Elicit: Mechanism of Ocrelizumab in B-Cell Depletion

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Mechanism of Ocrelizumab in B-Cell Depletion

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May 5, 2026

How does ocrelizumab target CD20-positive B cells to drive B-cell depletion?

Ocrelizumab binds to CD20 on B cells and drives their depletion primarily through antibody-dependent cellular cytotoxicity mediated by monocytes via Fcγ receptor pathways, achieving rapid and sustained reduction of circulating B cells while also depleting CD20-positive T-cell subsets.

Abstract

Ocrelizumab is a humanized anti-CD20 monoclonal antibody that targets the same CD20 epitope as rituximab but achieves B-cell depletion primarily through antibody-dependent cellular cytotoxicity (ADCC) rather than complement-dependent cytotoxicity (CDC). The antibody was engineered with amino acid modifications to enhance binding to Fcγ receptor IIIa, resulting in two- to five-fold greater ADCC activity compared to rituximab. Monocytes serve as the dominant effector cells mediating B-cell depletion through FcγRI and FcγRIII-dependent pathways, achieving rapid onset within hours and near-complete depletion (>95%) of circulating B cells by week 2. However, depletion efficiency varies across anatomical compartments, with secondary lymphoid organs retaining resistant B-cell subpopulations and bone marrow showing the weakest reduction. Ocrelizumab also co-depletes CD20-positive T-cell subsets and novel dual-expressor lymphocytes expressing both T-cell and B-cell receptors.

B-cell recovery begins in bone marrow and spleen before appearing in blood, with median time to repletion of 72 weeks. The phenotype of reconstituting B cells varies by immunological context: in settings with ongoing antigen stimulation, recovery is characterized by expansion of differentiated, myelin-reactive B cells, whereas absence of stimulation favors naive B-cell reconstitution. Treatment induces secondary immunological changes including increased BAFF levels and decreased sTACI, which may enhance regulatory plasma cell development and contribute to therapeutic efficacy. The ADCC-predominant mechanism provides more sustained depletion than CDC-predominant antibodies while potentially offering better tolerability.

Methods

We analyzed 10 sources from an initial pool of 200, using 6 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Mechanistic Focus on CD20/B-cells, Specific Depletion Mechanisms, Study Type and Mechanistic Data, Mechanistic Content Present, CD20/B-cell Relevance, Study Rigor and Detail

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How does ocrelizumab target CD20-positive B cells to drive B-cell depletion?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract detailed information about how ocrelizumab targets CD20-positive B cells, including:

Extract all mechanisms by which ocrelizumab induces B-cell death after CD20 binding, including:

Extract detailed patterns of B-cell depletion caused by ocrelizumab, including:

Extract timing and kinetics of B-cell depletion and recovery with ocrelizumab, including:

Extract molecular and structural features of ocrelizumab that influence its mechanism of action, including:

Extract direct comparisons of ocrelizumab’s mechanism to other anti-CD20 antibodies, including:

Results

Characteristics of Included Studies

Study

Full text retrieved?

Study focus

Key features

J. Uchida et al., 2004

Yes

Mouse model of anti-CD20 therapy

Developed mouse anti-mouse CD20 antibody panel to study B-cell depletion mechanisms

B. Cree et al., 2025

Yes

Review of anti-CD20 antibody evolution

Comprehensive comparison of ocrelizumab, ofatumumab, ublituximab, and rituximab

Pacheco-Fernández et al., 2018

No

In vitro CDC comparison

Compared ofatumumab and ocrelizumab CDC activity using B-cell lines

Samantha Ho et al., 2023

Yes

B-cell regulating factors

Examined effects on BAFF-APRIL system over 2.5 years

Schneider-Hohendorf et al., 2025

No

B- and T-cell receptor repertoires

Analyzed adaptive immune changes in 35 MS patients on ocrelizumab

Alice Willison et al., 2025

Yes

Immune cell dynamics comparison

Compared ocrelizumab (n=34) and ofatumumab (n=25) with flow cytometry

A. Bar-Or et al., 2021

Yes

Clinical perspectives review

Detailed molecular and pharmacological attributes of four anti-CD20 antibodies

Darius Häusler et al., 2018

Yes

EAE model of B-cell recovery

Characterized B-cell depletion and reconstitution in murine MS model

S. A. Kornilov et al., 2024

No

Multi-omic characterization

Analyzed plasma proteome, metabolome, and lipidome changes in 14 RRMS patients

Prajita Paul et al., 2025

No

Dual-expressor lymphocytes

Identified novel TCR/IgM dual-expressor cells susceptible to anti-CD20 therapy

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Pageof

The studies employed diverse methodological approaches, including murine models, in vitro cell line assays, human longitudinal cohort studies, and comprehensive reviews. Full-text availability varied, with six studies providing complete manuscripts and four available only as abstracts. Study populations ranged from mouse models to human RRMS cohorts, with sample sizes for human studies ranging from 14 to 35 patients.

Mechanisms of CD20 Targeting and B-cell Depletion

CD20 Binding Characteristics

Ocrelizumab is a humanized anti-CD20 monoclonal antibody that binds to the same epitope on the large extracellular loop of CD20 as rituximab. The antibody is classified as a type I anti-CD20 agent capable of crosslinking CD20 tetramers. Enhanced binding to low-affinity variants of Fcγ receptor IIIa distinguishes ocrelizumab from rituximab, contributing to its enhanced effector function. The antibody was specifically engineered through amino acid modifications in its Fc region to increase antibody-dependent cellular cytotoxicity (ADCC) activity.

Cytotoxic Mechanisms

Ocrelizumab depletes B cells through multiple cytotoxic pathways, with ADCC as the primary mechanism. The antibody exhibits two- to five-fold greater ADCC activity compared to rituximab, while demonstrating three- to five-fold lower complement-dependent cytotoxicity (CDC) activity. Both ocrelizumab and ofatumumab can induce ADCC and CDC, though ofatumumab demonstrates stronger CDC effects, particularly at low CD20 expression levels. In vitro comparisons using human B-cell lines revealed that ocrelizumab induced less cell lysis through CDC compared to ofatumumab, with the strongest differentiation observed when complement was added 8 hours after antibody washing, showing the hierarchy: ofatumumab > rituximab > ocrelizumab.

The dominance of ADCC over CDC in ocrelizumab’s mechanism of action has important implications. Studies in mouse models demonstrated that B-cell depletion is completely dependent on effector cell Fc receptor expression, with monocytes serving as the dominant effector cells. The depletion pathway utilizes both FcγRI and FcγRIII-dependent mechanisms, while genetic deficiency in complement components (C3, C4, or C1q) did not prevent B-cell elimination, confirming the primacy of ADCC over CDC in vivo.

Patterns of B-cell Depletion

B-cell Subpopulations

Ocrelizumab targets CD20-positive B cells, resulting in significant reductions across multiple B-cell subsets including naive B cells, memory B cells, transitional B cells, and plasmablasts. Plasma cells, which lack CD20 expression, remain resistant to depletion, as do CD20-negative B-cell precursors and hematopoietic stem cells. An expansion of CD5+CD19+CD20− B cells was observed following treatment, indicating a population resistant to depletion. Circulating B-cell counts showed almost complete depletion by week 2, remaining depleted through week 96 with infusions every 24 weeks.

Non-B-cell Targets

Beyond conventional B cells, ocrelizumab co-depletes CD20-positive T-cell subsets. Significant reductions occurred in double-negative (CD3+CD4−CD8−) T cells, which express higher CD20 levels compared to CD4 or CD8 positive T cells. Depletion was evident in highly expanded T-cell receptor beta chain and Vδ2+ T-cell receptor delta chain clonotypes. The treatment also targets novel dual-expressor cells (DEs) that co-express T-cell receptors and surface IgM, with up to 95% of DEs in cerebrospinal fluid expressing CD20. These DEs were found at significantly higher frequencies in RRMS patients compared to healthy controls and showed robust responses to myelin autoantigens.

Anatomical Compartments

B-cell depletion occurs across multiple anatomical compartments but with varying efficiency. In blood, depletion is nearly complete, with more than 95% reduction in circulating B cells. However, B cells in lymphatic organs and the CNS are not depleted to the same extent as those in blood. Secondary lymphoid organs including the spleen and lymph nodes retain a subpopulation of CD20-positive B cells resistant to depletion, with splenic B cells showing more than 93% reduction. The bone marrow demonstrates the weakest reduction in B-cell numbers, with persistence of CD20-negative B-cell precursors and plasma cells. CSF B cells are substantially reduced following treatment, with tissue-resident memory cells in cerebrospinal fluid showing initial therapy resistance.

Kinetics of Depletion and Recovery

Depletion Timeline

The onset of B-cell depletion is rapid, occurring within 1 hour in mouse models, with CD19+ cell counts in humans almost completely depleted by week 2 after the first dose. Maximal depletion persists for extended periods, with B cells remaining extensively depleted through week 96 when infusions are administered every 24 weeks. In longitudinal human studies, persistent depletion of B cells and CD20+ T cells was observed for up to 2.5 years. Depletion at 6 months was associated with reduction in B-cell associated proteins.

Recovery Patterns

B-cell recovery demonstrates complex kinetics varying by anatomical compartment and B-cell phenotype. In mouse models, bone marrow and spleen show simultaneous recovery, with B cells reappearing at 6 weeks and fully restored by 9 weeks, substantially preceding reappearance in blood. Lymph nodes and blood recover more slowly, with full repletion not achieved until 12 weeks. In humans, B-cell levels increase to baseline or the lower limit of normal in 90% of patients within 2.5 years after the last infusion, with a median time to repletion of 72 weeks.

The phenotype of reconstituting B cells depends on disease context. In experimental autoimmune encephalomyelitis (EAE) models involving activated B cells (MOG protein 1-117), recovery was characterized by expansion of mature, differentiated cells containing high frequencies of myelin-reactive B cells with restricted B-cell receptor gene diversity. These cells served as efficient antigen-presenting cells for myelin-specific T-cell activation. In contrast, in purely T-cell-mediated EAE models (MOG peptide 35-55), reconstituting B cells exhibited a naive phenotype without efficient antigen-presenting capacity. Human studies showed a bimodal distribution six months after initial depletion: some patients had few B cells with high somatic hypermutation consistent with incomplete depletion of differentiated B cells, while others had higher numbers of less differentiated B cells indicating reconstitution from germline. Extended interval dosing was associated with higher percentages of naive, transitional, and regulatory B cells, suggesting these populations recover first.

Fast B-cell repopulation starting around 6 months was associated with higher MRI activity and worsening disability, indicating clinical relevance of recovery kinetics. Studies showed that 26% of patients had B-cell repletion at 6 months.

Comparative Analysis with Other Anti-CD20 Antibodies

Mechanistic Distinctions

Ocrelizumab exhibits distinct mechanistic properties compared to other anti-CD20 antibodies. While all anti-CD20 agents induce both ADCC and CDC, their relative reliance on these pathways differs substantially. Ocrelizumab and ublituximab primarily use ADCC for B-cell depletion, whereas rituximab and ofatumumab rely more heavily on CDC. Ofatumumab demonstrates greater CDC potency compared to ocrelizumab, which may explain its efficacy at lower doses. Ublituximab shows more pronounced ADCC effects than rituximab, ocrelizumab, and ofatumumab due to its glycoengineered Fc segment.

The enhanced ADCC activity of ocrelizumab, achieved through amino acid engineering to increase binding to Fcγ receptor IIIa, is two- to five-fold greater than rituximab, while its CDC activity is three- to five-fold lower. This mechanistic profile has important clinical implications, as ADCC-mediated depletion is associated with fewer infusion-related reactions compared to CDC-mediated pathways.

Depletion Efficiency and Clinical Impact

Ocrelizumab achieves more complete and sustained B-cell depletion compared to rituximab. The extent of B-cell depletion may affect clinical efficacy, with higher median B-cell levels potentially associated with higher rates of disability progression. Ocrelizumab shows slower B-cell repletion rates compared to ofatumumab, which may contribute to differences in dosing requirements. The antibody is administered intravenously at 300 mg initially, followed by 300 mg two weeks later, then 600 mg every 6 months, compared to ofatumumab’s low-dose subcutaneous regimen enabled by its stronger CDC activity.

Secondary Effects on Immune Regulation

BAFF-APRIL System Modulation

Ocrelizumab treatment induces profound changes in the B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) system. The treatment persistently enhances BAFF levels while reducing the endogenous soluble receptor and decoy sTACI in both serum and CSF. Levels of sTACI negatively correlate with BAFF levels, with reduction of sTACI associated with formation of sTACI-BAFF complexes. The pronounced increase in circulating BAFF following CD20 depletion represents a tentative compensatory mechanism that may contribute to reconstitution of targeted B cells.

Since sTACI is a decoy for APRIL, its reduction may enhance local APRIL activity, thereby promoting regulatory IgA+ plasma cells and astrocytic IL-10 production. This mechanism may contribute to the beneficial effects of anti-CD20 therapy, as exogenous sTACI (atacicept) worsened MS. The therapeutic effects of ocrelizumab extend beyond CD20-mediated B-cell lysis to implicate metabolic reprogramming.

Broader Immunological Changes

B-cell depletion is accompanied by decreases in B-cell receptor and cytokine signaling pathways. At 6 months, treatment reduces plasma abundance of cytokines and cytotoxic proteins, markers of neuronal damage, and biologically active lipids including ceramides and lysophospholipids. This is followed by upregulation of numerous signaling and metabolic pathways at 12 months.

Ocrelizumab treatment also elevates exhaustion markers (CTLA-4, PD-1, TIGIT, TIM-3) across T, B, NK, and NKT cells. Regulatory T-cell numbers increase, especially in ocrelizumab-treated patients. Digital cytometry identified a putative increase in myeloid cells and a pro-inflammatory subset of T cells. These immune profile changes correlate with clinical parameters, suggesting pathophysiological relevance in RRMS.

Synthesis

The mechanism by which ocrelizumab drives B-cell depletion involves multiple coordinated processes that vary by context, anatomical location, and B-cell phenotype. The apparent heterogeneity in depletion efficiency and recovery patterns across different compartments and studies can be reconciled through several key considerations.

Mechanistic Heterogeneity by Anatomical Context

The differential depletion efficiency across anatomical compartments reflects distinct microenvironmental factors rather than conflicting findings. Blood shows near-complete depletion (>95%), whereas secondary lymphoid organs retain resistant subpopulations and bone marrow shows the weakest reduction. This pattern is mechanistically consistent with the ADCC-dependent mechanism of ocrelizumab. Blood provides optimal access for effector monocytes, while follicular B cells in lymphoid tissues may be protected by anatomical barriers or local factors that limit monocyte access. The persistence of plasma cells across all compartments reflects their lack of CD20 expression rather than resistance to the antibody’s cytotoxic mechanism.

Recovery Phenotype Determined by Disease Activity

The divergent phenotypes of reconstituting B cells—ranging from highly mutated memory-like cells to naive populations—reflect the immunological context at recovery rather than inconsistent biology. In models where B cells are actively engaged in antigen presentation (MOG protein 1-117 EAE), recovery populations are enriched for mature, myelin-reactive cells with efficient APC capacity. In purely T-cell-mediated models (MOG peptide 35-55 EAE), naive phenotypes predominate. This suggests that ongoing antigenic stimulation during the recovery phase selectively expands differentiated B-cell clones that survive in lymphoid tissues, while absence of such stimulation favors reconstitution from naive precursors.

The bimodal distribution in human patients at 6 months—with some showing few highly mutated cells and others showing numerous less differentiated cells—likely represents these two extremes occurring within a single patient population. Patients with incomplete depletion of tissue-resident differentiated B cells will show the first pattern, while those with more complete depletion will demonstrate the second. The association of fast repopulation with worse clinical outcomes supports the pathogenic relevance of early-recovering differentiated populations.

ADCC vs CDC Balance and Clinical Optimization

The comparative studies showing stronger CDC activity for ofatumumab versus ocrelizumab do not indicate superior efficacy, but rather different optimization strategies. Ofatumumab’s potent CDC enables low-dose subcutaneous administration, while ocrelizumab’s enhanced ADCC (2-5 fold greater than rituximab) allows for less frequent dosing and potentially better tolerability, as ADCC generates fewer infusion-related reactions than CDC. Both mechanisms achieve effective B-cell depletion; the choice reflects different balancing of efficacy, safety, and convenience.

Compensatory Mechanisms and Long-term Effects

The increase in BAFF following B-cell depletion represents a predictable homeostatic response rather than a treatment failure. The simultaneous decrease in sTACI may actually enhance therapeutic benefit by allowing more free APRIL to promote regulatory plasma cells and IL-10 production. This explains the apparent paradox where exogenous sTACI (atacicept) worsened MS, despite its theoretical B-cell depleting effects. The therapeutic window for anti-CD20 therapy thus involves balancing B-cell depletion against preservation of regulatory mechanisms, with the BAFF-APRIL system serving as a key mediator of this balance.

References

J. Uchida, Y. Hamaguchi, J. Oliver, J. Ravetch, J. Poe, and 2 more\ (2004).The Innate Mononuclear Phagocyte Network Depletes B Lymphocytes through Fc Receptor–dependent Mechanisms during Anti-CD20 Antibody Immunotherapy. Journal of Experimental Medicine

B. Cree, Joseph R Berger, Benjamin Greenberg\ (2025).The Evolution of Anti-CD20 Treatment for Multiple Sclerosis: Optimization of Antibody Characteristics and Function. CNS Drugs

Thalia Pacheco-Fernández, Ismahane Touil, C. Perrot, G. Elain, D. Leppert, and 2 more\ (2018).Anti-CD20 Antibodies Ofatumumab and Ocrelizumab Have Distinct Effects on Human B-cell Survival (S52.003). Neurology

Samantha Ho, E. Oswald, Hoi Kiu Wong, A. Vural, V. Yılmaz, and 8 more\ (2023).Ocrelizumab Treatment Modulates B-Cell Regulating Factors in Multiple Sclerosis. Neurology: Neuroimmunology & Neuroinflammation

T. Schneider-Hohendorf, Christian Wünsch, A. Schulte-Mecklenbeck, Lisa Revie, Catarina Raposo, and 7 more\ (2025).B- and T cell receptor sequencing elucidates characteristics of lymphocyte depletion by ocrelizumab. iScience

Alice Willison, Ramona Hagler, M. Weise, S. Elben, Niklas Huntemann, and 16 more\ (2025).Effects of Anti-CD20 Antibody Therapy on Immune Cell Dynamics in Relapsing-Remitting Multiple Sclerosis. Cells

A. Bar-Or, S. M. O’Brien, M. Sweeney, E. Fox, Jeffrey A. Cohen\ (2021).Clinical Perspectives on the Molecular and Pharmacological Attributes of Anti-CD20 Therapies for Multiple Sclerosis. CNS Drugs

Darius Häusler, Silke Häusser-Kinzel, L. Feldmann, Sebastian Torke, G. Lepennetier, and 5 more\ (2018).Functional characterization of reappearing B cells after anti-CD20 treatment of CNS autoimmune disease. Proceedings of the National Academy of Sciences of the United States of America

S. A. Kornilov, Nathan D. Price, Richard Gelinas, Juan Acosta, M. Brunkow, and 10 more\ (2024).Multi-Omic characterization of the effects of Ocrelizumab in patients with relapsing-remitting multiple sclerosis. Journal of Neurological Sciences

Prajita Paul, Marjan Behzadirad, Rafid Al Hallaf, Susana C. Dominguez-Peñuela, Carlos A. Pardo, and 3 more\ (2025).Myelin-Reactive TCR/IgM Dual-Expresser Lymphocytes in Multiple Sclerosis: Linking Pathogenesis to Anti-CD20 Therapy. Immunological Investigations

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The Evolution of Anti-CD20 Treatment for Multiple Sclerosis: Optimization of Antibody Characteristics and Function

B. Cree, Joseph R Berger, Benjamin Greenberg

CNS Drugs·

2025·

6 citations

SourceDOI

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CD20 Targeting

- CD20 epitope specificity: Ocrelizumab binds the same epitope on the large loop of CD20 as rituximab. - Binding affinity: Not specifically mentioned in terms of Kd values. - Antibody structure features: Humanized mAb with enhanced ADCC activity. - Comparison to other anti-CD20 antibodies: Ocrelizumab has higher ADCC activity compared to rituximab. - Dose-response relationships: Ongoing clinical trials to investigate higher doses for efficacy.

Cytotoxic Mechanisms

- Complement-dependent cytotoxicity (CDC) activity: Occurs, but not the primary mechanism. - Antibody-dependent cellular cytotoxicity (ADCC) activity: Primary mechanism; two to five times higher than rituximab. - Direct apoptosis induction: Not specifically mentioned for ocrelizumab. - Other cytotoxic pathways: Antibody-dependent cell phagocytosis (ADCP) mentioned, but not specifically for ocrelizumab. - Quantitative comparisons of mechanism potency: Not provided. - Time course of cytotoxicity: Not provided. - Genetic or phenotypic factors affecting cytotoxic efficacy: FcγRIIIa polymorphisms; glycoengineering may overcome these differences.

Depletion Patterns

- Specific B-cell subpopulations targeted: CD19+CD20+ B cells, CD20 dim T cells - B-cell populations resistant to depletion: Not specifically mentioned - Anatomical compartments affected: Blood, bone marrow, spleen, lymph nodes, CNS/brain - Degree of depletion: Variable; significant reduction in CSF B cells - Co-depletion: CD20 dim T cells - Factors influencing depletion efficiency: Dose, patient characteristics (body weight), genetic polymorphisms (FcγR)

Depletion Kinetics

- Time to initial B-cell depletion: Not explicitly mentioned, but implied to be rapid. - Duration of maximal depletion: At least 6 months. - Recovery timeline for different B-cell populations: Fast repopulation starts around 6 months. - Recovery timeline for different anatomical compartments: CSF B cells are substantially reduced following treatment. - Sequence of B-cell reconstitution: Naïve, transitional, and regulatory B cells recover first. - Phenotype of reconstituting B-cells: Naïve, transitional, and regulatory B cells. - Differences in recovery kinetics compared to other anti-CD20 antibodies: Implied to be distinct due to humanized nature and glycoengineering.

Molecular Determinants

- Antibody type: Humanized - Fc region modifications: Aminoacid-engineered to increase ADCC - Dosing regimen: Every 6 months - Route of administration: Both IV and subcutaneous - Engineering: Designed to enhance ADCC

Comparative Mechanisms

- Ocrelizumab has enhanced ADCC activity, two to five times higher than rituximab, due to amino acid engineering. - Ocrelizumab and ublituximab primarily use ADCC for B-cell depletion, whereas rituximab and ofatumumab rely more on CDC. - The enhanced ADCC activity of ocrelizumab may contribute to its clinical efficacy and safety profile compared to other anti-CD20 agents. - The extent of B-cell depletion may affect clinical efficacy, with higher median B-cell levels potentially associated with higher rates of disability progression. - The route and dose of administration may impact hypogammaglobulinemia, a consideration for ocrelizumab and other anti-CD20 therapies.

B-cell depletion with CD20-targeted agents is commonly used for treatment of multiple sclerosis (MS), other autoimmune diseases, and certain hematologic malignancies. Initial apparent success with rituximab in MS and neuromyelitis optica spurred development of the anti-CD20 monoclonal antibody (mAb) therapies ocrelizumab, ofatumumab, and ublituximab as well as the anti-CD19 mAb inebilizumab. While each are effective at targeting and depleting B cells, structural differences translate into different mechanisms of action affecting maintenance of B-cell depletion and safety and tolerability. Although the anti-CD20 mAbs differ in degree of human versus mouse sequences as well as target CD20 epitope, these properties do not appear to substantially affect activity or tolerability. In contrast, an antibody-dependent cell-mediated cytotoxicity (ADCC) versus a complement-dependent cytotoxicity mechanism of action as well as subcutaneous versus intravenous administration may provide improved tolerability. Glycoengineering of the mAbs ublituximab and inebilizumab enhances ADCC and can overcome the reduced responses to mAb-mediated B-cell depletion associated with certain genetic polymorphisms. Other strategies for therapeutic targeting of CD20, including brain shuttle antibodies (e.g., RO7121932), bispecific antibodies, chimeric antigen receptor T-cell therapies, and antibody–drug conjugates, are in active clinical development and may be future treatment approaches in MS and other B-cell-mediated autoimmune diseases.

1Introduction

A sea change in multiple sclerosis (MS) therapy occurred with the introduction of anti-CD20 monoclonal antibodies (mAbs). Early work with rituximab suggested that depleting B cells might be highly efficacious in MS and other inflammatory diseases of the central nervous system (CNS) such as neuromyelitis optica (NMO) [1] [2] [3][4][5]. A phase II trial with rituximab in relapsing-remitting MS (RRMS) showed a surprisingly powerful impact on new lesion formation and relapses and laid the groundwork for the seminal clinical trials with ocrelizumab, a humanized anti-CD20 mAb that showed superiority over thrice-weekly understanding of mAb structure and function, the engineering of anti-CD20 therapeutics has evolved to achieve more complete and sustained B-cell depletion, tolerability, and facility of administration. The subsequent development of a subcutaneously administered formulation of ofatumumab for RMS provided the option for anti-CD20 treatment for patients who either had poor IV access, were unable to access local infusion centers, or preferred selfadministration [9]. The development of the glycoengineered ublituximab, with its 1-h, twice-yearly infusion, offered patients an even greater degree of tolerability compared with either rituximab or ocrelizumab [10,11]. The objective of this article is to review the differences in chimeric, humanized, and human mAbs; distinctions in antibody class; brain shuttle mechanisms; the impact of glycoengineering on receptor binding and complement activation; and the prospects of further development of B-cell target therapies using bispecific antibodies and chimeric antigen receptor (CAR) T cells. How molecular engineering of these products translates directly into patient care is emphasized. This narrative review with expert opinion was developed following a comprehensive literature search.

2CD20 Role and Expression in the Immune System

CD20 is a 33-37 kDa non-glycosylated protein that is expressed on the surface of normal and malignant B cells; it is also dimly expressed on a small subset of T cells [12][13][14]. It is unclear whether CD20-expressing T cells transcribe CD20 or whether these cells acquire CD20 protein by trogocytosis, an exchange of plasma membrane portions between cells, through contact with B cells [15]. The CD20 protein functions as a calcium channel and comprises four hydrophobic transmembrane domains, one intracellular domain, and two extracellular domains (large and small loops); both amino-and carboxy-termini reside within the cytosol (Fig. 1 ) [14,16]. On B cells, CD20 is physically coupled to major histocompatibility complex (MHC) class II, CD40, the B-cell receptor (BCR), and the C-terminal-Src-kinase-binding protein. CD20 contributes to B-cell activation and proliferation and may be required for efficient BCR signaling in B cells, optimal T-independent humoral immunity, and immune response to T-dependent antigens [14,17]. CD20 expression on B cells is more restricted than that of the B-cell-specific marker CD19: CD20 appears later in B-cell development, at the pre-B-cell stage, and is absent on plasmablasts and plasma cells (Fig. 2 ) [18]. In contrast, CD19 is expressed on the first B-cell lineage cell, at the pro-B-cell stage, and is present on all B-cell types except for long-lived plasma cells. CD20 is highly expressed in the plasma membrane at most B-cell stages, typically remains on the cell surface even when cross-linked with mAb, and is not shed from the surface [17,19]. These features make CD20 a unique candidate for mAb-mediated targeting of B cells without directly impacting stem cells or plasma cells.

3Anti-CD20 Mechanisms in MS

Anti-CD20 mAbs are designed to bind to and cause depletion of CD20 + cells [10]. The primary target of anti-CD20 mAbs are CD19 + CD20 + B cells [10,20]. Depletion of B cells via this mechanism is thought to reduce antigen presentation and activation of pathogenic T cells, arrest inflammatory cytokines produced by B cells, and create a shift away from an activated inflammatory immune environment to one with increased immune downregulation with less activated or less mature T and B cells [21][22][23].

A subset of T cells, both CD4 and CD8 T cells, also expresses low levels of CD20; these cells are often termed CD20 dim T cells and are depleted by anti-CD20 mAbs [10,22,24]. While CD20 dim T cells make up a small fraction of T cells in the blood, they are implicated in MS pathogenesis [24][25][26][27][28][29]. CD20 dim T cells have a proinflammatory phenotype and produce high levels of interferon-γ (IFN-γ), tumor necrosis factor (TNF)-ɑ, and granulocyte-macrophage colony-stimulating factor [21,24,28]. CD20 dim T cells express higher levels of adhesion molecules than CD20 -T cells, suggesting an increased potential for migration into the CNS [27,28]. CD20 dim T cells are found at increased frequency in people with MS versus in healthy controls [21,28]. Lastly, CD20 dim T cells are enriched in the cerebrospinal fluid (CSF) of people with MS [28,29], and in one study, the percentage of CD20 dim T cells in the CSF correlated with RMS disease severity [28].

Thus, treatment effects associated with anti-CD20 mAbs may result from the depletion of both B cells and CD3 + CD20 + T cells [10]. In contrast, anti-CD19 mAbs are B-cell specific because T cells do not express CD19 [30,31]. Both anti-CD20 and anti-CD19 mAbs can be designed to be non-depleting, binding antibodies that interfere with B-cell activation, proliferation, and differentiation [32].

Recent studies demonstrated that treatment with ocrelizumab also impacts CD20 -T cells. More specifically, B-cell depletion results in a decrease in memory CD8 + T cells and alters both phenotype and function of these lymphocytes [33,34]. A study in treatment-naïve people with MS demonstrated an indirect effect on the frequency of effector memory CD4 and CD8 T-cell populations that preferentially migrate into the CNS following treatment with anti-CD20 therapy [35]. Moreover, in addition to impacts on T-cell populations, depletion of B cells in people with primary progressive MS (PPMS) increases the number of monocytes expressing PD-L1 that may promote immune tolerance by suppressing inflammatory T-cell activity [36]. Other studies also implicate a role for B-cell depletion in amelioration of MS disease activity via effects on antigen-specific T-cell populations, including myelin-specific CD8 + T cells and Epstein-Barr virus (EBV)-specific T cells that are postulated to drive progression and play essential roles in pathogenesis [26,37,38]. Taken together, these studies highlight the broad immunomodulatory impact of B-cell-depleting therapies.

A perspective that may be important to understanding the impact of anti-CD20 mAbs beyond direct effects on B-cell function is that neither T cells nor B cells evolved separately from each other; rather, they evolved to function together as critical components of adaptive immunity. A more sophisticated understanding of how depletion of B cells perturbs the adaptive immune system could provide further insights into biomarkers relevant to MS pathogenesis and treatment efficacy. Further, given the complexity of immune cascades invoked by anti-CD20 and anti-CD19 mAbs, differences in mAb potency and predominant mechanisms of action (e.g.,

4.1Initial Anti-CD20 mAb Development and Indications

The first anti-CD20 mAb, rituximab, was approved in 1997 for the treatment of follicular lymphoma [39]. Since then, millions of people have been treated with anti-CD20 mAbs for oncologic and autoimmune indications, with long-term data (published and congress abstracts) demonstrating a favorable benefit/risk profile [7,[39][40][41][42][43].

4.2History of Anti-CD20 mAb use in MS and Neuromyelitis Optica Spectrum Disorder (NMOSD)

Rituximab was the original anti-CD20 mAb used in MS and NMO. The first case report of rituximab in RRMS and case series in NMO were in 2005 [2, 44]. In 2006, a small study of rituximab showed favorable outcomes in MS patients who experienced suboptimal responses to other disease-modifying therapies [3]. In 2008, a phase I study (N = 26) in active RRMS provided initial evidence of an apparent effect of rituximab on gadolinium-enhancing (Gd+) lesions on magnetic resonance imaging (MRI) and clinical relapses along with a reasonable safety profile despite a high rate of infusion-related reactions (IRRs) [1]. In the same year, the randomized, placebo-controlled, phase II HERMES study (N = 104) showed that RMS participants who received rituximab 1000 mg experienced reduced inflammatory lesions and relapses compared with placebo [4]. A high rate of IRRs (78%) occurred in this study. Rituximab was evaluated in PPMS in the phase II/ III OLYMPUS study (N = 439), a 96-week, randomized, controlled trial in which participants received either two 1000 mg doses of rituximab 2 weeks apart every 24 weeks or placebo. Although the overall study failed to meet its primary endpoint (time to confirmed disease progression), it suggested that efficacy might be apparent in younger (< 51 years of age) participants with Gd+ lesions on baseline MRI [45]. Although not approved by the US Food and Drug Administration (FDA) for MS, rituximab is widely used off-label to treat people with MS [42].

The first case series evaluating rituximab to treat NMO found an apparent reduction in attack rate and improvement in neurologic function comparing pretreatment with posttreatment epochs [2] . A subsequent case series of 25 participants refractory to other NMOSD empiric treatments replicated and expanded upon these observations [5].

Subsequently, rituximab became widely used as an off-label NMO treatment. Rituximab was approved for use in Japan in 2022 on the basis of the results of the RIN-1 study (N = 38), a 72-week, randomized, controlled trial in anti-aquaporin-4 antibody-positive (AQP4-Ab+) NMOSD participants who received rituximab (375 mg/m 2 ) every week for 4 weeks followed by 1000 mg every 6 months versus placebo. The study demonstrated a significant reduction in relapses as well as an improvement in quantification of optic nerve and spinal cord impairment scores [46,47]. Interestingly, both the HERMES and RIN-1 trials showed that rituximab impacts relapsing disease activity in RMS and NMOSD, respectively; in contrast, the OLYMPUS study failed to reduce confirmed disease progression in PPMS despite a larger cohort size, longer study duration, and substantially higher cumulative rituximab dose. These observations suggest that there are fundamental differences in therapeutic efficacy that depend on the underlying disease state rather than simple dosage effects.

4.3Evolution of Anti-CD20 mAb Use in MS and Anti-CD19 mAb Use in NMOSD

Anti-CD20 therapy evolved through an improved understanding of mAb characteristics and design. Currently, each of the four anti-CD20 therapies used in MS (rituximab, ocrelizumab, ofatumumab, and ublituximab) exhibits unique molecular characteristics (Table 1 ). The structural differences among the antibodies are responsible for different tolerability profiles and differences in the kinetics of B-cell depletion. Figure 3 illustrates the development timeline of additional anti-CD20 mAbs. How these different aspects contribute to mAb design, structure, function, and clinical applications and effects will be presented next.

5Scaffold: Chimeric Versus Humanized Versus Fully Human mAbs

Therapeutic mAbs can be chimeric, humanized, or fully human. Chimeric antibodies are nonhuman (often murine) mAbs engineered to have their constant regions replaced by human sequences; thus, most of the fragment antigen-binding (Fab) region is mouse-derived, while the fragment crystallizable (Fc) is human [48]. In humanized mAbs, all sequences are human except the complementarity determining regions (CDR) of the variable domains. Murine-sequence-derived CDRs are engrafted onto human-sequence-derived variable regions, while the remaining Fab and Fc components are fully human. Fully human mAbs contain exclusively human sequences and are generated in mice with humanized immune systems [48,49]. Ofatumumab is a fully human antibody produced by immunizing HCo7 and KM mice with a murine cell line (NS0) transfected with human CD20 [50,51]. Rituximab and ublituximab are chimeric mAbs, and ocrelizumab is a humanized mAb [52,53].

The rationale for increasing the proportion of human sequences in mAbs is to reduce immunogenicity, hypersensitivity reactions, and antidrug antibodies (ADAs) that have the potential to reduce efficacy [48]. ADAs can either be non-neutralizing, binding the drug but do not affect the drug-target interaction, or neutralizing, inhibiting the biological activity of a drug [54]. While humanization of anti-CD20 mAbs reduces immunogenicity, a clear effect on tolerability/IRRs was not observed (discussed further below).

Levels of ADAs are higher with rituximab and ublituximab, both chimeric mAbs, than with other anti-CD20 mAbs, [10,163] as noted in published reports and congress abstracts [55,56]. However, development of ADAs does not impact safety or efficacy outcomes. In a large observational study, there was no difference in efficacy, incidence, or severity of IRRs or adverse events (AEs) with rituximab between ADA groups (positive versus negative) [56]. Furthermore, there was no association between ADA titers and IRRs or AEs. In a congress presentation of data from the ULTIMATE studies, treatment-emergent ADAs with ublituximab were observed in most participants but were generally transient and had no observable impact on B-cell depletion, annualized relapse rate (ARR), the number of new/enlarging T2 lesions, or tolerability [55].

6Fc Engineering

Anti-CD20 mAb activity is determined by both the specific antigen binding of the Fab region and the effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), activated by the Fc region [57]. The Fc region of immunoglobulin G1 (IgG1) antibodies expresses binding sites for Fc receptors, including Fc gamma receptor IIIa (FcγRIIIa) on immune cells, and for the C1q component of complement.

Fc engineering can involve either glycoengineering or amino acid engineering to increase the strength of binding to Fc gamma receptors (FcγRs). Reduced fucosylation with glycoengineering enhances affinity to FcγRIIIa in particular [58] and can enhance natural killer (NK) cell effector functions [57,[59][60][61]. This is because the core fucose of Fc-linked oligosaccharides inhibits the ADCC of anti-CD20 mAbs by sterically blocking interaction with the FcγRIIIa receptor, thereby reducing affinity [62,63]. Exclusion of fucose from the Fc region allows for closer interaction and increased affinity with FcγRIIIa receptors without altering antigen binding or complement-dependent cytotoxicity (CDC) [57,62,63]. As such, mAbs with low fucose content exhibit significantly higher ADCC activity than those with high fucose content (Fig. 4 ) [57,[59][60][61].

Clinical advantages of glycoengineering can only be observed in head-to-head studies, and while no head-tohead studies have been conducted in MS to date, studies in oncology have evaluated obinutuzumab, a glycoengineered anti-CD20, versus rituximab [64,65]. In a phase III study, obinutuzumab in combination with venetoclax (a blocker of the anti-apoptotic B-cell lymphoma protein) demonstrated improved progression-free survival (PFS) and more robust disease eradication versus rituximab in combination with venetoclax in chronic lymphocytic leukemia [64,66]. The proportion of participants in the trial achieving undetectable residual disease (the most sensitive measure of residual B cells) was greatly improved with obinutuzumab versus rituximab. Similarly, in a phase III trial in advanced CD20 + follicular lymphoma, first-line obinutuzumab-based chemotherapy with obinutuzumab maintenance therapy provided significantly longer PFS than rituximab-based chemotherapy with rituximab maintenance therapy [67].

Glycoengineering of anti-CD19 mAbs showed similar improvements in effector function. MDX-1342, an afucosylated anti-CD19 mAb, was compared with its fucosylated parental mAb and showed higher ADCC and more potent B-cell depletion in preclinical studies [68]. Inebilizumab is an afucosylated anti-CD19 IgG1 mAb approved for the treatment of AQP4-Ab+ NMOSD [69]. Similar to MDX-1342, when compared with its fucosylated parental mAb, inebilizumab had higher affinity to FcγRIIIa and stronger ADCC. When compared with rituximab, in vitro and in vivo B-cell depletion was higher with inebilizumab [70,71]. Inebilizumab was evaluated in a phase I study in people with RMS and showed rapid and robust B-cell depletion and a trend toward a reduction in MRI lesions [30]. Inebilizumab is indicated for treatment of AQP4-Ab+ NMOSD and IgG4-mediated disease and is under investigation in generalized myasthenia gravis [69,72]. Neither obinutuzumab nor inebilizumab are in development for MS.

In MS, ublituximab was glycoengineered to have a low fucose content in the Fc region [52,73], resulting in greater in vitro ADCC activity compared with rituximab, ocrelizumab, and ofatumumab, as reported in a congress presentation [74]. Ocrelizumab was aminoacid-engineered to increase its ADCC, which is two to five times higher relative to rituximab [75].

7Pharmacogenetics: FcγRIIIa Polymorphisms

Humans have six different FcγRs that vary in their affinities for IgG Fc as well as their signaling activities. The expression of different FcγRs by immune cells, in addition to the genomic complexity of the FcγR family, allows for fine tuning of immune responses [76]. Genetic diversity in FcγRs is in part due to single-nucleotide polymorphisms that can have pharmacogenetic effects influencing clinical outcomes. Polymorphisms of FcγRIIIa on NK cells modulate the strength of interaction with the lower hinge region of IgG1 and therefore determine the strength of IgG1 binding (Fig. 5A ) [77][78][79]. The FcγRIIIa 158F polymorphism, expressed in approximately 40% of healthy individuals, has weaker IgG binding than the FcγRIIIa 158V polymorphism [80]. NK cells with 158F have less effective ADCC compared with NK cells with the 158V variant [77].

FcγRIIIa polymorphisms are associated with variable clinical response to rituximab. In a study of people with NMOSD (N = 100), the 158F variant was associated with a greater risk of insufficient memory B-cell depletion and relapse during rituximab treatment [81]. In a retrospective study of people with rheumatoid arthritis (N = 212), there was a significantly higher rate of clinical response to rituximab in participants with the 158V polymorphism (89%) compared with the 158F polymorphism (64%) [82]. In a systemic lupus erythematosus (SLE) study (N = 262), participants homozygous for 158V had a statistically significant 2.5-fold improvement in odds of a clinical response with rituximab compared with those with 158F [83]. In the oncology setting, the 158V polymorphism was associated with higher response rates to rituximab in people with lymphoma across multiple trials [84]. In people with MS, a presentation at a recent congress showed that, among 45 people treated with ocrelizumab, there was no significant correlation between FcγRIIIa polymorphisms and disease activity over 18 months. However, all nine participants who experienced early B-cell repopulation carried at least one F allele at position 158, further supporting a link between this genetic polymorphism and ADCC activity [85].

Glycoengineering may overcome the differential effects of FcγRIIIa polymorphisms. In contrast to what was observed in studies with rituximab, the activity of the glycoengineered anti-CD19 mAb inebilizumab was not impacted by FcγRIIIa polymorphisms in a study of people with NMOSD. B-cell depletion was similar in participants with either FcγRIIIa 158F or 158V variants, and there was no significant difference in the risk of relapse, ARR, or worsening Expanded Disability Status Scale score between the groups [86]. Ublituximab, which is similarly glycoengineered, has enhanced affinity for all variants of the FcγRIIIa receptor, with the highest binding and relative affinity for FcγRIIIa 158V and FcγRIIIa 158F variant receptors compared with other, non-glycoengineered anti-CD20 mAbs (reported in congress presentation) (Fig. 5B ) [52,73,74].

8Type I versus Type II Antibodies

The difference between type I and type II antibodies pertains to their relative abilities to translocate antibody-bound CD20 into lipid rafts [53]. Lipid rafts are lipid-protein microdomains that are important for signal transduction via colocalization of receptors and effector molecules [87]. Type I antibodies promote CD20 translocation to lipid rafts [53]. Rituximab, ocrelizumab, ofatumumab, and ublituximab are all type I antibodies [52,53]. In contrast, type II antibodies do not aggregate CD20 in lipid rafts and include obinutuzumab and tositumomab, both used to treat hematologic malignancies [53].

Clustering of CD20 with type I antibodies stabilizes CD20 on lipid rafts, leading to stronger C1q binding and higher CDC, with limited induction of programmed cell death (PCD) [53,88]. Conversely, because type II antibodies do not stabilize CD20 in lipid rafts, their ability to activate complement is reduced. However, type II antibodies can induce PCD independent of complement activation. Neurotherapeutics, volume 17. pp. 1768-1784. Copyright 2020, the American Society for Experimental NeuroTherapeutics, Inc. [79]. B Ublituximab, due to its glycoengineering, has enhanced affinity for all variants of the FcγRIIIa receptor, with the highest binding and relative affinity for FcγRIIIa 158V and FcγRIIIa 158F variant receptors compared with other, non-glycoengineered anti-CD20 mAbs [74]. ADCC antibody-dependent cell-mediated cytotoxicity, CD cluster of differentiation, Fc fragment crystallizable, FCGR Fc gamma receptor gene, FcγR Fc gamma receptor, IgG immunoglobulin G, K a association constant, K D equilibrium dissociation constant, mAb monoclonal antibody, NK natural killer, SNP single-nucleotide polymorphism Both type I and type II antibodies induce ADCC (Fig. 6 ) [87,88]. Type I antibodies are capable of binding substantially more CD20 receptors than type II antibodies, which may increase CD20 internalization and cause CD20 degradation and downregulation of CD20 expression [87]. All therapeutic anti-CD20 mAbs thus far used in MS are type I [52,53].

9CD20-Antigen Binding

Anti-CD20 mAbs also bind different CD20 epitopes and differ in CD20-binding affinity [53]. Rituximab and ocrelizumab bind the same epitope on the large loop of CD20, ublituximab binds a different epitope (with some overlap with rituximab/ocrelizumab) on the large loop, and ofatumumab binds portions of the small loop and a distinct epitope on the large loop (Fig. 1 ) [20]. mAb epitopebinding location and angle influences cell depletion [19]. Binding of the small loop (ofatumumab only) is associated with higher CDC due to oblique positioning of the antibody, which encourages IgG-IgG interaction (facilitating C1q recruitment), increases Fc proximity to cell membrane, and promotes CD20 translocation into lipid rafts. Fig. 6 Type I and type II mAb-mediated cell lysis. Type I mAbs are able to engage CDC and ADCC but do not elicit efficient direct cell death, whereas type II mAbs can mediate direct cell death through a lysosomal pathway and can engage ADCC but not CDC. Reprinted from Beers SA, Chan CHT, French RR, Cragg MS, Glennie MJ. CD20 as a target for therapeutic type I and II monoclonal antibodies.

Seminars in Hematology, volume 47, issue 2. pp. 107-114. Copyright 2010, with permission from Elsevier [164]. ADCC antibody-dependent cell-mediated cytotoxicity, C1q complement component 1q, CD cluster of differentiation, CDC complement-dependent cytotoxicity, Fc fragment crystallizable, mAb monoclonal antibody, PCD programmed cell death

10Mechanisms of Cell Death

Anti-CD20 mAbs induce target cell lysis through different mechanisms: CDC, ADCC, antibody-dependent cell phagocytosis (ADCP), and PCD/direct apoptosis [88]. CDC and ADCC are thought to be the most common mechanisms for anti-CD20 mAb-mediated cell lysis [20,89]. CDC involves antibody-mediated activation of the complement pathway and occurs when IgG antibodies coat a target cell [20]. The Fc region of the anti-CD20 mAb is bound by C1q of the complement system, resulting in the formation of the membrane attack complex and subsequent lysis of the target cell [20,88]. ADCC is independent of the complement system and is largely mediated by interactions between the Fc region of the anti-CD20 mAb and FcγRIIIa, an IgG-binding FcγR, on NK cells [59,90]. This interaction initiates a series of signaling pathways in NK cells, including release of cytolytic compounds such as granzyme B and perforin, leading to B-cell lysis [53,87,88,91]. In ADCP, FcγRs on macrophages bind to anti-CD20 mAbs that are attached to target B cells, leading to antibody-dependent cellular phagocytosis of the B cells by the macrophages; FcγRIIa is thought to be the main FcR involved in ADCP by macrophages [88]. Lastly, type II antibodies can induce direct cell death (i.e., PCD) upon binding to CD20 in an Fc-independent manner [53,88].

Of interest, the relative contribution of CDC and ADCC to mediating B-cell lysis varies depending on the specific anti-CD20 mAb [10]. For rituximab and ofatumumab, CDC predominates over ADCC, whereas for ocrelizumab and ublituximab, ADCC is the primary mechanism, although CDC also occurs [10,20]. Inebilizumab does not fix complement and depletes B cells exclusively by ADCC and ADCP. In preclinical studies, ublituximab had the strongest ADCC of all four anti-CD20 mAbs, resulting from its glycoengineered Fc region, as discussed above [10,74].

11Antibody Structure and Infusion-Related Reactions

The mechanistic cause of infusion reactions explains why differences in antibody scaffold (i.e., level of "humanness") do not appear to have a clinically meaningful effect on IRRs with anti-CD20 mAbs. Infusion reactions are most correlated with cytokine release events and/or complement activation. Cytokine release is the most common cause of IRRs with mAbs, including with anti-CD20 mAbs, and this is distinct from allergic reactions mediated by IgE (hypersensitivity reactions) [92]. During mAb-mediated cell lysis, both the target cells and the immune effector cells release cytokines into the circulation [92,93]. Symptoms of anti-CD20 mAb cytokine release syndrome (CRS) are generally mild to moderate in severity, usually occur within the first several hours postinfusion (most often with the first infusion) [92], and tend to decrease with subsequent doses due to the reduced number of target cells, which is the pattern of IRRs observed with anti-CD20 mAbs in MS [6,94]. Cytokine release reactions may be managed by infusion interruptions, use of histamine blockers with or without corticosteroids, and slowed infusion rate [92].

In oncology, an increase in inflammatory cytokines, including interleukin (IL)-6, TNF-ɑ, IL-8, and IFN-γ, follows anti-CD20 mAb infusion [95][96][97][98]. Levels of inflammatory cytokines positively correlate with the amount of circulating malignant B cells [97,98] as well as with the prevalence and severity of IRR symptoms [96].

Some mAbs, however, can directly activate complement, releasing complement 3a (C3a), 5a (C5a), and 5b-9 (C5b-9), leading to production of vasoactive mediators by mast cells, basophils, and other immune cell types, a phenomenon termed "complement activation-related pseudoallergy" (CARPA) [93]. Rituximab causes both CARPA and CRS [93,99]. Similar to cytokine-release-mediated IRRs, CARPA occurs most frequently at the first infusion and can be reduced with pre-medications and slower infusion rates [93]. IV-infused anti-C20 mAbs used in MS have recommended pre-medications to reduce IRRs [10,11,20,100]. In contrast, pre-medications are not needed for subcutaneous ofatumumab, presumably related to the route of administration and lower dose, because IRR rates with IV ofatumumab were higher than those seen with other IV anti-CD20 mAbs [101][102][103].

Due to the predominant mechanism of cytokine-releasemediated IRRs, the mAb scaffold (chimeric, humanized, or fully human) appears to not have a substantial effect on infusion/injection reactions in clinical practice.

In contrast, the predominance of an ADCC-versus CDCmediated mechanism may have clinical relevance because CDC has a potential role in IRRs [20,104,105]. Activation of complement generates C3a and C5a, which are potent inflammatory mediators and anaphylatoxins that can affect vasodilation; stimulate macrophages, neutrophils, and eosinophils; cause histamine release; and activate antigenpresenting cells to produce IL-12 and IL-13 [106]. Clinical data from people with lymphoma treated with rituximab support an association between CDC and IRRs: following the first rituximab dose, there was a correlation between the amount of CDC activation (C3b/c levels) and IRR severity [105]. Supporting the notion that IRR frequency may be complement-mediated is the observation that the afucosylated anti-CD19 mAb, inebilizumab, which does not activate complement, is not associated with a higher rate of IRRs compared with placebo-although, as with MS, premedications are used with inebilizumab infusions [31].

12Rationale for Improving Anti-CD20 mAbs for MS

Anti-CD20 mAb therapy revolutionized the treatment of MS; however, opportunities to improve treatment outcomes with anti-CD20 mAbs remain.

12.1B-Cell Depletion/Repletion

In some people treated with anti-CD20 mAbs, B-cell depletion is incomplete, and repletion between infusions occurs. Nearly all data are from blood tests, representing levels of circulating B cells. In an observational study, 26% of people with MS treated with ocrelizumab (N = 155) had B-cell repletion at 6 months, and fast repopulation (B-cell repletion at 6 months) was associated with significantly higher MRI activity at 12 months [107]. In the OPERA I and II and ORATORIO studies, population pharmacokinetic analyses found that body weight impacted ocrelizumab exposure and B-cell depletion, and that > 30% of participants in the lowest exposure quartile had incomplete B-cell depletion at week 96 [108]. Additionally, higher B-cell counts pretreatment and greater body mass index (BMI) appear to contribute to early reconstitution of B cells [107,109]. These data are in accordance with results from the OPERA and ORATORIO studies. Beyond clinical factors inf luencing early repopulation, the kinetics of B-cell repletion in different immune compartments is not well understood. A study using the experimental autoimmune encephalomyelitis (EAE) model investigated B-cell repopulation following ocrelizumab treatment and demonstrated reconstitution of B cells in the spleen and bone marrow prior to their appearance in the blood. The data suggest that certain subpopulations of B cells might differ in their response to ocrelizumab treatment and contribute to the early repopulation reported in some patients [110].

The extent of B-cell depletion may affect clinical efficacy. A post hoc analysis of OPERA I and II and ORATORIO revealed a nonsignificant trend toward an association of higher median B-cell levels with higher rates of 24-week confirmed disability progression during the 96-week double-blind period and open-label extension [111]. An observational study of 108 ocrelizumab-treated people with MS corroborated data from the clinical trials [109]. Fast B-cell repopulation was associated with higher BMI, and people with fast B-cell repopulation had numerically lower rates of no evidence of disease activity-3 and no evidence of progression or active disease and experienced worsening disability. However, these observations do not exclude the possibility that higher BMI might be associated with higher disease activity independently from reduced B-cell depletion. Clinical trials investigating the pharmacodynamics, efficacy, and safety of higher doses of ocrelizumab are underway in RMS (NCT04544436) and PPMS (NCT04548999).

Studies extending dosing intervals with rituximab or ocrelizumab did not find an association between B-cell levels and clinical activity [112][113][114][115]. In one study that evaluated B-cell repopulation following extended interval dosing, no associations with inflammatory activity were found, and higher percentages of naïve, transitional, and regulatory B cells were associated with extended interval dosing versus standard interval dosing [116]. The WINDOCRE trial (NCT05999604) will investigate noninferiority of yearly dosing of ocrelizumab versus standard 6-month dosing in people with MS who have been stable on therapy for 2 years. This study will provide insight into whether extended dosing intervals for anti-CD20 therapies can result in increased safety profiles without compromising efficacy. Whether extended dosing and corresponding repopulation of B cells translates into decreased efficacy long term is not known.

The extent of B-cell depletion in peripheral blood does not necessarily reflect the extent of B-cell depletion in lymphoid tissues. Only 2% of lymphocytes circulate in peripheral blood, whereas lymphoid tissues house the remainder of the population [117]. In people with MS, B cells are also found in tertiary lymphoid structures (TLS) within the meninges and perivascular spaces of the CNS [118]. It is presumed that TLS are the intrathecal source of antibodies detected as oligoclonal bands and IgG index elevation in CSF. Further, it is hypothesized that these CNSresident B cells are directly involved in MS pathogenesis because cortical demyelination is topographically associated with meningeal TLS [119].

The extent of B-cell depletion in tissues by anti-CD20 mAb treatment was studied in people treated with rituximab across different disease states [120][121][122]. These studies found that B cells are depleted to a variable extent in spleen, lymph nodes, and bone marrow, with a greater depletion in spleen and bone marrow than in lymph nodes.

Studies of B-cell depletion in the CSF of people with MS treated with anti-CD20 mAbs are limited but generally show that CSF B cells are substantially reduced following anti-CD20 mAb treatment [3,123]. A more recent study evaluated the effects of ocrelizumab on CSF lymphocytes in people with PPMS and reported a significant reduction of total B cells as well as CD4 + CD20 dim CD45RA -memory T cells. Interestingly, CD8 + CD20 dim T cells were not substantially depleted in the CSF [124].

In a small observational study of people with progressive MS and leptomeningeal-enhancing (LME) lesions, intrathecal rituximab resulted in a reduction of CSF B-cell counts at 2 weeks after the first dose, with recovery at 8 weeks despite a second dose at week 2 [125]. There were no changes in the number or appearance of LME lesions or development of new LME lesions. A small study of people with MS and LME lesions initiating ocrelizumab reported that there was no significant reduction in the number or volume of LME lesions after approximately 1 year of treatment [126].

Evidence for B-cell depletion in the CNS with anti-CD20 mAb treatment also comes from preclinical studies. One study using a transgenic human CD20 EAE model showed no effect on B-cell depletion in the spinal cord with rituximab treatment [127]. Another study using a transgenic human CD20 EAE model found that rituximab depleted only dense perivascular B-cell infiltrates but not parenchymal B-cell infiltrates [128]. In contrast, obinutuzumab, a glycoengineered type II mAb, depleted both perivascular and intraparenchymal B-cell infiltrates [128,129]. Compared with rituximab, obinutuzumab has stronger in vitro ADCC activity and can also trigger PCD; therefore, these preclinical data suggest that anti-CD20 mAbs with non-CDC mechanisms of B-cell lysis might have higher CNS activity. However, findings about depletion in CNS appear to be model dependent; results using a different, humanized CD20 mouse model of secondary progressive MS showed similar reductions in CNS B cells when administering either rituximab or obinutuzumab [130].

Data on whether anti-CD20 mAb route of administration (IV versus subcutaneous) affects B-cell depletion in secondary lymphoid organs come from animal studies. In cynomolgus monkeys, there was slightly higher depletion of B cells in distant (non-draining) lymph nodes with subcutaneous versus IV rituximab treatment (57% versus 42% depletion of B cells, respectively), although numbers were small [131]. In a larger imaging study of mice expressing human CD20, improved lymph node targeting was observed with subcutaneous administration of ofatumumab or ocrelizumab versus IV administration; however, similar depletion of B cells in spleen and lymph nodes occurred regardless of antibody or route of administration [132].

12.2Administration Experience

Administration of the different anti-CD20 mAbs can affect people's treatment experiences, and the anti-CD20 mAbs used in MS have different administration protocols. Ofatumumab is self-administered monthly by subcutaneous injection [9]. A formulation of ocrelizumab administered every 6 months by subcutaneous infiltration over 10 min by a healthcare professional recently became available [133,134].

IV administration of ocrelizumab and ublituximab is every 6 months and every 24 weeks, respectively, following the initial dose(s) [11,100], with a similar cadence typically used for rituximab in MS [56]. In clinical trials of rituximab, the minimum infusion time was 4.5 h for the first dose and 3 h for subsequent doses [4,41]. The ocrelizumab IV infusion rate is ≥ 2.5 h for the initial dose and ≥ 2 h for subsequent doses [100]. The ublituximab IV infusion rate is 4 h for the initial dose on day 1 and 1 h for subsequent doses [11]. Postinfusion, an observation period of ≥ 1 h is required for ocrelizumab [100], whereas for ublituximab, a 1-h postinfusion observation period is only required for people who experienced an IRR during the first two infusions [11]. Therefore, modifications in anti-CD20 mAb structure and formulation may impact patient experience and convenience.

The route and dose of administration might also impact hypogammaglobulinemia. Initial data from the ASCLEPIOS (and open-label extension) studies demonstrated immunoglobulin levels above the lower limit of normal [135]. The decreased rates of hypogammaglobulinemia in people treated with ofatumumab was attributed to the idea that subcutaneous administration of a lower, more frequent dose of antibody would result in a more specific (i.e., targeted to the lymph node) and readily reversible mechanism of B-cell depletion. However, a subsequent study presented at a recent conference showed equal rates of hypogammaglobulinemia in people treated with ocrelizumab versus ofatumumab (9.9% and 8.9%, respectively) [136]. Further comparative studies are needed to determine whether ofatumumab reduces the risk of hypogammaglobulinemia compared with other anti-CD20 mAbs.

13Continuing Evolution of Strategies to Target CD20

Brain shuttle antibodies, bispecific antibodies, CAR T-cell (CAR T) therapies, and antibody-drug conjugates are also being designed and evaluated for therapeutic targeting of B cells.

13.1Brain Shuttle Antibodies

Transferrin is transported across the blood-brain barrier using transferrin receptors [137]. This receptor system can be leveraged to move mAbs across the blood-brain barrier by coupling mAbs to transferrin. A newer anti-CD20 mAb, RO7121932, uses brain shuttle technology to increase brain and CNS penetrance and is in development for MS (NCT05704361) [138].

Although the specific characteristics of the mAb are not yet detailed, it is presumed that this molecule is based on the type II antibody obinutuzumab, which uses PCD rather than requiring complement activation to affect B-cell lysis.

13.2Bispecific Antibodies

Bispecific antibodies co-targeting CD3 and B-cell antigens activate and engage T cells against CD19-or CD20-expressing cells; cytotoxicity occurs in an MHC-independent fashion [139]. As an example, a CD20 × CD3 bispecific antibody binds to CD20 expressed on the surface of B cells and to the CD3 receptor expressed on the surface of T cells, causing T-cell activation and proliferation, secretion of cytokines, and B-cell lysis [140]. Bispecific antibodies can be engineered in multiple ways: CD20 × CD3 bispecific antibodies can have one or more CD20-binding Fab arms, varying arrangement of CD20-and CD3-binding sites, or bind different CD20 epitopes (Table 2 ) [139].

Bispecific antibodies are divided into two major classes [139,141]: (1) antibodies containing an Fc region, conferring an Ig-like structure, and (2) antibodies that lack an Fc region, an example of which are the bispecific T-cell engagers (BiTEs), which contain only the variable regions of antibodies in the form of antigen-binding fragments connected by linker peptides.

The first bispecific antibody to be approved by the FDA was blinatumomab, a CD19 × CD3 BiTE comprising two single-chain antibody fragments, one targeting CD19 and one targeting CD3, connected by a linker [139,142]. Blinatumomab is approved for CD19 + acute lymphoblastic leukemia [142] and was used off-label in autoimmune diseases with some success. Compassionate use of blinatumomab in people with refractory rheumatoid arthritis reduced disease activity, improved synovitis, reduced Reprinted from Falchi L, Vardhana SA, Salles GA. Bispecific antibodies for the treatment of B-cell lymphoma: promises, unknowns, and opportunities. Blood, volume 141, issue 5. pp. 467-480. Copyright 2023, with permission from the American Society of Hematology [139] C1q complement component 1q, CD cluster of differentiation, Fab fragment antigen-binding, Fc fragment crystallizable, FcγR Fc gamma receptor, FcR Fc receptor, Ig immunoglobulin, scFv single-chain variable fragment * These Fc-silencing mutations do not abolish the binding of bispecific antibodies to neonatal FcR autoantibodies, and depleted activated memory B cells [143]. Currently, there are three CD3 × CD20 bispecific antibodies that received accelerated approval in the USA: glofitamab and epcoritamab for relapsed or refractory diffuse large B-cell lymphoma and mosunetuzumab for relapsed or refractory follicular lymphoma [140,144,145]. These target the same or overlapping CD20 epitopes as approved anti-CD20 mAbs [139] (Table 2 ). There are also bispecific antibodies targeted against B-cell activating factor or inflammatory cytokines in clinical development for autoimmune and/or inflammatory conditions [146,147].

13.3CAR T-Cell Therapies

CD19-directed CAR T therapies showed impressive success in treating B-cell malignancies [148,149] and offer the potential for a single-dose treatment with long-term efficacy [150]. Sustained beneficial effects of CD19 CAR T cells were observed in people with SLE, idiopathic inflammatory myositis, and systemic sclerosis [151]. KYV-101 is a CD19 CAR T therapy designed to improve tolerability over earlier CD19 CAR Ts that is being evaluated in people with MS and myasthenia gravis (studies NCT06451159, NCT06384976, NCT06138132, and NCT06193889). In an initial study, an acceptable safety profile was observed in two participants with progressive MS who received KYV-101 [152].

CD20 is also a target of CAR T therapies [149] and is being evaluated in clinical trials of hematologic malignancies, including combinations of CD20 with other B-cell antigens, such as dual CD20/CD19 and CD20/CD22, or trivalent CAR T therapies, including CD19/CD20/CD22 [153].

Currently, all approved CAR T therapies use autologous cells; however, "off the shelf" allogeneic CAR T therapies are in clinical development. CAR Ts targeting CD19 (azercabtagene zapreleucel; ALLO-501A; CTX110) and CD20 (PBCAR20A) have phase II trials completed or ongoing [154].

Because anti-CD19 CAR T therapies result in the depletion of all B cells, excluding long-lived plasma cells, efforts are ongoing to develop chimeric autoantibody receptor (CAAR) T cells, in which T cells are genetically modified with a chimeric receptor containing a target autoantigen with the aim of targeting specific B-cell populations that express autoantibodies to the target autoantigens [148]. Therapeutic efficacy using CAAR T cells was demonstrated in preclinical models of pemphigus vulgaris [155] and muscle-specific tyrosine kinase (MuSK) myasthenia gravis [156]. A clinical trial of MuSK-CAART is underway (NCT05451212). A preclinical EAE study evaluated a novel CAAR T cell containing part of the myelin basic protein autoantigen. The CAAR T cells induced higher B-cell lysis than controls and increased proliferation and production of inflammatory cytokines when co-cultured with autoreactive B cells, providing initial proof of concept that CAAR T cells might have activity in MS [157].

13.4Antibody-Drug Conjugates

Antibody-drug conjugates utilize the capacity of mAbs to target specific cells with an attached cytotoxic agent, typically microtubule inhibitors or DNA-damaging agents [158,159]. Upon binding of the mAb to the cell surface antigen followed by internalization of the mAb-drug complex, the cytotoxic payload is delivered selectively to the target cell and causes cell death or apoptosis [159]. The Fc-mediated immune effector functions may also play a pivotal role by inducing ADCC, CDC, and ADCP [159,160]. The first antibody-drug conjugates were developed for use in the treatment of various cancers such as loncastuximab tesirine (ADCT-402), a CD19-targeted antibody-drug conjugate used to treat diffuse large B-cell lymphoma [161].

14Conclusion

Since the initial experience of rituximab in MS and NMOSD, engineering of anti-CD20 mAbs has evolved. These changes in antibody structure and function were made to improve clinical efficacy and safety. The adaptation of antibody scaffolds from chimeric to humanized or fully human altered the immunogenicity of anti-CD20 mAbs but did not translate into meaningful differences in tolerability or efficacy, as supported by data from studies with rituximab and ublituximab, two chimeric antibodies with good tolerability profiles. Rather, tolerability with anti-CD20 therapy, which is an important consideration for patients and healthcare providers when choosing a medication, may be a consequence of the predominant pathway of B-cell depletion: either ADCC or CDC. Ublituximab and ocrelizumab mediate B-cell depletion predominately via ADCC, whereas rituximab and ofatumumab rely on complement activation for efficient lysis of target cells. These different mechanisms of cell lysis can translate into differences in tolerability and IRRs for patients. Deeper and sustained peripheral B-cell depletion could be an important predictor of disease activity suppression and may be influenced by mAb-binding epitope and mechanism and strength of cell lysis, as well as individual characteristics such as body weight and FcγR polymorphisms. Glycoengineered antibodies, such as ublituximab and inebilizumab, correlate with stronger binding affinities and more efficient cell lysis, even when polymorphisms in the FcRs of effector cells are present. Whether this translates to improved long-term efficacy or impacts safety is still being evaluated. The role of genetic polymorphisms and pharmacogenetics is a new concept in MS and could open the door to potentially more personalized approaches as novel treatment options become available. Further elucidation of the characteristics that drive therapeutic success for anti-CD20 mAbs could pave the way for the development of new treatments, including bispecific antibodies and CAR T-cell therapies, and improve the overall quality of life for people living with MS.

Code Availability Not applicable.

Data Availability Data sharing is not applicable to this article, as no

Declarations

Funding Funding for the open access fee, medical writing and editorial support was provided by TG Therapeutics.

Acknowledgements

Acknowledgments Medical writing (literature searching, drafting manuscript outline, and revisions) and editorial support (copyediting and journal styling) were provided by Britt Anderson, PhD, and Kimberly Church of Apollo Medical Communications, part of Helios Global Group, and funded by TG Therapeutics.

Funding

He has received grant funding from NIH, Anokion, and Regeneron.He serves as an unpaid member of the board of the Siegel Rare Neuroimmune Association.He has equity in Clene and GenrAb.He receives royalties

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