(592h) Multivalent Soluble Antigen Arrays Target Antigen Presenting B Cells and Dampen Antigen-Specific Signaling to Promote Therapeutic Efficacy in Multiple Sclerosis

Multivalent soluble antigen
arrays target antigen presenting B cells and dampen antigen-specific signaling
to promote therapeutic efficacy in multiple sclerosis

Brittany L. Hartwell, Chad
J. Pickens, Martin Leon, Laura Northrup, Francisco Martinez-Becerra, Cory
Berkland

The University of Kansas

Many current therapies for autoimmune diseases such as
multiple sclerosis (MS) act through nonspecific targeting of the immune
response, rendering global immunosuppression, poor efficacy, and adverse side
effects. To address a pressing need for safer and more effective therapies, we developed
multivalent soluble antigen arrays (cSAgAs) to induce
antigen-specific tolerance in MS. cSAgA_PLP:LABL
consists of a flexible hyaluronic acid (HA) linear polymer backbone click-conjugated
with multiple copies of autoantigen (PLP­) and adhesion inhibitor (LABL)
peptides. We hypothesized that multivalent presentation of both peptides would
enable antigen-specific targeting and enhanced binding with autoreactive
antigen-presenting cells (APCs), thereby interrupting signaling between APCs
and T cells to halt propagation of an autoimmune response.
In vivo studies established that cSAgA_PLP:LABL is therapeutically effective against experimental
autoimmune encephalomyelitis (EAE), a murine model of relapsing-remitting MS.
To elucidate the moleculeÕs therapeutic cellular mechanism, flow cytometry binding
and calcium flux signaling assays were performed alongside live cell imaging to
evaluate binding, specificity, and signaling modulation in different cell
populations.

Studies performed in vitro in a model B cell system revealed that cSAgA_PLP:LABL exhibited enhanced binding over polymer alone (cHA) and polymer conjugated with PLP only (cHA_PLP) or LABL only (cHA_LABL).
Furthermore, cSAgA_PLP:LABL exhibited high
avidity antigen-specific binding, targeting the B cell receptor (BCR) to dampen
BCR-mediated calcium signaling. These observations suggested the moleculeÕs
action at the cell surface led to B cell anergy, a state of antigen
unresponsiveness induced by continuous BCR binding and marked by reduced
calcium flux signaling.

cSAgA_PLP:LABL binding and signaling were then
evaluated ex vivo in mixed primary
splenocytes isolated from EAE and healthy mice to determine which immune cell
subsets were targeted and whether this resulted in an anergic phenotype. Flow
cytometry binding studies were performed in splenocytes antibody-labeled for B
cells (CD19⁺), T cells (CD3⁺), dendritic cells (CD11c⁺),
and autoimmune-associated B cells (CD19⁺CD11c⁺, ABCs), a
subclass of B cells that act as potent antigen presenting cells in the spleen
during autoimmune disease. cSAgA_PLP:LABL
exhibited significantly higher binding in B cells, particularly ABCs, over
other cell types (Figure 1AB). Additionally, B cell binding was significantly
higher in EAE cells than healthy cells, suggesting disease-specific targeting
that was not present in T cells (Figure 1CD). These results support our
hypothesis that cSAgA_PLP:LABL targets B
cells and APCs over other cell types in a disease-specific manner.

In addition to reduced
calcium flux signaling, another result of B cell anergy is down-regulation of
costimulatory signals (i.e., CD80 and CD86) whose presentation is essential for
autoreactive T cell activation. Thus, we investigated CD80/CD86 expression in
splenocytes under two conditions: (1) harvested from EAE mice at peak of
disease and treated in vitro during
simultaneous PLP antigen challenge to evaluate the effect on costimulatory
signaling, and (2) harvested from mice treated in vivo and re-challenged with PLP antigen ex vivo to evaluate lasting tolerance. First, in vitro treatment with cSAgA_PLP:LABL
caused significant down-regulation of CD86 in EAE splenocytes harvested from
mice at peak of disease, causing B cells and ABCs to signal more like healthy
cells rather than activated APCs (Figure 2AB). Furthermore, B cells and ABCs
from mice treated with cSAgA_PLP:LABLin vivo (Figure 2C) did not respond to ex vivo PLP rechallenge with a
significant increase in CD80/CD86 expression, again resembling healthy cells
(Figure 2DE). Splenocytes from the treated mice also exhibited elevated levels
of regulatory cytokines IL-10 and IL-4. Combined, these results indicate that
treatment with cSAgA_PLP:LABL achieved
efficacy by inducing PLP-specific anergy in B cells, resulting in reduced APC
capacity and lasting tolerance to autoantigen. We conclude that cSAgA_PLP:LABL offers a promising option for antigen-specific
immunotherapy to repress autoimmune disease.

Figure 1. Maximum steady state (max. SS) binding in antibody-labeled splenocytes
isolated from EAE and healthy mice determined by flow cytometry, comparing
binding of fluorescently-labeled molecules in (A) CD19⁺ B cells, (B)
CD19⁺CD11c⁺ autoimmune-associated B cells (ABCs), (C) CD19⁺ B cells, and (D) CD3⁺ T cells.
Splenocytes were isolated from mice at peak of disease and cultured for 72 hr prior to the experiment. Statistical significance was
determined by ANOVA followed by TukeyÕs post hoc test
with p<0.05 and n=3 (*p<0.05, **p<0.01, ***p<0.001,
****p<0.0001).

Figure 2. Costimulatory marker expression was evaluated in
antibody-labeled EAE splenocytes following in vitro or in vivo treatment
with cSAgA_PLP:LABL, compared to EAE and
healthy controls. (A/B) Splenocytes isolated from EAE mice at peak of
disease were treated with cSAgA_PLP:LABL for
72 hr in vitro in the presence or
absence of PLP. CD86 expression (anti-CD86 mean fluorescence intensity, MFI)
was evaluated by flow cytometry in (A) CD19⁺ B cells and (B)
CD19⁺CD11c⁺B220⁺ autoimmune-associated B cells
(ABCs) to evaluate APC costimulatory signaling. (C) EAE mice were treated in vivo with cSAgA_PLP:LABL versus PBS negative control on days 4, 7,
and 10, where higher clinical score indicated greater disease severity (n=5). (D/E) Splenocytes isolated from mice on
day 25 following in vivo treatment in (C) were rechallenged with/without PLP for 72 hr. CD86 expression
was evaluated by flow cytometry in (C) CD19⁺ B cells and (D)
CD19⁺CD11c⁺B220⁺ autoimmune-associated B cells
(ABCs) to evaluate lasting tolerance to autoantigen. Statistical significance
(compared to the negative PBS control) was determined by ANOVA followed by TukeyÕs post hoc test with p<0.05 and n=3 (*p<0.05,
**p<0.01, ***p<0.001, ##/****p<0.0001).