Dawn K. Stetsko

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

Blockade of CD28 signals results in the up-regulation of 2B4 on primary CD8+ effectors and plays a critical role in controlling antigen-specific CD8+ T cell responses.

Small molecule antagonist of leukocyte function associated antigen-1 (LFA-1): structure-activity relationships leading to the identification of 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonan-7-yl)nicotinic acid (BMS-688521).

Leukocyte function-associated antigen-1 (LFA-1), also known as CD11a/CD18 or alpha(L)beta(2), belongs to the beta(2) integrin subfamily and is constitutively expressed on all leukocytes. The major ligands of LFA-1 include three intercellular adhesion molecules 1, 2, and 3 (ICAM 1, 2, and 3). The interactions between LFA-1 and the ICAMs are critical for cell adhesion, and preclinical animal studies and clinical data from the humanized anti-LFA-1 antibody efalizumab have provided proof-of-concept for LFA-1 as an immunological target. This article will detail the structure-activity relationships (SAR) leading to a novel second generation series of highly potent spirocyclic hydantoin antagonists of LFA-1. With significantly enhanced in vitro and ex vivo potency relative to our first clinical compound (1), as well as demonstrated in vivo activity and an acceptable pharmacokinetic and safety profile, 6-((5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro-[4.4]nonan-7-yl)nicotinic acid (2e) was selected to advance into clinical trials.

A Monovalent Anti-Human CD28 Domain Antibody Antagonist: Preclinical Efficacy and Safety

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4–mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell–dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.

An LFA-1 (αLβ2) Small-Molecule Antagonist Reduces Inflammation and Joint Destruction in Murine Models of Arthritis

LFA-1 appears to play a central role in normal immune responses to foreign Ags. In autoimmune or inflammatory diseases, there is increased expression of LFA-1 and/or its counterligand, ICAM-1. Others have demonstrated that the targeted disruption of LFA-1:ICAM interactions, either by gene deletion or Ab treatment in mice, results in reduced leukocyte trafficking, inflammatory responses, and inhibition of inflammatory arthritis in the K/BxN serum transfer model. However, there has been little success in finding a small-molecule LFA-1 antagonist that can similarly impact rodent models of arthritis. In this paper, we present the first reported example of an LFA-1 small-molecule antagonist, BMS-587101, that is efficacious in preclinical disease models. In vitro, BMS-587101 inhibited LFA-1–mediated adhesion of T cells to endothelial cells, T cell proliferation, and Th1 cytokine production. Because BMS-587101 exhibits in vitro potency, cross-reactivity, and oral bioavailability in rodents, we evaluated the impact of oral administration of this compound in two different models of arthritis: Ab-induced arthritis and collagen-induced arthritis. Significant impact of BMS-587101 on clinical score in both models was observed, with inhibition comparable or better than anti-mouse LFA-1 Ab. In addition, BMS-587101 significantly reduced cytokine mRNA levels in the joints of Ab-induced arthritis animals as compared with those receiving vehicle alone. In paws taken from the collagen-induced arthritis study, the bones of vehicle-treated mice had extensive inflammation and bone destruction, whereas treatment with BMS-587101 resulted in marked protection. These findings support the potential use of an LFA-1 small-molecule antagonist in rheumatoid arthritis, with the capacity for disease modification.

Selective IRAK4 Inhibition Attenuates Disease in Murine Lupus Models and Demonstrates Steroid Sparing Activity

The serine/threonine kinase IL-1R–associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.

B cells from African American lupus patients exhibit an activated phenotype

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers. We found that B cells expressing the activation markers CD86, CD80, PD1, and CD40L, as well as CD19+CD27-IgD- double-negative B cells, were enriched in African American patients vs. patients of European ancestry. In addition to increased expression of CD40L, surface levels of CD40 on B cells were lower, suggesting the engagement of the CD40 pathway. In vitro experiments confirmed that CD40L expressed by B cells could lead to CD40 activation and internalization on adjacent B cells. To conclude, these results indicate that, compared with European American patients, African American SLE patients present with a particularly active B cell component, possibly via the activation of the CD40/CD40L pathway. These data may help guide the development of novel therapies.

IL-12 and IL-23 in health and disease

IL-12 and IL-23 are both members of the IL-6 family of cytokines. Both of these cytokines are important mediators of immune-mediated inflammatory diseases [1–4]. IL-12 and IL-23 are both heterodimeric cytokines, consisting of two disulfide-linked subunits, with one of the subunits being common in both cytokines. IL-12 is composed of IL-12p40 and IL-12p35, whereas IL-23 is composed of IL-12p40 and Il-23p19. IL-12p35 and IL-23p19 are closely related at the structural level. The sequence of the IL-12p35 subunit is homologous to IL-6 and granulocyte colony-stimulating factor (G-CSF), with the typical four α-helix bundle structure found in cytokines. The sequence of IL-12p40 is homologous to the extracellular portion of the receptor in the hemopoietin receptor family. The expression of each subunit for IL-12 is regulated independently. Since the genes for each subunit are found on different chromosomes, protein expression is regulated independently of each subunit. The p35 subunit of IL-12 is expressed ubiquitously and constitutively at low levels, whereas the p40 subunit is regulated at the transcription level and is induced by microbial products. The p40 subunit is produced by antigen-presenting cells, such as dendritic cells (DCs), monocytes, macrophages, neutrophils and, to a lesser extent, B cells. In order for a functional protein to be produced, both the p40 and p35 subunits of IL-12 must be produced in the same cell. The p19 subunit of IL-23 is also produced by antigenpresenting cells, such as macrophages and DCs, and also has sequence homology to IL-6 and G-CSF. As with IL-12, both subunits of IL-23 must be expressed in the same cell for the production of a functional protein [1–3,5–7]. The high affinity receptors for IL-12 and IL-23 are both heterodimeric proteins that are composed of a common subunit, IL-12Rβ1, which binds to IL-12p40. The IL-12p35 subunit binds to IL-12Rβ2 and the IL-23p19 binds to IL-23R. The receptors for IL-12 are found on activated T cells, natural killer (NK) cells, neutrophils, macrophages and DCs. The IL-23 receptors are found on activated memory T cells, NK cells, macrophages, neutrophils and DCs. For functional signaling to occur, the cytokine must bind to both subunits of the receptor. Once binding has occurred, there is an activation of Janus kinases and STAT signaling molecules; however, where IL-12 signaling induces a strong phosphorylation of STAT4 and a weak phosphorylation of STAT3, the reverse is true for IL-23 [5–10].