates the expression of B-cell maturation antigen.

Infections are common in plasma cell cancer multiple myeloma (MM) due to disease-related immune deficiencies and cancer treatment. Myeloma cells express Toll-like receptors (TLRs), and TLR activation has been shown to induce proliferative and pro-survival signals in cancer cells. MM is a complex and heterogeneous disease, and expression levels of TLRs as well as downstream signaling components are likely to differ between patients. Here, we show that in a large cohort of patients, TLR1, TLR4, TLR6, TLR9, and TLR10 are the most highly expressed in primary CD138+ cells. Using an MM cell line expressing TLR4 and TLR9 as a model, we demonstrate that TLR4 and TLR9 activation promoted the expression of well-established pro-survival and oncogenes in MM such as MYC, IRF4, NFKB, and BCL2. TLR4 and TLR9 activation inhibited the efficacy of proteasome inhibitors bortezomib and carfilzomib, drugs used in the treatment of MM. Inhibiting the autophagosome-lysosome protein degradation pathway by hydroxychloroquine (HCQ) diminished the protective effect of TLR activation on proteasome inhibitor-induced cytotoxicity. We also found that TLR signaling downregulated the expression of TNFRSF17, the gene encoding for B-cell maturation antigen (BCMA). MYC, BCL2, and BCL2L1 were upregulated in approximately 50% of primary cells, while the response to TLR signaling in terms of TNFRSF17 expression was dichotomous, as an equal fraction of patients showed upregulation and downregulation of the gene. While proteasome inhibitors are part of first-line MM treatment, several of the new anti-MM immune therapeutic drugs target BCMA. Thus, TLR activation may render MM cells less responsive to commonly used anti-myeloma drugs.

Abstract CT067: The anti-BCMA antibody-drug conjugate HDP-101 with a novel amanitin payload shows promising initial first in human results in relapsed multiple myeloma

Abstract Background: HDP-101 is a new antibody drug conjugatetargeting B-cell maturation antigen (BCMA) that carries a synthetic amanitin payload with a new mode of action. Amanitin inhibits RNA polymerase II, effectively stopping transcription and inducing apoptosis in tumor cells, regardless of their proliferation status. It's shown cytotoxicity in vitro against BCMA-positive myeloma cell lines and non-proliferating primary CD138+ cells from refractory myeloma patients withefficacy even in cells with low BCMA density. Clinical Study: HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial in patients with multiple myeloma whose disease has progressed or is refractory, aiming to determine the Maximum Tolerated Dose and/or the Recommended phase 2 Dose in Phase 1. Dose escalation is guided by an adaptive Bayesian logistic regression model with overdose control. The primary objective in phase 2 is to assess anti-tumor activity. Study Progress: As of November 2023, 18 patients (7 females, 11 males) were enrolled in 5 consecutive dose cohorts of 20, 30, 60, 80, and 100µg/kg. Patients had a median age of 70 years (48-82), were heavily pre-treated and multidrug-resistant, and had a median of 6,5 prior treatments (2-15). Study Results: Preliminary data shows that pharmacokinetics of HDP-101 aligns with expectations, and exposure to HDP-101 is dose proportional. Free payload was not detected in serum at a limit of detection of 30 ng/mL, and no anti-drug antibodiesor immunogenic reactions were noted. 17 of 18 patients were evaluable for dose limiting toxicities (DLT) in the 5 treatment cohorts. Initial cohorts were well tolerated, without any DLTs, including no signs of hepatic and renal toxicities, infusions reactions, or ocular disorders. All patients in Cohort 5 had Grade 1-4 transient thrombocytopeniawith platelet reductions starting on Cycle 1/Day 2 (C1/D2), a nadir on C1D5, and full recovery by C1D15 at the latest without any clinical sequelae or interventions. Notably, subsequent dosing with HDP-101 did not produce similarly deep episodes of thrombocytopenia, supporting the possibility that this event is not due to a direct cytotoxic effect against megakaryocytes. After Cohort 5, mitigation strategies are being considered and further dose and schedule optimization is planned with fractionated dosing and/or with premedication. Efficacy: Cohort3 has a patient with stable disease (SD) after 15 cycles. Cohort 5 has 4 ongoing patients with promising results after 3-4 cycles: 2 patients achieved PR, and 2 have SD. Updated data will be presented at the AACR2024 meeting. Citation Format: Jonathan Kaufman, Marc Raab, Shambavi Richard, Sebastian Grosicki, István Takács, András Strassz, Andreas Pahl, Michael Kulke, Thorsten Michael, Anette Last, Hajnalka Szabóki, Garrit Jentsch, Oliver Schönborn-Kellenberger, Robert Orlowski. The anti-BCMA antibody-drug conjugate HDP-101 with a novel amanitin payload shows promising initial first in human results in relapsed multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT067.

Fully Human Anti-CD19 CAR T Cells Derived from Systemic Lupus Erythematosus Patients Exhibit Cytotoxicity with Reduced Inflammatory Cytokine Production

KYV-101 is an autologous anti-CD19 chimeric antigen receptor (CAR)-T cell therapy under investigation for patients with B-cell driven autoimmune diseases. Hu19-CD828Z is a fully human anti-CD19 CAR designed and demonstrated to have a favorable clinical safety profile. Since anti-CD19 CAR T cells target and kill B cells in both circulation and tissues, the treatment with Hu19-CD828Z CAR T cells offers great potential in depleting autoreactive B cells. Demonstrate that Hu19-CD828Z CAR T cells manufactured from cryopreserved leukaphereses from patients with systemic lupus erythematosus (SLE) exhibit CAR-mediated and CD19-dependent cytokine release, proliferation and cytotoxicity when co-cultured with autologous primary B cells. T cells were enriched from cryopreserved leukaphereses from SLE patients or healthy donors (HD). CAR T cells were generated by transducing these cells with a lentiviral vector encoding Hu19-CD828Z. CAR-mediated and CD19-dependent activity was monitored in vitro in a set of cytotoxicity, cytokine release, and proliferation studies, in response to autologous primary CD19+ B cells, a CD19+ cell line (NALM-6), or a CD19- cell line (U937). Hu19-CD828Z CAR T cells produced from SLE patients or HD induced greater proliferation and dose-dependent cytotoxicity against both autologous primary B cells and the CD19+ NALM-6 cells than nontransduced control T cells or co-cultures with a CD19- cell line. Interestingly, there was lower inflammatory cytokine production from SLE patient-derived CAR T cells compared to HD donor-derived CAR T cells with either CD19+ cells or primary B cells. Hu19-CD828Z CAR T cells generated from SLE patient lymphocytes demonstrate CAR-mediated and CD19-dependent activity against autologous primary B cells with reduced inflammatory cytokine production supporting KYV-101 as a novel potential therapy for the depletion of pathogenic B cells in SLE patients.

Abstract 47: Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy

Abstract Introduction: Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown substantial anti-tumor efficacy in hematological malignancies during recent years. As the field evolves to novel target epitopes, longer CAR structures are needed to reach membrane proximal epitopes for improved efficacy. Our approach to reach such epitopes is to have (non-functional) SIRPalpha based extracellular extension, spacer, to our CAR structure. We have shown, in this proof-of-concept study, that stability of the extracellular spacer of a CAR plays a pivotal role to improve in vivo anti-tumor efficacy. Various SIRPalpha-based CARs, FiCARs, targeting CD19 was studied for their function. Protein stability of the CD19 CAR structures were characterized and subsequently the killing efficacy of target cells and cytokine release in in vitro as well as in vivo anti-tumor efficacy was evaluated. Methods & Results: In vitro co-culture assays with primary CD19 CAR T and CD19 positive cancer cells had shown no significant differences between the different spacer containing FiCAR structures. A dose dependent killing efficacy was observed in all the tested structures with up to >90% killing of NALM6 and Raji cells. Comparable results for IL-2, IFN-gamma, TNF-alpha, IL-4, IL-13 and granzyme B response were seen in co-cultured assay. In the protein stability studies, modifications in the extracellular part of the CAR structure led to an increase in thermal stability (over 2 °C) and reduced/halted multimer formation. To study the FiCAR T cell function in an in vivo tumor model, NSG mice were injected i.v with Raji cells and few days later mice were injected i.v with FiCAR T cells. Anti-tumor efficacy and overall survival were studied at the end of the study (60 days), the enhanced stability of FiCAR T structure translated into improved anti-tumor efficacy and survival. Interestingly, none of the mice survived in the least stable structure, 40% survival with intermediate stability structures and over 70% survival was observed in most stable structures. Conclusion: A stabilized SIRPalpha-based CD19 FiCARs dramatically enhance anti-tumor efficacy and survival in Raji tumor model. The stabilization is critical for improved efficacy, especially in the in vivo setting. This data suggests that, in addition to the length of the CARs stability of the CAR structure appear to play a crucial role in the efficacy of the CAR T cells. The modifications presented here enhanced CD19-targeting CAR T cell efficacy. This approach requires further investigation to improve the efficacy of currently approved CAR T cells, especially CD19-CAR T cells. Citation Format: Henrik Paavilainen, Jan Koski, Veera Nikoskelainen, Manar Elmadani, Endrit Elbasani, Can Hekim, Emmi-Leena Ihantola, Virginie Kergourlay, Pirjo Vehmaan-kreula, Marie Nyman, Kiira Kalke, Markus Nurmi, Hector Monzo, Paivi Ojala, Anu Autio, Matti Korhonen, Anil Thotakura. Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 47.

Identification of COQ2 as a regulator of proliferation and lipid peroxidation through genome-scale CRISPR-Cas9 screening in myeloma cells.

Multiple myeloma (MM) is the second most common malignant haematological disease with a poor prognosis. The limit therapeutic progress has been made in MM patients with cancer relapse, necessitating deeper research into the molecular mechanisms underlying its occurrence and development. A genome-wide CRISPR-Cas9 loss-of-function screening was utilized to identify potential therapeutic targets in our research. We revealed that COQ2 plays a crucial role in regulating MM cell proliferation and lipid peroxidation (LPO). Knockout of COQ2 inhibited cell proliferation, induced cell cycle arrest and reduced tumour growth invivo. Mechanistically, COQ2 promoted the activation of the MEK/ERK cascade, which in turn stabilized and activated MYC protein. Moreover, we found that COQ2-deficient MM cells increased sensitivity to the LPO activator, RSL3. Using an inhibitor targeting COQ2 by 4-CBA enhanced the sensitivity to RSL3 in primary CD138+ myeloma cells and in a xenograft mouse model. Nevertheless, co-treatment of 4-CBA and RSL3 induced cell death in bortezomib-resistant MM cells. Together, our findings suggest that COQ2 promotes cell proliferation and tumour growth through the activation of the MEK/ERK/MYC axis and targeting COQ2 could enhance the sensitivity to ferroptosis in MM cells, which may be a promising therapeutic strategy for the treatment of MM patients.

Allogeneic CAR-T cells with of HLA-A/B and TRAC disruption exhibit promising antitumor capacity against B cell malignancies

BackgroundAlthough chimeric antigen receptor T (CAR-T) cells have been proven to be an effective way of treating B cell malignancies, a lot of patients could not benefit from it because of failure in CAR-T cell manufacturing, disease progression, and unaffordable price. The study aimed to explore universal CAR-T cell products to extend the clinical accessibility.MethodsThe antitumor activity of CRISPR/Cas9-edited allogeneic anti-CD19 CAR-T (CAR-T19) cells was assessed in vitro, in animal models, and in patients with relapsed/refractory (R/R) acute B cell lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma.ResultsB2M−/TRAC− universal CAR-T19 (U-CAR-T19) cells exhibited powerful anti-leukemia abilities both in vitro and in animal models, as did primary CD19+ leukemia cells from leukemia patients. However, expansion, antitumor efficacy, or graft-versus-host-disease (GvHD) was not observed in six patients with R/R B cell malignancies after U-CAR-T19 cell infusion. Accordingly, significant activation of natural killer (NK) cells by U-CAR-T19 cells was proven both clinically and in vitro. HLA-A−/B−/TRAC− novel CAR-T19 (nU-CAR-T19) cells were constructed with similar tumoricidal capacity but resistance to NK cells in vitro. Surprisingly, robust expansion of nU-CAR-T19 cells, along with rapid eradication of CD19+ abnormal B cells, was observed in the peripheral blood and bone marrow of another three patients with R/R B-ALL. The patients achieved complete remission with no detectable minimal residual disease 14 days after the infusion of nU-CAR-T19 cells. Two of the three patients had grade 2 cytokine release syndrome, which were managed using an IL-6 receptor blocker. Most importantly, GvHD was not observed in any patient, suggesting the safety of TRAC-disrupted CAR-T cells generated using the CRISPR/Cas9 method for clinical application.ConclusionsThe nU-CAR-T19 cells showed a strong response in R/R B-ALL. nU-CAR-T19 cells have the potential to be a promising new approach for treating R/R B cell malignancies.

Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma

Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM. Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138 + cells were obtained from bone marrow aspirations followed by MACS ® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified. Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138 + MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co-cultures with MM cells and primary CD138 + MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated. Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.

Hdp-101, an Anti-BCMA Antibody-Drug Conjugate with a Novel Payload Amanitin in Patients with Relapsed Multiple Myeloma, Initial Findings of the First in Human Study

Introduction Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of malignancies. The vast majority of these are based on a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins which impact proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as multiple myeloma. Thus, new compounds with alternative modes of action and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. HDP-101 an Amanitin based antibody drug conjugate HDP-101 is a new ADC targeting BCMA carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma The cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. HDP-101-01 Clinical study HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial with HDP-101 in patients with myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended Phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. An adaptive Bayesian logistic regression model with overdose control principle is used to guide the dose escalation steps. An Interim Analysis is planned after each cohort is completed. The design of the study ensures a safe and adaptive dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. HDP-101-01 Study progress The study started enrollment in February, 2022. As of 10th of July, 2023, eight (2 female and 6 male) patients were dosed in 3 consecutive dose cohorts. The median age of the patients was 70 years, ranging between 50 and 80. All 8 patients were heavily pre-treated and multidrug-resistant. The median previous lines of treatment were 7 (5 to 15). Table 1 HDP-101-01 Results Seven of 8 patients were evaluable for dose limiting toxicities (DLTs) in the first 3 treatment cohorts. The initial 3 cohorts were well tolerated, without any DLTs, there were no signs of liver and kidney toxicity, no infusion reaction was detected. No reports of keratopathy or visual acuity loss were observed. Free payload was not detected in any of the available pharmacokinetic samples. Based on the limited data, the PK of HDP-101 was in line with our expectation based on the preclinical observations, exposure to HDP-101 is dose proportional. Anti-drug antibody (ADA) was not detected and there's no sign of immunogenicity. Objective responses were not reported in these initial cohorts however in Cohort #3 (60μg/Kg) there was one patient ongoing after 8 cycles of treatment with SD. This patient had a slow decrease in M-protein levels. Image 1 The initial dose cohorts showed good tolerability in late stage relapsed and/or refractory multiple myeloma patients. The study continues to enroll patients to higher dose cohorts at selected sites in the US, Germany, Poland and Hungary. Currently the study is enrolling patients in Cohort #4 at the dose of 80μg/Kg. Additional updates will be provided at the 2023 ASH Annual Meeting.

Targeting RBM39-MEK5 Axis Synergizes with Bortezomib to Inhibit Multiple Myeloma Malignancy

Multiple myeloma (MM) is a type of haematological malignancy characterised by the accumulation and expansion of malignant plasma cells in the bone marrow (BM). Despite recent advances in chemotherapy, MM remains an incurable disease for most patients and therefore requires novel therapies. Abnormal alternative splicing is a hallmark of cancer and is associated with poor survival and an ultra-high-risk population in MM (Bauer et. al. Haematologica 2021). Targeting splicing is a promising therapeutic strategy in MM. Our previous study showed that RBM39 is highly expressed in primary MM cells and cell lines and correlates with poor overall survival (Tong et. al. Haematologica 2020). To investigate the underlying role of RBM39 in MM, we knocked down RBM39 in MM cell lines. We found that knockdown of RBM39 inhibited MM cell proliferation, induced apoptosis and cell cycle arrest at the G2/M phase, and inhibited subcutaneous tumour growth. Indisulam has been reported to induce RBM39 degradation via the proteasomal pathway by acting as a molecular glue to link RBM39 to the E3 ubiquitin ligase DCAF15 (Han et. al. Science 2017). In our study, Indisulam treatment in MM cells was shown to degrade RBM39 via DCAF15. Indisulam could induce apoptosis and G2/M phase arrest in MM cell lines and inhibite tumour growth in vivo. It also significantly decreased MM cell viability in a co-culture system with MM BM-derived mesenchymal stromal cells. Indisulam was also able to inhibit primary CD138+ cells isolated from newly diagnosed MM patients. To explore the mechanistic role of RBM39 in promoting MM malignancy, we performed RNA immunoprecipitation (RIP)-Seq and RNA sequencing in RBM39 knockdown MM cells to investigate the alternative splicing targets of RBM39. KEGG pathway analysis of differentially expressed and alternatively spliced genes revealed the enrichment of the MAPK pathway. We then identified MEK5, a non-redundant member of the MAPK family, as a potential target of RBM39, which is highly expressed in MM. Full-length MEK5 contributes to increased proliferation and survival of MM cells. We further demonstrated that MEK5 is bound and alternatively spliced by RBM39 and that splicing of full-length MEK5 is dependent on RBM39. Given the importance of the RBM39-MEK5 axis in MM, we also found that inhibition of RBM39 with Indisulam or MEK5 with the inhibitor BIX02189 increased the cytotoxicity of bortezomib in vivo and in vitro. In conclusion, we have demonstrated that the RBM39-MEK5 axis, in which RBM39 maintains splicing of full-length MEK5, is essential for MM cell survival and proliferation. Mechanistically, shRNA-mediated knockdown or Indisulam-mediated degradation of RBM39 caused extensive altered splicing, including mis-splicing of MEK5. Targeting RBM39 or MEK5 could not only inhibit MM malignancy, but also enhance the cytotoxicity of bortezomib for MM.

MSC.sensor: Capturing cancer cell interactions with stroma for functional profiling.

Mesenchymal stromal cells (MSCs) contribute to the microenvironment regulating normal and malignant hematopoiesis, and thus may support subpopulations of cancer cells to escape therapeutic pressure. Here, we engineered bone marrow MSCs to express a synthetic CD19-sensor receptor to detect and display interacting primary CD19+ leukemia cells in coculture. This implementation provides a versatile platform facilitating ex vivo drug response profiling of primary CD19+ leukemia cells in coculture with high-sensitivity and scalability.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma

BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies.MethodsWe performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt’s and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment’ synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy.ResultsWe identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects.ConclusionsThe present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Abstract 2907: Development of a CAR-NK cellular platform for cancer immunotherapy

Abstract Although Chimeric Antigen Receptor (CAR)-T cell therapy has proven to be successful in treating multiple hematological malignancies, their use is limited due to the potential life-threatening adverse events, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). CAR natural killer (CAR-NK) cells are rapidly emerging as a promising alternative cellular therapeutic with improved efficacy and reduced adverse effects. Moreover, due to the intrinsic characteristics of NK cells, whereby they lack antigen-specific cell surface receptors, CAR-NK cells offer the additional advantage of being an “off-the-shelf” product, thus satisfying the requirement for large-scale production for cancer immunotherapy, giving it the potential to reach more patients at a reduced cost. Here we outline the method to generate, expand and enrich human primary CD19 CAR-NK cells, and demonstrate their efficacy in-vitro against CD19-expressing target cancer cells. Human primary NK cells were isolated from healthy donors and transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV, as an empty vector transduced control. Transduction efficiency greater than 10% or 20% was achieved following transduction and expansion over a 7-day period in response to CD19 CAR LV or GFP LV respectively. Following sufficient expansion, cytotoxicity of the CD19 CAR-NK cells was tested in-vitro in a Tumor Killing Assay (TKA) against CD19+ (Raji) or CD19- (SKOV-3) target cells. As expected, CD19 CAR-NK cells displayed increased cytotoxicity towards CD19+ target cells when compared to non-transduced or GFP transduced controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting specific CD19 CAR mediated killing. Moreover, CD19 CAR-NK cells showed increased degranulation and IFNꝩ secretion when compared to both non-transduced and GFP-transduced controls, suggesting increased NK cell activation and cytotoxicity. Additionally, when degranulation of CD19 CAR positive and CD19 CAR negative NK populations was directly compared, the level of NK cell degranulation was analogous between the two populations, suggesting that CD19 CAR NK cells can enhance the cytotoxic activity of neighboring non-transduced cells, resulting in enhanced killing of CD19+ target cells by the total NK population present. Taken together these data demonstrate the generation, expansion and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This culture system provides the scientific community a platform to screen novel CAR-constructs and to test novel immunomodulators which could enhance NK cell expansion or functional potential to further develop new, more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Ravindra Mode, Kaitlin Mitchell, David Cobeta-Lopez, Sabrina de Munnik, Rene McLaughlin, Gemma Moiset, Louise Brackenbury, Lauren Schewitz-Bowers, Robert Nunan. Development of a CAR-NK cellular platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2907.

Luteolin inhibits the TGF-β signaling pathway to overcome bortezomib resistance in multiple myeloma

Multiple myeloma (MM) is an incurable condition and the second most common hematological malignancy. Over the past few years, there has been progress in the treatment of MM, but most patients still relapse. Multiple myeloma stem-like cells (MMSCs) are believed to be the main reason for drug resistance and eventual relapse. Currently, there are not enough therapeutic agents that have been identified for eradication of MMSCs, and thus, identification of the same may alleviate the issue of relapse in patients. In the present study, we showed that luteolin (LUT), a natural compound obtained from different plants, such as vegetables, medicinal herbs, and fruits, effectively inhibits the proliferation of MM cells and overcomes bortezomib (BTZ) resistance in them in vitro and in vivo, mainly by decreasing the proportion of ALDH1+ cells. Furthermore, RNA sequencing after LUT treatment of MM cell lines and an MM xenograft mouse model revealed that the effects of the compound are mediated through inhibition of transforming growth factor-β signaling. Similarly, we found that LUT also significantly reduced the proportion of ALDH1+ cells in primary CD138+ plasma cells. In addition, LUT could overcome the BTZ treatment-induced increase in the proportion of ALDH1+ cells, and the combination of LUT and BTZ had a synergistic effect against myeloma cells. Collectively, our findings suggested that LUT is a promising agent that manifests MMSCs to overcome BTZ resistance, alone or in combination with BTZ, and thus, is a potential therapeutic drug for the treatment of MM.

Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) targeting BCMA and GPRC5D have demonstrated very encouraging responses in heavily pre-treated multiple myeloma (MM). Recent studies interrogating T cells repertoires and fitness correlated resistance to these immune based therapies with exhausted T cell states. While in lymphoma, antigen escape with CD19 loss is observed in up to 30% of patients with acquired resistance to adaptive therapies, in MM TNFRSF17 or GPRC5D structural mutations are thought to be less prevalent in recipients of BCMA or GPRC5D-targeting CARs or TCEs . Bi-allelic loss (n=2) and a missense truncating mutation (n=1) of TNFRSF17 with CAR T cells, as well as one case of biallelic loss in TNFRSF17 with TCE are reported to date with no reported cases of bi-allelic loss of GPRC5D. Further studies of the role TNFRSF17 or GPRC5D structural or point mutations to the immune escape to these adaptive therapies are needed. Serial BM aspirates were collected from patients treated with BCMA or GPRC5D-targeting TCE or CARs prior to therapy and at relapse. MM cells were profiled with single cell mRNA (scRNAseq), single cell copy number (scCNVseq) using the 10x Genomics GemCode system and whole genome sequencing (WGS). Sequenced reads were aligned to hg38. Single cell reads were processed with CellRanger and downstream analyses with Seurat R package. In order to define the prevalence of TNFRSF17 or GRPC5D structural variants and mutations in MM, we performed WGS (n=19, 14 pre-CAR/TCE, 5 post-CAR/TCE) as well as scCNVseq (n=33, 16 RRMM, 7 pre-CAR/TCE, 8 post-CAR/TCE) on sorted CD138+ cells from relapsed MM patients. WGS (100x coverage) identified at baseline a subclonal deletion at the TNFRSF17 gene locus in only 1 patient; this small subclone subsequently drove the progression post BCMA CAR T cell therapy with an acquired biallelic loss of TNFRSF17. Mono-allelic large and focal deletions of GPRC5D (chr.12p) were detected by WGS in 7 cases (5 deletions, 2 copy neutral LOH). scCNVseq studies identified more frequent subclonal monoallelic copy number losses of TNFRSF17 in 11.2% of the cells (27884 MM cells) prior to anti-BCMA therapy exposure with the deletions ranging from 200 kb to 2.3 Mb. A homozygous deletion of TNFRSF17 was noted in 2 patients at the time of disease progression, one post CAR T and the other post BCMAxCD3 TCE. Structural mutations at the GPRC5D locus were also identified by scCNVseq with monoallelic and biallelic deletions in 11.9% and 2.7% of the cells, respectively. Clonal biallelic loss of GPRC5D post GPRC5DxCD3 TCE was detected in one patient who harbored a monoallelic loss in 79.2% of the cells pre-TCE. With regard to point mutations, in one patient progressing from a durable CR on BCMA TCE, we observed an acquired mono-allelic loss of TNFRSF17 (scCNV) coupled with de novo mutation in TNFRSF17 extracellular domain c.81G>C [p.(R27P)]. This point R27P mutation is predicted to be damaging to BCMA (Polyphen) with disruption of several H-bonds between R27 and C24, Q25, S29, S30 and T32 of BCMA extracellular aa residues. Importantly, molecular dynamic simulations of WT vs. mutant R27P using publicly deposited crystal structural of BCMA bound to anti-BCMA Ab (J22.9-xi) (PBD ID 4ZFO) demonstrated that this mutation disrupts all H-bonds between BCMA and the light chain of chimeric mouse/human anti-BCMA Ab (J22.9-xi). Functionally, primary CD138+ cells with mutant BCMA [p.(R27P)] as well as K562 cells transduced with mutant BCMA [p.(R27P)] were completely resistant to BCMAxCD3 cytotoxicity with teclistamab and elranatamab compared to cells with WT BCMA. While structural alterations of TNFRSF17 and GPRC5D remain relatively rare, we have identified biallelic loss of TNFRSF17 post BCMA CAR T and BCMAxCD3 TCE therapy and herein reported the first case of biallelic loss of GPRC5D post GPRC5DxCD3 TCE treatment. Importantly, we also identified a monoallelic loss of TNFRSF17 (del16p) coupled with BCMA extracellular domain point mutation [p.(R27P)] that confers resistance to two BCMAxCD3 TCEs in MM. Therefore, in addition to structural alterations (biallelic loss of TNFRSF17 or GPRC5D), antigen escape resulting from non-truncating missense point mutations in BCMA extracellular domain also mediate resistance to targeted-immunotherapies.

EGFR and HDAC Dual-Target Inhibitor CUDC-101 Enhances the Anti-Myeloma Effects of Bortezomib By Regulating the G2/M Cell Cycle Arrest

Background Mutiple myeloma (MM) has become the second most prevalent tumor in hematologic diseases, with the age of its onset decreasing year by year. Proteasome inhibitors, immunomodulators and monoclonal antibodies are the most commonly used agents for treating MM. Although the treatment effect and survival time of MM patients has significantly improved with therapies, MM remains an incurable disease, and most MM patients during treatment inevitably undergo primary or secondary drug resistance, leading to disease recurrence and progression, and even death. Therefore, exploring new agents for MM treatment can have significant clinical benefits. CUDC-101 is a multiple inhibitor that directly blocks the HDAC, EGFR and HER2 signaling pathways. In recent years, many studies have shown that CUDC-101 has strong antitumor effects on many tumor types through inducing caspase-dependent apoptosis, regulating proliferation and migration. In addition, CUDC-101 has been reported to exert a synergistic effect with a lot of conventional drugs, for example, Gemcitabine, Arsenic trioxide, or Carfilzomib. However, it was not reported whether CUDC-101 has an anti-myeloma effect or a synergistic effect when applied with other drugs for MM treatment. Methods CCK8 assay was used to detect the inhibitory effect of drugs on the cell proliferation of six human myeloma cell lines. TUNEL assay and Flow cytometry was used to detect the apoptosis and cell cycle. Western bolt was used to detect the expression of apotosis-related proteins and signaling pathway. MM xenografts model was used to indicate the efficacy in vivo. Results In this paper, we identified the potential efficacy of CUDC-101 in MM treatment and elaborated the underlying mechanisms. Our results suggest that CUDC-101 can induce cell cycle arrest to significantly inhibit the proliferation of as well as induce cell apoptosis in MM cell lines or primary CD138 positive MM cells through the inhibition of EGFR/PI3K and HDAC signaling pathways. Meanwhile, CUDC-101 also showed marked growth inhibition in the MM xenografts model. In addition, we confirmed the synergistic effect between CUDC-101 and Bortezomib, one of the drugs most commonly used drugs for MM treatment. Using MM cell lines and xenograft models, we demonstrated the remarkable inhibitive effect of these drugs on cell proliferation and tumor growth. Further investigation revealed that the G2/M phase arrest is a pivotal mechanism to achieve this effect, which involves the upregulation of P21 and P27. And these data prove that CUDC-101 is an effective agent in MM therapy. Conclusion In summary, we have confirmed the efficacy of CUDC-101 in MM treatment as a single drug or in combination with Bortezomib, and our findings offer a new strategy for MM therapy.

Hdp-101, an Anti-BCMA Antibody-Drug Conjugate with a Novel Payload Amanitin in Patients with Relapsed Multiple Myeloma, Initial Findings of the First in Human Study

Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of malignancies. Vast majority of these ADCs are based on a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins targets proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as multiple myeloma. Thus, new compounds with alternative mode of actions and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. HDP-101 is a new ADC targeting BCMA (B cell maturation antigen) carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma. Furthermore, the cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial with HDP-101 in patients with multiple myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. An adaptive Bayesian logistic regression model with overdose control principle is used to guide the dose escalation steps. An Interim Analysis is planned after each cohort is completed. The design of the study ensures a safe and adaptive dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. The study started enrollment in February 2022. As of 12th of July 2022 four (1 female and 3 male) patients were dosed in 2 consecutive dose cohorts. The median age of the patients was 63.5 years, ranging between 51 and 80. All 4 patients were heavily pre-treated and multidrug-resistant. The median previous lines of treatment were 11 (5 to 16). Three of 4 patients were evaluable for dose limiting toxicities (DLT). The initial 2 cohorts were well tolerated, without any reports on DLTs. No reports of keratopathy or visual acuity loss were observed. Free payload was not detected in any of the available pharmacokinetic samples. In line with our expectation objective responses also were not reported in these initial cohorts. The initial dose cohorts showed good tolerability in late stage relapsed and/or refractory multiple myeloma patients. The study continues to enroll patient to higher dose cohorts. An updated dataset will be presented at the ASH2022 meeting.

Abstract LB101: Novel Drug-responsive domain (DRD)-based regulation technology enables tightly controlled activity of potent membrane-bound IL12 in adoptive cell therapies

Abstract Interleukin 12 (IL12) is an attractive cancer immunotherapeutic known to be extremely potent against solid tumors preclinically. However, the clinical utility of IL12 has been limited by toxicities stemming from high systemic cytokine exposure. Thus, armoring cellular therapies such as chimeric antigen receptor T cells (CAR-Ts) or tumor infiltrating lymphocytes (TILs) with IL12 will require technologies that provide tight control of IL12 expression and localization. Herein, we describe a tightly regulated version of IL12 for use in cellular therapies. First, we show that tethering IL12 to the membrane increases the activity of IL12 at the tumor site in vivo while reducing potential systemic toxicities. Membrane bound (mbIL12) was compared to secreted IL12 in the syngeneic CD8 gp100 TCR transgenic PMEL model. In this model, PMEL T cells transduced with mbIL12 demonstrated similar reduction in B16 tumor outgrowth as secreted IL12. Like secreted IL12, mbIL12 enhanced the expansion of PMEL T cells and retained T cell extrinsic activities, such as activation of myeloid cells and remodeling the tumor microenvironment. However, mbIL12 showed reduced toxicity signals as compared to secreted IL12, including a reduction in serum IFNy. Likewise, in a xenograft system, CD19-CARTs expressing human mbIL12 demonstrated enhanced potency against Raji tumors. To enhance the safety of mbIL12 further we sought to regulate its expression using Obsidian’s cytoDRIVE® technology. This platform consists of small, fully human protein sequences called drug responsive domains (DRD)s, such as carbonic anhydrase 2, that enable regulation of expression of a fused target protein under the control of FDA-approved, orally bioavailable small molecule ligands, such as, acetazolamide. In the absence of ligand (the “off-state”), the fusion protein is unfolded and degraded. In the presence of ligand (the “on-state”), the DRD is stabilized, allowing for protein expression and function. Thus, the cytoDRiVE® system acts as a titratable and reversible rheostat for on-demand protein activity. While a single DRD can enable some regulation of mbIL12 levels, we found that adding a modulation hub that increases the multiplicity of DRDs greatly improves regulation. Indeed, these modifications enabled off-state levels of mbIL12 that were indistinguishable from untransduced controls in HEK and Jurkat cell lines as well as primary human CD19 CAR-T-cells using flow cytometry. These modifications also enhanced mbIL12 regulation, leading up to a 30-fold dynamic range of mbIL12 abundance. Regulated mbIL12 was demonstrated to be active by phosphorylation of STAT4 in bystander NK cells in vitro. Combining DRDs with modulation hubs and membrane tethering enables the control of highly potent, previously clinically intractable cytokines, such as IL12, for use in enhancing cell therapies. Citation Format: Sean Gregory Smith, James A. Storer, Dexue Sun, Dan Jun Li, Benjamin Primack, Theresa Ross, Scott LaJoie, Jeremy Tchaicha, Dhruv Sethi, Jan ter Meulen, Michelle Ols. Novel Drug-responsive domain (DRD)-based regulation technology enables tightly controlled activity of potent membrane-bound IL12 in adoptive cell therapies [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 LB101.

Individualized regimen of low-dose rituximab monotherapy for new-onset AChR-positive generalized myasthenia gravis.

Generalized AChR-MG is an archetype of B cell-mediated autoimmune disorders, and use of biologic agent rituximab (RTX) for B cell depletion is generally limited to immunosuppressive therapy-refractory cases. However, benefit of RTX monotherapy and individualized regimen with optimal dosage in early stage of new-onset generalized AChR-MG still remains to be elucidated. In this retrospective study, we explore the efficacy and safety of personalized regimen of 100mg low-dose rituximab monotherapy in treating new-onset generalized AChR-MG. Thirteen new-onset generalized AChR-MG patients were enrolled for the study, initiating RTX treatment from November 2017 to August 2020. The individualized low-dose RTX monotherapy protocol consisted of 100mg induction treatment weekly with no more than three circles, followed by reinfusion (100mg once) sequentially according to whether achieving primary endpoint and peripheral CD19 + B-cell repopulation ≥ 1% of total lymphocytes at each visit (every 3months). Outcome measures included MGFA-PIS Minimal Manifestation (MM) or better status (primary endpoint), changes in QMG, MMT, MG-ADL and MGQOL-15 scores (secondary endpoint), as well as cholinesterase inhibitors dosage. All 13 patients achieved the primary endpoint in parallel with significant improvement of QMG, MMT, MG-ADL MGQOL-15 scores, and reduction of cholinesterase inhibitors dose. A total of 52 visits were performed during follow-up, and only 10 assessments presenting peripheral CD19 + B-cell repopulation (≥ 1%) without "MM or better status" were followed by RTX reinfusions (100mg once) for clinical remission. The total dosage of RTX was only 346.15 ± 96.74mg (including 269.23 ± 63.04mg for induction and 76.92 ± 59.91mg for reinfusion), which seemed to be much lower than those dosages used in new-onset generalized AChR-MG as described previously. Moreover, compared with patients without thymoma, thymectomy markedly delayed initiation of RTX for patients with thymoma (log-rank test, p = 0.0002), but the delaying treatments showed no influence on the time for achieving primary outcome (log-rank test, p = 0.2517). Our study firstly showed that individualized regimen of low-dose RTX monotherapy is effective and safe for early treatment of new-onset generalized AChR-MG, and practicable for directing RTX reinfusion and withdrawal. Moreover, the monotherapy protocol was also indicated to be extensively applicable in both new-onset AChR-MG with thymoma (thymectomy) and without thymoma.