A new perspective on therapies involving B-cell depletion in autoimmune diseases.

It has been rediscovered in the last fifteen years that B-cells play an active role in autoimmune etiology rather than just being spectators. The clinical success of B-cell depletion therapies (BCDTs) has contributed to this. BCDTs, including those that target CD20, CD19, and BAFF, were first developed to eradicate malignant B-cells. These days, they treat autoimmune conditions like multiple sclerosis and systemic lupus erythematosus. Particular surprises have resulted from the use of BCDTs in autoimmune diseases. For example, even in cases where BCDT is used to treat the condition, its effects on antibody-secreting plasma cells and antibody levels are restricted, even though these cells are regarded to play a detrimental pathogenic role in autoimmune diseases. In this Review, we provide an update on our knowledge of the biology of B-cells, examine the outcomes of clinical studies employing BCDT for autoimmune reasons, talk about potential explanations for the drug's mode of action, and make predictions about future approaches to targeting B-cells other than depletion.

Switching from natalizumab to an anti-CD20 monoclonal antibody in relapsing remitting multiple sclerosis: A systematic review.

BACKGROUND: Use of natalizumab (NTZ) is precluded in many Multiple Sclerosis (MS) patients by the risk of progressive multifocal leukoencephalopathy (PML). Regardless, some patients may commence natalizumab for short term disease control in spite of being seropositive, and others may seroconvert whilst on treatment. In these circumstances, discontinuation of NTZ should not occur until a clear exit strategy is established to prevent post-NTZ disease reactivation, which often exceeds the severity of disease activity prior to NTZ treatment. The objective of this systematic review was to summarise the available evidence for CD20-monoclonal antibodies (CD20mAb) as a suitable NTZ exit strategy, and to identify whether a superior switch protocol can be established. METHODS: In accordance with PRISMA guidelines, a total of 2393 references were extracted from a search of three online databases (PubMed, Scopus, MEDLINE). Following the application of inclusion/exclusion criteria, a total of 5 studies representing 331 patients were included. RESULTS: The overall incidence of clinical relapse during washout periods ranging from 4.4-10.7 weeks was 0 %. The incidence of clinical relapse during two-year follow-up ranged from 1.8 % to 10 % for switches to all types of CD20 monoclonal antibody. The weighted mean for clinical relapse at 12 months was 8.8 %. Three studies reported an annualised relapse rate (ARR) ranging from 0.02-0.12 with a weighted mean ARR of 0.07. The overall incidence of PML during washout was 0 % and the overall incidence of PML within 6 months follow-up was 0.6 %. CONCLUSIONS: This systematic review provides the first attempt at identifying a superior switch protocol in patients at risk of PML transitioning from NTZ to a CD20mAb. Our results indicate that CD20mAb's are a suitable transitional option for patients who discontinue NTZ, with our cohort demonstrating very low rates of carryover PML and low rates of clinical relapse. The most appropriate washout period is unclear due to confounding factors but is likely between 4 and 12 weeks.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators.

The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.

Drugs Targeting CD20 in Multiple Sclerosis: Pharmacology, Efficacy, Safety, and Tolerability.

Currently, there are four monoclonal antibodies (mAbs) that target the cluster of differentiation (CD) 20 receptor available to treat multiple sclerosis (MS): rituximab, ocrelizumab, ofatumumab, and ublituximab. B-cell depletion therapy has changed the therapeutic landscape of MS through robust efficacy on clinical manifestations and MRI lesion activity, and the currently available anti-CD20 mAb therapies for use in MS are a cornerstone of highly effective disease-modifying treatment. Ocrelizumab is currently the only therapy with regulatory approval for primary progressive MS. There are currently few data regarding the relative efficacy of these therapies, though several clinical trials are ongoing. Safety concerns applicable to this class of therapeutics relate primarily to immunogenicity and mechanism of action, and include infusion-related or injection-related reactions, development of hypogammaglobulinemia (leading to increased infection and malignancy risk), and decreased vaccine response. Exploration of alternative dose/dosing schedules might be an effective strategy for mitigating these risks. Future development of biosimilar medications might make these therapies more readily available. Although anti-CD20 mAb therapies have led to significant improvements in disease outcomes, CNS-penetrant therapies are still needed to more effectively address the compartmentalized inflammation thought to play an important role in disability progression.

Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

BACKGROUND: Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated cytotoxicity independent of T-cell/major histocompatibility complex interaction. Phase I results in patients with R/R B-NHL demonstrated that odronextamab monotherapy could achieve deep and durable responses with a generally manageable safety profile (ELM-1; NCT02290951). As part of a biomarker analysis of the same study, we investigated potential biomarkers and mechanisms of resistance to odronextamab. METHODS: Patients with R/R B-NHL enrolled in ELM-1 received one time per week doses of intravenous odronextamab for 4×21 day cycles, then doses every 2 weeks thereafter. Patient tumor biopsies were obtained at baseline, on-treatment, and at progression. Immune cell markers were analyzed by immunohistochemistry, flow cytometry, single-cell RNA sequencing, and whole genome sequencing. RESULTS: Baseline tumor biopsies showed that almost all patients had high proportions of B cells that expressed the CD20 target antigen, whereas expression of other B-cell surface antigens (CD19, CD22, CD79b) was more variable. Responses to odronextamab in patients with diffuse large B-cell lymphoma were not related to the relative level of baseline CD20 expression, cell of origin, or high-risk molecular subtype. A potential link was observed between greater tumor programmed cell death-ligand 1 expression and increased likelihood of response to odronextamab. Similarly, a trend was observed between clinical response and increased levels of CD8 T cells and regulatory T cells at baseline. We also identified an on-treatment pharmacodynamic shift in intratumoral immune cell subsets. Finally, loss of CD20 expression through inactivating gene mutations was identified as a potential mechanism of resistance in patients who were treated with odronextamab until progression, as highlighted in two detailed patient cases reported here. CONCLUSIONS: This biomarker analysis expands on clinical findings of odronextamab in patients with R/R B-NHL, providing verification of the suitability of CD20 as a therapeutic target, as well as evidence for potential mechanisms of action and resistance.

5 signature genes revealed by single-cell profiling identified unique immune subtypes affecting the prognosis of ovarian cancer.

OBJECTIVE: Ovarian cancer (OC) ranks among the most prevalent gynecological malignancies, with surgery, chemotherapy, and immunotherapy constituting primary treatment modalities. However, despite advancements, immunotherapy, particularly immune checkpoint inhibitors, has yielded suboptimal outcomes. The pressing need to identify biomarkers predictive of clinical prognosis underscores our objective. We aim to discern gene signatures and establish prognostic subgroups, specifically in the context of immunotherapy and chemotherapy, guiding clinical decision-making. MATERIALS AND METHODS: We used the Tumor Immunotherapy Gene Expression Resource (TIGER) and The Cancer Genome Atlas (TCGA) databases to extract signature genes of prognostic significance. Unsupervised consensus clustering was employed to classify patients based on these signature genes. The Tumor Immune Estimation Resource (TIMER) database, along with the R packages "maftools" and "ESTIMATE" facilitated immune infiltration estimation. Gene set variation analysis (GSVA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were implemented to probe immune-related cell signaling pathways among distinct subtypes. The Tumor Immune Dysfunction and Exclusion (TIDE) database was used to assess immunotherapy effects, while the R package "OncoPredict" evaluated drug sensitivity differences among subtypes. RESULTS: We identified five prognostically influential genes in ovarian cancer: IGFBP7, JCHAIN, CCDC80, VSIG4, and MS4A1. Utilizing these signature genes, we categorized TCGA-OV patients into five clusters, each associated with varying clinical prognoses. Notably, 2 clusters exhibited superior prognoses, accompanied by enhanced immune cell infiltration. KEGG enrichment analysis revealed their heightened enrichment in cellular immunity and immune cell interaction pathways. Given the elevated expression levels of multiple immune checkpoint molecules, these clusters may substantially benefit from immune checkpoint inhibitor therapy. Additionally, chemotherapy sensitivity analysis indicated their favorable responses to first or second-line chemotherapy regimens. CONCLUSIONS: We subclustered ovarian cancer patients by 5 signature genes obtained from the Single-cell RNA sequencing (scRNA-seq) dataset, which demonstrated a good typing effect. Patients in the two molecular subtypes showed better survival, higher immune cell infiltration, and higher drug sensitivity. This meticulous typing may help clinicians to quickly assess the prognosis of patients and the response to immunotherapy and chemotherapy.

Relevance of CD20 antigen expression among paediatric patients with B-lineage acute lymphoblastic leukaemia.

Literature regarding prognostic relevance of CD20 antigen expression among paediatric B-lineage acute lymphoblastic leukaemia (B-ALL) patients is sparse and contradictory. We analysed clinical laboratory parameters and survival characteristics pertinent to CD20 expression among 224 treatment-naïve paediatric B-ALL patients. 50% patients had CD20 expression (CD20+ B-ALL). There was no difference in the clinical & laboratory presentation and end of induction measurable residual disease (EOI-MRD) status according to CD20 expression. As compared to CD20- B-ALL patients, CD20+ B-ALL patients had two times more relapse (16% vs. 29%, p = 0.034), inferior relapse-free survival (79% vs. 66%, p = 0.025) but no difference in overall survival (75% vs. 69%, p = 0.126). Similar to high-risk NCI status and EOI-MRD positivity, CD20 expression was an independent predictor for inferior relapse-free survival (HR: 1.860, 95% CI: 1.008-3.432, p = 0.047). Compared to baseline, there was a significant increase in CD20-expressing EOI-residual blasts among CD20- B-ALL patients (5% vs. 13%, p = 0.001). EOI residual blasts of both CD20+ and CD20- patients had three times increased normalized CD20 expression intensity (nCD20), with the intensity among CD20- B-ALL patients reaching the pretreatment nCD20 of CD20+ B-ALL patients (4.9 vs. 3.6, p = 0.666). Rituximab can be considered in managing EOI-MRD-positive CD20- B-ALL patients as the residual blasts of these patients have quantitative and qualitative increases in CD20 expression.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

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.

Neutropenia following immune-depletion, notably CD20 targeting, therapies in multiple sclerosis.

Neutropenia serves as a risk factor for severe infection and is a consequence of some immune-depleting immunotherapies. This occurs in people with multiple sclerosis following chemotherapy-conditioning in haematopoietic stem cell transplantation and potent B cell targeting agents. Whilst CD52 is expressed by neutrophils and may contribute to early-onset neutropenia following alemtuzumab treatment, deoxycytidine kinase and CD20 antigen required for activity of cladribine tablets, off-label rituximab, ocrelizumab, ofatumumab and ublituximab are not or only weakly expressed by neutrophils. Therefore, alternative explanations are needed for the rare occurrence of early and late-onset neutropenia following such treatments. This probably occurs due to alterations in the balance of granulopoiesis and neutrophil removal. Neutrophils are short-lived, and their removal may be influenced by drug-associated infections, the killing mechanisms of the therapies and amplified by immune dyscrasia due to influences on neutropoiesis following growth factor rerouting for B cell recovery and cytokine deficits following lymphocyte depletion. This highlights the small but evident neutropenia risks following sustained B cell depletion with some treatments.

A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system.

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli-based in vitro coupled transcription-translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

Obinutuzumab-Based Drug-Free Macromolecular Therapeutics Synergizes with Topoisomerase Inhibitors.

Drug-free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen-crosslinking-induced apoptosis in target cells. DFMT is a two-component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab-MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA-(MORF2)x ). The two components recognize each other via the Watson-Crick base pairing complementation of their respective MORFs. One HSA-(MORF2)x molecule can hybridize with multiple Ab-MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink-inducing mechanism of action. Herein, various anti-neoplastic agents in combination with the anti-CD20 Obinutuzumab (OBN)-based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou-Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN-based DFMT. It is determined that OBN-based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN-based DFMT with etoposide in vivo are demonstrated using a human xenograft non-Hodgkin's lymphoma mouse model.

Loss of CD20 expression as a mechanism of resistance to mosunetuzumab in relapsed/refractory B-cell lymphomas.

ABSTRACT: CD20 is an established therapeutic target in B-cell malignancies. The CD20 × CD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHLs). Because target antigen loss is a recognized mechanism of resistance, we evaluated CD20 expression relative to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsy specimens collected before treatment at predose, during treatment, or upon progression. Before treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, in 16 of 293 patients (5.5%) the proportion was <10%. Analyses of paired biopsy specimens from patients on treatment revealed that CD20 levels were maintained in 29 of 30 patients (97%) vs at progression, where CD20 loss was observed in 11 of 32 patients (34%). Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effects of CD20 variants identified in clinical samples on reduction of CD20 expression and missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands the knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to include CD20-targeting bispecific antibodies and elucidates mechanisms of reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also confirm the utility of readily available IHC staining for CD20 as a tool to inform clinical decisions. This trial was registered at www.ClinicalTrials.gov as #NCT02500407.

Systemic administration of anti-CD20 indirectly reduces B cells in the inflamed meninges in a chronic model of central nervous system autoimmunity.

Anti-CD20 B cell depleting therapies have demonstrated that B cells are important drivers of disease progress in Multiple Sclerosis, although the pathogenic mechanisms are not well understood. A population of B cells accumulates in the inflamed meninges in MS and also some chronic animal models of disease, typically adjacent to demyelinating lesions. The role of these meningeal B cells in disease is not known, nor is their susceptibility to anti-CD20 therapy. Here, we administered anti-CD20 to 2D2 IgHMOG spontaneous experimental autoimmune encephalomyelitis mice in the chronic phase of disease, after the establishment of meningeal B cell clusters. Compared to the circulation, lymph nodes, and spleen, B cell depletion from the meninges was delayed and not evident until 7d post-administration of anti-CD20. Further, we did not find evidence that anti-CD20 accessed meningeal B cells directly, but rather that depletion was indirect and the result of ongoing turnover of the meningeal population and elimination of the peripheral pool from which it is sustained.

Cross-Reactivity of Antibodies to Rituximab with Other Therapeutic Anti-CD20 Antibodies.

One reason for a lack of response to rituximab as well as infusion-related anaphylactic adverse events is the development of antidrug Abs to rituximab. Besides rituximab, a number of other therapeutic Abs targeting CD20 are nowadays available as alternatives. In this study, we investigated the potential cross-reactivity of (human) anti-rituximab Abs to three other anti-CD20 mAbs: ofatumumab, obinutuzumab, and ocrelizumab. In 25 cases of anti-rituximab Abs, cross-reactivity was examined using both direct binding assays and inhibition immunoassays. Although no cross-reactivity was observed to ofatumumab or obinutuzumab, 8 of 25 samples also showed reactivity toward ocrelizumab in at least one of the two assays. Furthermore, in three cases of anti-ocrelizumab Abs, cross-reactivity to rituximab was observed in an inhibition immunoassay, albeit not in a direct binding assay. Our results suggest that obinutuzumab or ofatumumab are safe anti-CD20 alternatives in case of the presence of anti-rituximab Abs. It is advisable to proceed cautiously if switching from rituximab to ocrelizumab (or vice versa) is considered in case these alternatives may not be available.

Ocrelizumab and ofatumumab, but not rituximab, trigger complement induction in vitro.

The clinical and adverse effects of the therapeutic monoclonal antibodies (mAb) ocrelizumab, ofatumumab and rituximab in multiple sclerosis (MS) are presently subject to extensive study. While the two former are approved for MS, the older and less costly rituximab is used off label, and adverse effect profiles are important in their evaluation. The three mAbs all induce B cell depletion, with complement-dependent cytotoxicity (CDC) as one of several mechanisms of action. Complement activation is also postulated to underlie adverse reactions related to infusion/injection. Such administration-related reactions are associated with all three mAbs, but comparisons have so far been indirect, resting on incidence reports from separate clinical trials. The objective of this study was to perform head-to-head comparison of complement activation by ofatumumab, ocrelizumab and rituximab. In vitro experiments were performed in whole blood from healthy donors. The complement-activating potential of the three mAbs was analyzed after 30 min of exposure to 0.3 mg/mL or 0.9 mg/mL of each drug, and compared with those of the well-known TNF inhibitory mAbs adalimumab and infliximab, the latter with recognized potential for infusion reactions. Ofatumumab, ocrelizumab, and infliximab, but not rituximab and adalimumab, triggered statistically significant complement activation measured as increased levels of terminal C5b-9 complement complex (TCC), a sensitive marker of such activation. While results demand careful interpretation, they provide an indication of distinct complement-inducing potential among anti-CD20 mAbs currently used to treat MS.