# Inhibiting CD40 Ligand in Multiple Sclerosis: A Review of Emerging Therapeutic Potential

Patrick Vermersch, D. Wagner, Lennart T. Mars, R. Noelle, G. Giovannoni

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Patrick Vermersch, D. Wagner, Lennart T. Mars, R. Noelle, G. Giovannoni. Inhibiting CD40 Ligand in Multiple Sclerosis: A Review of Emerging Therapeutic Potential. Current Treatment Options in Neurology, 2025, Curr. Treat. Options Neurol., 27 (7), 10.1007/s11940-024-00818-2. hal-04918330

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Distributed under a Creative Commons Attribution 4.0 International License
# Inhibiting CD40 Ligand in Multiple Sclerosis: A Review of Emerging Therapeutic Potential

Patrick Vermersch<fnref n="1" /> · David Wagner<fnref n="2" /> · Lennart T. Mars<fnref n="3" /> · Randolph Noelle<fnref n="4" /> · Gavin Giovannoni<fnref n="5" />

Accepted: 29 October 2024

© The Author(s) 2024

## Abstract

**Purpose of Review** Current high-efficacy disease-modifying therapies for multiple sclerosis (MS) primarily target adaptive immune cells in peripheral tissues, controlling focal inflammation (acute relapses and magnetic resonance imaging [MRI] activity). However, there is a need for new, safe therapies that address disease progression and disability accumulation in relapsing and progressive MS. The CD40/CD40L pathway, which regulates adaptive and innate immunity, is implicated in MS pathogenesis, making it a potential therapeutic target. Early clinical trials of anti-CD40L antibodies showed encouraging efficacy in autoimmune indications but were discontinued due to thromboembolic risk. Recent therapeutic advancements now allow researchers to leverage this pathway while reducing safety risks.

**Recent Findings** Frexalimab is a humanized anti-CD40L immunoglobulin-G1 monoclonal antibody, Fc-engineered to overcome thromboembolic risk. It is the first second-generation anti-CD40L antibody being investigated in MS. In a randomized-controlled phase 2 trial, frexalimab was well-tolerated, showing rapid and sustained reduction in disease activity (assessed by MRI) while preserving lymphocyte levels.

**Summary** Recent clinical findings strengthen the rationale for targeting CD40L in MS, supporting further development of anti-CD40L antibodies as potential high-efficacy, non-lymphocyte-depleting MS therapy. Further research is needed to understand the role of this pathway in MS pathogenesis and explore CD40L inhibition to address neuroinflammation and neurodegeneration, where unmet medical needs exist.

**Keywords** Anti-CD40L Monoclonal Antibody · Adaptive Immunity · High-Efficacy Disease-Modifying Therapy · Innate Immunity · Multiple Sclerosis

## Introduction

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It is characterized by inflammation and neurodegeneration, leading to varied neurological symptoms that progress to increased disability in people with MS [1, 2]. In MS, adaptive immunity, characterized by antigen-specific B and T cells, inadvertently targets neural antigens, triggering an inflammatory cascade that leads to demyelination, oligodendrocyte loss, and axonal damage in the focal parenchymal plaques. The onset of disease is driven by lymphocytes primed in the secondary lymphoid organs [2, 3]. During neuroinflammation, autoreactive CD4+T cells infiltrate the CNS and are re-activated after recognizing cognate antigens presented on myeloid-derived antigen-presenting cells (APCs) and activated B cells. This orchestrates the focal inflammatory response, which causes tissue damage through autoantibodies and cytokine release [2, 3]. Demyelinating

<footnote>Patrick Vermersch
patrick.vermersch@univ-lille.fr</footnote>

<footnote n="1">¹ Univ. Lille, Inserm U1172, Lille Neuroscience and Cognition, CHU Lille, FHU Precise, Lille, France</footnote>

<footnote n="2">² Webb Waring Center and Department of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, USA</footnote>

<footnote n="3">³ Laboratory of Neuroinflammation and Multiple Sclerosis, UMR-S1172, Univ. Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, Lille, France</footnote>

<footnote n="4">⁴ Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA</footnote>

<footnote n="5">⁵ Queen Mary University of London, London, UK</footnote>
lesions can lead to breakdown of the blood-brain barrier (BBB), facilitating the infiltration of innate immune cells, such as dendritic cells (DCs) and macrophages, into the CNS parenchyma where they present antigens to T cells to initiate and propagate tissue-specific damage [2, 3]. CNS-resident immune cells, including microglia, border-associated macrophages, and astrocytes are activated in response to elevated cytokine levels. They play a critical role throughout the disease course, from initial immune infiltration and demyelination to chronic neuroinflammation and neurodegeneration at later stages [2, 4]. The crosstalk between the adaptive and CNS-resident innate immune systems is critical throughout MS pathogenesis [5].

Although the treatment landscape for relapsing MS is evolving, there remains a significant unmet need to treat progressive disease, which is characterized by disability accumulation with or without relapses [6, 7]. The processes driving disability accumulation from underlying smoldering pathology begin early in the disease course, with CNS-resident cells playing a major role. This underscores the need for early and effective treatments targeting neurodegenerative mechanisms [3, 8–10]. Current high-efficacy disease-modifying therapies (DMTs), including anti-CD20 antibodies, focus on adaptive immune system, particularly B cells in the periphery, but exhibit modest or temporary effects on CNS-compartmentalized chronic neuroinflammation and neurodegeneration [8]. These treatments are effective in controlling acute relapses and accumulation of focal lesions visible on magnetic resonance imaging (MRI) scans, but their impact on disability accumulation remains moderate and may carry long-term risks due to chronic B-cell depletion. Thus, novel mechanisms of action should be identified that are safe and offer comprehensive disease control by addressing both adaptive and innate immunity [9, 11–13].

The impact of cluster of differentiation 40 (CD40)/CD40 ligand (CD40L) costimulatory signaling pathway on immune responses and its contribution to the onset of autoimmune diseases have established it as a potential therapeutic target. This pathway is being investigated as a prospective solution to address the unmet need in the treatment of various autoimmune diseases, including MS [14, 15]. In this review, we explore the pivotal role of CD40/CD40L interactions in both adaptive and innate immune responses and in the pathophysiology of MS, the rationale behind targeting CD40L in relapsing and progressive forms of MS, and the current drugs in development that target this pathway.

## Role of the CD40/CD40L Pathway in Immune Cell Signaling

The costimulatory receptor CD40 and its natural ligand CD40L (CD154) are transmembrane proteins that belong to the tumor necrosis factor (TNF) gene superfamily [16].

Human CD40 was first recognized as an antigen expressed in urinary bladder carcinoma cells [17]. It was parallelly identified as a receptor on resting B cells and is functionally expressed on myeloid APCs (DCs, monocytes, and macrophages) as well as non-immune cells (fibroblasts, epithelial, endothelial, and neurons) [16, 18]. CD40L is mainly expressed on antigen-activated T cells, and to a lesser extent on activated B cells, mast cells, basophils, eosinophils, natural killer cells, platelets, endothelial, epithelial, and smooth muscle cells [19]. It also exists as soluble CD40L (sCD40L), which is a non-membrane-bound form primarily produced by activated T cells and platelets. It is released into circulation, where it can bind to CD40 and promote the expression of proinflammatory cytokines [20–23].

CD40 engagement by CD40L initiates a complex signaling cascade, including recruitment of specific TNF-receptor-associated factors (TRAFs) and activation of various signal transduction pathways, including nuclear factor kappa B (NFkB) pathway, c-Jun N-terminal kinase pathway, and p38 mitogen-activated protein kinase (MAPK) pathway. Additionally, it activates various protein tyrosine kinases and transcription factors, leading to production of cytokines and chemokines [14, 24].

## Impact of the CD40/CD40L Pathway on Humoral Immune Response

It has been established that the interaction of CD40 expressed on B cells with CD40L on antigen-activated T cells controls T-cell-dependent humoral immune responses. Mutations in the gene encoding CD40L can lead to X-linked hyper IgM syndrome (HIGM), resulting in severely impaired T-cell-dependent antibody response, lack of B-cell memory, and reduced levels of circulating immunoglobulins (IgG, IgA, and IgE). This makes patients more susceptible to opportunistic infections, highlighting the importance of CD40/CD40L interaction in isotype switching and anamnestic B-cell responses [25]. CD40L-knockout mice exhibit a phenotype similar to that of patients with HIGM syndrome [26].

Generally, humoral responses originate in the secondary lymphoid organs called germinal centers (GCs), wherein B cells undergo isotype switching and affinity maturation through somatic hypermutation of the complementarity-determining regions in the immunoglobulin variable domain [27]. The intrinsic risk of autoimmunity imposed by these processes requires tight regulation by a dedicated T-cell subset called follicular helper T cells (Tfh), which orchestrate the GC B-cell response. A key aspect of this regulation is the ability of B cells to capture antigens using their receptors. This allows B cells

<figure><img src="image_2.png" /></figure>
to present these antigens as peptide-major histocompat-
ibility complex (MHC) complexes, which enable the acti-
vation of Tfh cells that transiently express CD40L. The
interaction between CD40 on B cells and CD40L on Tfh
cells is crucial for B-cell activation in response to T-cell-
dependent antigens [14]. CD40-signaling provides a sec-
ondary signal to B cells, which along with the primary
signal from antigen-bound membrane immunoglobulins,
leads to B-cell activation, proliferation, and differentiation.
The interaction between CD40 on B cells and CD40L on
Tfh cells promotes GC formation and supports the sur-
vival of antigen-stimulated B cells in GCs and ectopic
follicles. These B cells then proliferate and differentiate
into immunoglobulin-secreting plasma cells or long-lived
memory B cells, and with additional signals, Tfh drive
isotope switching [27–30].

Impact of the CD40/CD40L Pathway
on Cell-Mediated Immune Response

The CD40/CD40L pathway has several essential cellular
functions beyond regulating antibody response, including
T-cell priming in the draining lymph nodes and orchestrat-
ing T-cell driven cellular immune response [31, 32]. Naive
T cells require both primary and secondary signals for
activation. The primary signal comes from binding of the
T-cell receptor (TCR) to an antigen presented by MHC. The
secondary signal involves CD40/CD40L interaction, which
upregulates CD80 and CD86 on “licensed” APCs, which
then bind to CD28 receptors on T cells. This interaction
promotes differentiation of CD4+ T cells into effector T-cell
subsets (helper T [Th]1, Th2, or regulatory T [Treg] cells),
and is essential for the priming of cytotoxic CD8+ T cells
[14, 16, 20, 33]. CD40L-expressing CD4+ T cells license
APCs via CD40-signaling, which activates CD8+ T cells.
Antigen-specific CD8+ T cells can also express CD40L, pro-
moting their own proliferation and differentiation through
DCs [34, 35].

Impact of the CD40/CD40L Pathway on Innate
Immune Response

**Dendritic Cells** DCs serve as a bridge between innate and adaptive immunity, as they link two processes, i.e., anti-gen-mediated priming of T cells and orientation of effector T-cell functions [14, 36]. DCs are critical for T-cell prim-ing owing to their ability to pinocytose the antigens in tis-sues and migrate to the T-cell zones in secondary lymphoid organs, where T cells reside and provide optimal antigen presentation and co-stimulation for antigen-specific naive T cells [36]. Engagement of CD40 on DCs by CD40L on T cells upregulates the expression of costimulatory molecules, CD80/B7-1 and CD86/B7-2 on DCs, which serve as ligands

for CD28 on T cells. This results in DC maturation, proper
licensing of DCs and effective T-cell priming. [36, 37]. In
DCs, TRAF6 is recruited downstream of CD40 ligation,
leading to activation of p38-MAPK and NFKb pathways.
Activation of these pathways promotes survival and matura-
tion of DCs, and secretion of polarizing cytokines, including
IL-12 and IL-6 [14, 24, 38–40].

**Macrophages/Microglia and other CNS-Resident Cells** The CD40/CD40L pathway is important for initiating immune responses by functional phenotype of macrophages, enhancing their antigen presentation and proinflammatory activities. Antigen presentation by macrophages to Th1 cells leads to CD40L expression and interferon gamma (IFNγ) release by Th1 cells. This interaction also increases expression of CD40 and TNF-receptor levels on macrophages [41]. Subsequent ligation of CD40 on monocytes and macrophages activates these cells and induces production of proinflammatory cytokines and chemokines. It also upregulates the expression of MHCII and costimulatory molecules, CD80 and CD86 [41].

Microglia are the resident macrophages of the CNS,
permanently located in the parenchyma and are involved
in antigen presentation and secretion of proinflammatory
cytokines. They engage in crosstalk with CNS-infiltrating T
cells and other components of innate immune system, facili-
tating the recruitment of adaptive immune cells into the CNS
[42, 43]. Under resting conditions, microglia express CD40
at relatively low levels in the CNS. However, proinflamma-
tory stimuli, such as IFNγ, TNFα, and lipopolysaccharides,
markedly increase CD40 expression in the microglia. This
CD40 upregulation plays a critical role in microglia activa-
tion and amplifies the inflammatory responses in the CNS
[42, 44]. Other CNS-resident cells, such as astrocytes and
neurons also contribute to the regulation of autoimmune
neuroinflammation in the CNS through CD40 expression
[20, 45].

Role of the CD40/CD40L Pathway
in Response to Infections and Autoimmunity

The CD40/CD40L pathway is essential for mediating B- and T-cell co-stimulation. This interaction is crucial for initiating adaptive immune responses, which are the host's defense mechanisms against specific pathogens. CD40 expressed on innate immune cells also interacts with CD40L on activated T cells and helps in immune activation during infections [46]. CD4+ helper T cells indirectly control pathogens by activating innate immune cells, assisting in the development of cytotoxic CD8+ T-lymphocyte responses, and contributing to B-cell maturation and antibody production [47, 48].
Due to the significant impact of the CD40 axis, it is essen-
tial to tightly regulate the CD40/CD40L expression to main-
tain appropriate immune responses. Dysregulation of this
pathway may lead to the development or maintenance of
pathogenic autoimmune responses [14]. CD40 plays a role
in T-cell-dependent autoimmune diseases at several levels.
In the thymus, it assists autoreactive T-cell clones during
T-cell selection; in secondary lymphoid organs, it is crucial
for T-cell priming; and in the target tissues, CD40-signaling
triggers the production of pro-inflammatory cytokines and
chemokines, contributing to tissue damage and inflamma-
tion [49–51].

Given the pleiotropic role of CD40L in regulating both
innate and adaptive immunity, it is critical to understand
how CD40L inhibition affects the host's defense mecha-
nisms. The CD40L-deficient mice exhibit a more severe
impact on their immune defense than those treated with an
anti-CD40L antibody. The genetic loss of CD40L expression
(as seen in HIGM syndrome) significantly affects the host's
defense [52]. However, in the absence of CD40L expression,
thymus-independent antigens (mainly from bacteria) can
still stimulate B cells to produce protective antibodies with-
out T-cell involvement. In mice treated with an anti-CD40L
antibody, responses to T-independent antigens remain intact
[53]. Similarly, while anti-viral responses are affected by
the absence of CD40L, antibody and cell-mediated immu-
nity remain functional against many viruses [54]. There-
fore, unlike the significant immune suppression seen with
B-cell depleting antibodies, CD40L antagonists may allow
the host to maintain immune responses to certain bacteria
and viruses since they modulate B-cell activity through a
non-lymphocyte depleting mechanism of action [11, 55].
Whether this approach will offer protection against infec-
tions while ensuring a favorable safety profile remains to be
explored, and phase 3 clinical trials and real-world data are
warranted to better understand long-term safety of CD40L
antagonists.

Role of the CD40/CD40L Pathway in MS
Pathophysiology

Targeting CD40/CD40L pathway, which regulates both
adaptive and innate immune responses, is a rationale thera-
peutic strategy for MS, supported by genetic evidence. The
significance of CD40-signaling in MS has been emphasized
through data-driven Mendelian randomization analysis,
showing that around 200 single-nucleotide polymorphisms
(SNPs) are linked to MS risk [56]. The CD40 gene has
been identified as a risk gene for MS, with SNPs rs1883832
and rs6074022 linked to increased MS risk [57–62]. SNP
rs1883832 and its reduced mRNA expression are associ-
ated with increased MS susceptibility [63–65]. Another SNP

called rs4810485 is also linked to MS risk and correlates
with lower CD40 expression on B cells [58, 61]. The MS
risk allele of CD40 is associated with reduced CD40 expres-
sion on B cells and polarized DCs in peripheral blood mono-
nuclear cells isolated from MS patients. CD40 levels are
significantly lower in B cells from MS patients compared to
healthy controls, regardless of the risk genotype [63].

Targeting CD40L to inhibit the CD40/CD40L pathway
reduces disease severity in animal models of MS. An anti-
CD40L antibody inhibited the induction of experimental
autoimmune encephalomyelitis (EAE) in mice, reduced the
severity of established disease, and resulted in fewer clinical
relapses [43, 66–70]. In EAE mice, relapses correlated with
increased expression of CD40 and CD40L [71]. In relapsing-
EAE mice, an anti-CD40L antibody completely prevented
the onset of acute disease after initial induction and reduced
disease progression, with relapse rates dropping to 30%
compared to 100% in control mice. Furthermore, administra-
tion of an anti-CD40L antibody at the time of EAE induction
prevents disease and if treatment is given at the peak of acute
disease, mice have fewer relapses, absence of detectable glial
scarring, and lesser demyelination. This may be attributed
to the CD40/CD40L interaction influencing CNS-infiltration
of T cells and affecting their effector function to activate
CNS-macrophages/microglia [68]. Treatment of EAE mice
with an anti-CD40L antibody at the acute disease peak or
remission inhibited Th1 differentiation and effector function
and reduced the disease incidence from 91% in the control
group to 50% in the anti-CD40L antibody group [68, 72].

The CD40/CD40L interaction is essential for EAE induc-
tion in the absence of CD28-mediated co-stimulation. While
CD28 does not regulate immunological anergy, it adjusts
the threshold of Th1 activation. CD40L can compensate for
the absence of CD28 by activating autoreactive T cells and
differentiating them into Th1 effector cells capable of medi-
ating EAE [73].

CD40 is upregulated in EAE mice, which along with
other markers, like CD80 and inducible nitric oxide syn-
thase (iNOS), is associated with the M1 (inflammatory)
phenotype and can be indicative of MS pathogenesis [74].
In EAE mice, CD40 deficiency in macrophages strongly
reduces disease severity [75]. In a marmoset MS model,
CD40 expression was localized to CNS-macrophages [76]
and anti-CD40 antibody (ch5D12) treatment, given early or
after neuroinflammation onset, effectively inhibited clini-
cal signs of EAE and reduced CNS-lesions in post-mortem
analysis [77, 78]. These in vitro and in vivo preclinical stud-
ies have provided insights into the putative mechanism of
action of an anti-CD40L antibody in MS (Fig. 1).

Clinical and pathological studies in MS patients have
indicated the involvement of the CD40/CD40L path-
way in disease pathophysiology [20, 67, 79–81]. Most
CD40L+ cells in the brains of MS patients are CD4+T cells,

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Proposed mechanism of action of an anti-CD40L antibody in multiple sclerosis. In the periphery, an anti-CD40L antibody may modify the activation and function of T cells, B cells, and innate immune cells (macrophages/microglia and dendritic cells). By binding to CD40L on peripheral T cells, it could prevent T-cell activation, Th cell differentiation, and subsequent CNS-infiltration of CD40L+T</figcaption></figure>

cells, which are key drivers of CD40-mediated inflammation. In the CNS, anti-CD40L antibody may indirectly block the effector function of activated T cells to stimulate CNS macrophages/microglia. BBB, blood-brain barrier; CD40, cluster of differentiation 40; CD40L, CD40 ligand; CNS, central nervous system; MHC, major histocompatibility complex; TCR, T-cell receptor; Th, T helper

and these patients have a higher frequency of CD40L+T cells than healthy controls. Treatment with IFNβ reversed the increased expression of CD40L on CD4+T cells [82]. Increased sCD40L concentrations have been reported in patients with active MS compared to those in healthy controls [83–86]. Treatment with an anti-CD40L antibody toralizumab (IDEC-131) was found to be efficacious in a phase 1 trial conducted in 12 patients with relapsing-remitting MS (RRMS), with no relapses observed during the 18-week study. However, further development was halted due to potential thromboembolic risks [87].

## Implications of the CD40/CD40L Pathway in Progressive MS

Progressive MS is characterized by a steady disability accumulation due to chronic neurodegeneration and neuroinflammation behind the BBB, driven by microglial and astrocyte activation, as well as leukocyte infiltration. Studies have shown that elevated CD40 expression on astrocytes and glial cells is required for EAE development, while lack of CD40 expression on CNS-resident microglia ameliorates EAE severity in mice and reduces leukocyte infiltration into

the CNS [20, 43, 44]. Blocking CD40/CD40L signaling is believed to decrease the adhesion of T cells to endothelial cells, which is a key step in migration of T cells across the BBB to sites of inflammation in the CNS [88]. In an adoptive transfer of activated T-cell-driven EAE model, treatment with an anti-CD40L antibody decreased the function of these T cells due to interference with CNS homing of the activated T cells or direct interference with other effector functions, or both [68].

Autopsy studies in MS patients revealed that active lesions in the CNS are characterized by mononuclear cell infiltrates, comprising of CD40-expressing monocytes, macrophages, and activated microglia [67]. CD40L-expressing T cells infiltrate the CNS and activate CD40-expressing microglia in the demyelinating lesions, resulting in release of cytokines, nitric oxide, and metalloproteinases by the microglia [80]. Blood sample analysis of MS patients revealed that the interaction of CD40 with CD40L on activated CD4+T cells markedly increases IL-12 secretion by antigen-presenting myeloid cells. This increase is significantly higher in patients with progressive MS than in healthy individuals (P = 0.003) and RRMS patients (P < 0.05). The increased secretion of IL-12 enhances IFNγ production in patients
with progressive MS, which is significantly higher than that in healthy controls (P < 0.001) [79]. Monocytes derived from blood samples of SPMS patients had higher CD40L expression and IL-12 secretion than those from RRMS patients or healthy controls [89]. In vitro studies suggest that the neurotoxicity of activated T cells can be neutralized by anti-human CD40 antibody, potentially preventing the neuronal damage observed in progressive disease [90].

Higher serum sCD40L levels have been linked to increased BBB permeability, suggesting that sCD40L plays a critical role in BBB disruption and the associated disease mechanisms in progressive MS [91, 92]. In a prospective study, CSF sCD40L was identified as a key marker for differentiating between SPMS and RRMS. The combination of monocyte chemoattractant protein-1 (MCP1; also known as chemokine [CC-motif] ligand 2 [CCL2]) and sCD40L effectively distinguished patients with SPMS from those with RRMS. Meanwhile, SPMS was most effectively differentiated from non-progressive benign MS by the combination of sCD40L and IFNγ [84]. In contrast, another prospective study using a combined approach with different biomarkers found that individuals with RRMS had a higher level of sCD40L compared to those with SPMS; however, firm conclusions could not be drawn due to the limited number of patients and controls in this study [93].

## CD40/CD40L Pathway in MS and its Link to the Epstein-Barr Virus

Epstein-Barr Virus (EBV) infection is believed to play a central role in the development and progression of MS by driving episodic peripheral inflammation that causes relapses and persistent CNS-compartmentalized inflammation within the CNS, leading to progressive damage [94]. Molecular mimicry between epitopes of EBV and neural antigens may explain this association, notably in case of overt mononucleosis [95, 96]. EBV primarily targets B cells and induces ectopic expression of CD40L on B cells to enhance B-cell survival and EBV-mediated transformation [97]. In MS, B cells expressing EBV proteins locate within the CNS, where they interact with T cells via CD40/CD40L pathway and contribute to inflammatory demyelination. Consequently, EBV+ B cells may exacerbate disease by increasing B-cell activation, enhancing CD40 expression, presenting neural autoantigens to T cells, inflammatory cytokine release, and promoting the longevity of B cells [98–103]. The CD40/CD40L pathway promotes the survival of EBV-infected T, B, and natural killer cells, indicating its potential utility in treating EBV-induced lymphoproliferation [98, 104]. In EAE mice infected with murine γHV-68 (murine homolog to EBV), the frequency of Treg cells decreased via a CD40-dependent mechanism, contributing to a robust Th1

response, and resulting in a more severe disease pathology [105].

## Overview of CD40 and CD40L Inhibitors in MS and Other Autoimmune Diseases

The CD40/CD40L pathway critically orchestrates the cellular and humoral immune responses. As such, it is actively explored as a potential solution to address the unmet need in treating various autoimmune conditions [14, 16, 106, 107].

The potential of blocking CD40/CD40L pathway has been confirmed in patients with proliferative lupus nephritis through a preliminary open-label phase 2 trial (NCT00001789) using the anti-CD40L antibody ruplizumab (BG9588). Although this study was small, it suggested that inhibiting CD40/CD40L pathway could modulate key clinical parameters in lupus, highlighting its immunomodulatory potential in autoimmunity. However, due to increased thromboembolic events in this study and in studies investigating other indications, the trials involving ruplizumab were terminated prematurely [108–111]. Another anti-CD40L antibody, toralizumab, was evaluated in phase 1 and 2 trials for systemic lupus erythematosus (SLE), MS and Crohn's disease. In a phase 1 RRMS trial, toralizumab did not exacerbate the disease, as assessed by clinical measures and MRI parameters. However, in SLE, it failed to improve disease activity. Development of toralizumab was discontinued across all indications due to the potential for developing thromboembolic complications, although no such events were reported in early trials [87, 112, 113].

The thromboembolic events associated with first-generation anti-CD40L antibodies likely resulted from platelet activation triggered via FcγRIIa stimulation by CD40L-mediated immune complexes [110, 114]. Novel Fc-engineering approaches have emerged to leverage this pathway for therapeutic benefits while mitigating the safety risks associated with platelet-expressed CD40L [115]. Notably, the second-generation anti-CD40 and anti-CD40L antibodies either lack an Fc domain or contain a modified Fc domain with low FcγRIIa binding, and thus, prevent platelet activation and eliminate the risk of Fc-mediated thromboembolism [55, 116–119].

Several trials of second-generation anti-CD40 and anti-CD40L antibodies are ongoing or have recently been completed for various autoimmune diseases (Table 1). Frexalimab is the first second-generation anti-CD40L monoclonal antibody being investigated for MS. In a phase 2 randomized controlled trial in participants with relapsing MS (NCT04879628), frexalimab met the primary endpoint, with an 89% (95% confidence interval, 62%–97%) reduction in the number of new gadolinium-enhancing T1-lesions with 1200-mg intravenous high-dose treatment versus placebo at

<figure><img src="image_5.png" /></figure>
<table><caption>**Table 1** Second-generation anti-CD40 antibodies and anti-CD40L antibodies under clinical investigation for autoimmune indications</caption><thead><tr><th>Target</th><th>Drug name</th><th>Mechanism of action</th><th>Disease population</th><th>Clinical trial phase</th><th>Clinical trial identifier</th></tr></thead><tbody><tr><td rowspan="10">CD40L</td><td rowspan="5">Frexalimab (SAR441344)</td><td rowspan="5">Humanized IgG1 mAb</td><td>RMS</td><td>Phase 3: Recruiting</td><td>NCT06141473</td></tr><tr><td>nrSPMS</td><td>Phase 2: Active (not recruiting)</td><td>NCT04879628</td></tr><tr><td>T1DM</td><td>Phase 3: Recruiting</td><td>NCT06141486</td></tr><tr><td>SLE</td><td>Phase 2: Recruiting</td><td>NCT06111586</td></tr><tr><td>Primary SjS</td><td>Phase 2: Completed</td><td>NCT05039840</td></tr><tr><td rowspan="2">Dapirolizumab pegol (CDP7657)</td><td rowspan="2">Fab' antibody fragment</td><td>SLE</td><td>Phase 3: Recruiting</td><td>NCT04976322</td></tr><tr><td></td><td>Phase 2: Completed</td><td>NCT02804763</td></tr><tr><td rowspan="5">Dazodalibep (VIB4920)</td><td rowspan="5">Non-antibody Tn3 fusion protein</td><td>SjS</td><td>Phase 3: Recruiting</td><td>NCT06245408, NCT06104124</td></tr><tr><td>RA</td><td>Phase 2: Completed</td><td>NCT04129164</td></tr><tr><td>LN</td><td>Phase 2: Recruiting</td><td>NCT05201469</td></tr><tr><td>Transplantation (Kidney)</td><td>Phase 2: Completed</td><td>NCT04046549</td></tr><tr><td>ALS</td><td>Phase 2: Completed</td><td>NCT04322149</td></tr><tr><td rowspan="7">Tegoprubart (AT-1501)</td><td rowspan="7">Humanized IgG1 mAb</td><td>Transplantation (Kidney)</td><td>Phase 2: Recruiting</td><td>NCT05983770</td></tr><tr><td>Transplantation (Islet cells)</td><td>Phase 1b: Recruiting</td><td>NCT05027906</td></tr><tr><td>IgAN</td><td>Phase 2: Withdrawn</td><td>NCT04711226</td></tr><tr><td>Organ transplant</td><td>Phase 1/2: Recruiting</td><td>NCT06305286</td></tr><tr><td>ITP</td><td>Phase 2: Terminated</td><td>NCT05125068</td></tr><tr><td>SjS</td><td>Planned</td><td>Not available</td></tr><tr><td>TNX-1500</td><td>Fc-modified human IgG1 mAb</td><td>Humanized IgG1 mAb</td><td>SLE</td><td>Phase 2: Completed</td><td>NCT02273960</td></tr><tr><td rowspan="15">CD40</td><td rowspan="2">Letolizumab (BMS-986004)</td><td rowspan="2">Fc-silent human IgG1 mAb</td><td>Phase 2 extension: Completed</td><td></td><td>NCT03905525</td></tr><tr><td>Graves' disease</td><td>Phase 2: Active (not recruiting)</td><td>NCT04541589</td></tr><tr><td rowspan="10">Iscalimab (CFZ533)</td><td rowspan="10">Humanized IgG1 mAb</td><td>SLE</td><td>Phase 2: Completed</td><td>NCT03656562</td></tr><tr><td>LN</td><td>Phase 2: Completed</td><td>NCT02713256</td></tr><tr><td>MG</td><td>Phase 2: Completed</td><td>NCT02565576</td></tr><tr><td>RA</td><td>Phase 1: Completed</td><td>NCT02089087</td></tr><tr><td>T1DM</td><td>Phase 2: Completed</td><td>NCT04129528</td></tr><tr><td>Transplantation (Kidney)</td><td>Phase 2: Completed</td><td>NCT03663335</td></tr><tr><td>Transplantation (de Novo Renal)</td><td>Phase 2: Completed</td><td>NCT02217410</td></tr><tr><td>Transplantation (Liver)</td><td>Phase 2: Terminated</td><td>NCT03781414</td></tr><tr><td>Ravagalimab (ABBV-323)</td><td>Humanized IgG1 mAb</td><td>UC</td><td>Phase 2a: Completed</td><td>NCT03695185</td></tr><tr><td rowspan="2">Abiprubart (KPL-404)</td><td rowspan="2">Humanized IgG4 mAb</td><td>SjS</td><td>Phase 2a: Completed</td><td>NCT05217472</td></tr><tr><td>RA</td><td>Phase 2: Completed</td><td>NCT05198310</td></tr><tr><td rowspan="3">Bleselumab (ASKP1240)</td><td rowspan="3">Humanized IgG4 mAb</td><td>Psoriasis</td><td>Phase 2a: Completed</td><td>NCT01585233</td></tr><tr><td>Transplantation (Kidney)</td><td>Phase 2: Completed</td><td>NCT01780844</td></tr><tr><td>FSGS</td><td>Phase 2: Completed</td><td>NCT02921789</td></tr><tr><td rowspan="4">BI-655064</td><td rowspan="4">Humanized IgG1 mAb</td><td>RA</td><td>Phase 1: Completed</td><td>NCT01751776</td></tr><tr><td>LN</td><td>Phase 2: Completed</td><td>NCT02770170</td></tr><tr><td>ITP</td><td>Phase 1: Completed</td><td>NCT03385564</td></tr><tr><td></td><td></td><td>Phase 1: Terminated</td><td>NCT02009761</td></tr></tbody></table>

Source: https://clinicaltrials.gov/. Accessed October 3, 2024

ALS, amyotrophic lateral sclerosis; CD, Crohn's disease; CD40, cluster of differentiation 40; CD40L, CD40 ligand; FSGS, focal segmental glomerulosclerosis; IgAN, immunoglobulin A nephropathy; IgG1, immunoglobulin G1; ITP, immune thrombocytopenia; LN, lupus nephritis; mAb, monoclonal antibody; MG, myasthenia gravis; nrSPMS, non-relapsing secondary progressive multiple sclerosis; RA, rheumatoid arthritis; RMS, relapsing multiple sclerosis; SjS, Sjogren's syndrome; SLE, systemic lupus erythematosus; T1DM, type 1 diabetes mellitus; UC, ulcerative colitis

12 weeks [55]. A sustained reduction in disease activity was observed, along with a 41% reduction in plasma neurofilament light chain (NfL) levels, a biomarker of neuroaxonal damage after 48 weeks in the open-label extension [120, 121].

Frexalimab was well-tolerated with no reports of thromboembolic events [55, 121]. Efficacy and safety of frexalimab is currently being investigated in phase 3 trials in relapsing MS (NCT06141473) and non-relapsing SPMS (NCT06141486).
Conclusions

Current high-efficacy DMTs for MS primarily target adaptive immune cells to control acute relapses, but do not fully address the progressive phase characterized by continuous disability accumulation. Therefore, it is crucial to identify novel mechanisms of action that address both neuroinflammatory and neurodegenerative aspects of MS.

The broad impact of the CD40/CD40L costimulatory pathway on immune responses, including antibody-mediated and cellular processes and its contribution to the onset of autoimmune diseases, has positioned it as a potential therapeutic target. Early clinical trials of anti-CD40L antibodies showed encouraging efficacy in MS but were halted due to thromboembolic complications. Novel Fc-engineering techniques are being utilized to harness this pathway, while mitigating the safety risks associated with platelet-expressed CD40L. Frexalimab is the first second-generation anti-CD40L monoclonal antibody being investigated in MS, with the potential to address both innate and adaptive immunity-related aspects of disease onset and progression. Positive phase 2 findings of frexalimab showed a rapid reduction in MS-related disease activity while preserving lymphocyte levels. This emerging evidence strengthens the rationale for targeting CD40L in MS and supports further development of anti-CD40L antibodies as potential high-efficacy, non-lymphocyte-depleting MS therapy. It is important to further elucidate the impact of this pathway in MS pathogenesis and explore the therapeutic potential of CD40L inhibition to address the full spectrum of neuroinflammation and neurodegeneration where unmet medical needs remain.

**Acknowledgements** The authors would like to thank Reema Patel and Christine Hwang, of Sanofi for their contribution to manuscript planning, review, and coordination. Medical writing support was provided by Akshada Deshpande and Akhilesh Vikram Singh, of Sanofi.

**Author Contributions** All the authors have contributed to the paper, according to ICMJE criteria.

**Funding** Open access funding provided by Centre Hospitalier Universitaire de Lille.

**Data Availability** No datasets were generated or analysed during the current study.

Declarations

**Conflict of Interest** Patrick Vermersch has received honoraria and consulting fees from Biogen, Sanofi, Novartis, Teva, Merck, Roche, Imcyse, AB Science, BMS-Celgene, and Ad Scientiam and research support from Novartis, Sanofi, and Roche.

David Wagner is the chief scientific officer and co-founder of Op-T, LLC.

Lennart T. Mars has received research support and honoraria as speaker from Merck and Novartis.

Randolph Noelle is the co-founder of ImmuNext, which has licensed an anti-CD40L to Sanofi that is in commercial development and is an inventor on anti-CD40L patents.

Gavin Giovannoni has received compensation over the last five years for receiving research support or serving as a consultant or speaker from AbbVie, Actelion, Atara Bio, Biogen, Canbex, Celgene, EMD Serono, Japanese Tobacco, Sanofi, Genentech, GlaxoSmithKline, GW Pharma, Merck, Novartis, Roche, Synthon BV, and Teva.

**Human and Animal Rights and Informed Consent** This article does not contain any studies with human or animal subjects performed by any of the authors.

**Competing Interests** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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