Anthony Shock

The mechanistic impact of CD22 engagement with epratuzumab on B cell function: Implications for the treatment of systemic lupus erythematosus

Epratuzumab is a B-cell-directed non-depleting monoclonal antibody that targets CD22. It is currently being evaluated in two phase 3 clinical trials in patients with systemic lupus erythematosus (SLE), a disease associated with abnormalities in B-cell function and activation. The mechanism of action of epratuzumab involves perturbation of the B-cell receptor (BCR) signalling complex and intensification of the normal inhibitory role of CD22 on the BCR, leading to reduced signalling and diminished activation of B cells. Such effects may result from down-modulation of CD22 upon binding by epratuzumab, as well as decreased expression of other proteins involved in amplifying BCR signalling capability, notably CD19. The net result is blunting the capacity of antigen engagement to induce B-cell activation. The functional consequences of epratuzumab binding to CD22 include diminished B-cell proliferation, effects on adhesion molecule expression, and B-cell migration, as well as reduced production of pro-inflammatory cytokines, such as IL-6 and TNF. Studies in patients treated with epratuzumab have revealed a number of pharmacodynamic effects that are linked to the mechanism of action (i.e., a loss of the target molecule CD22 from the B-cell surface followed by a modest reduction in peripheral B-cell numbers after prolonged therapy). Together, these data indicate that epratuzumab therapy affords a unique means to modulate BCR complex expression and signalling.

Discovery and evaluation of N-(triazin-1,3,5-yl) phenylalanine derivatives as VLA-4 integrin antagonists

SAR studies aimed at improving the rate of clearance of a series of VLA-4 integrin antagonists by the introduction of a 1,3,5-triazine as an amide isostere are described.

CD22 and Autoimmune Disease

CD22 is a 140-kDa member of the Siglec family of cell surface proteins that is expressed by most mature B-cell lineages. As a co-receptor of the B-cell receptor (BCR), it is known to contribute to the sensitive control of the B-cell response to antigen. Cross-linking of CD22 and the BCR by antigen triggers the phosphorylation of CD22, which leads to activation of signaling molecules such as phosphatases. Signal transduction pathways involving CD22 have been explored in a number of mouse models, some of which have provided evidence that in the absence of functional CD22, B cells have a “hyperactivated” phenotype, and suggest that loss of CD22 function could contribute to the pathogenesis of autoimmune diseases. Modulating CD22 activity has therefore been suggested as a possible therapeutic approach to such diseases. For example, the novel CD22-targeting monoclonal antibody epratuzumab is currently under investigation as a treatment for the connective tissue disorder systemic lupus erythematosus (SLE).

Squaric acid derivatives as VLA-4 integrin antagonists.

SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.

Epratuzumab inhibits the production of the proinflammatory cytokines IL-6 and TNF-α, but not the regulatory cytokine IL-10, by B cells from healthy donors and SLE patients

IntroductionCytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD).MethodsPeripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10+ B cells from patients with SLE and HD were analyzed.ResultsThe secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR- and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10–producing B cells in culture was not affected by epratuzumab.ConclusionsEpratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.

Dehydrophenylalanine derivatives as VLA-4 integrin antagonists

We describe a series of dehydrophenylalanine derivatives where the Z isomers are potent VLA-4 antagonists but are subject to rapid biliary clearance and the E isomers have poor activity but have a slower rate of clearance. These configurationally constrained molecules have led to the design of a novel class of benzodiazepine VLA-4 antagonists.

Targeting CD22 with the monoclonal antibody epratuzumab modulates human B-cell maturation and cytokine production in response to Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) signaling

BackgroundAbnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement.MethodsB-cell subsets were isolated from human tonsils and stimulated with F(ab′)2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry.ResultsPretreatment of phenotypically naïve CD19+CD10–CD27– cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10–CD27– B cells. CD10–CD27–IgD– cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27hiCD38hi cells. Emab significantly inhibited the activation and differentiation of CD10–CD27–IgD– B cells into plasma cells.ConclusionsEmab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27–IgD– tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.

N-(Pyrimidin-4-yl) and N-(Pyridin-2-yl) phenylalanine derivatives as VLA-4 integrin antagonists

The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based VLA-4 antagonists are described.

α4 integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation

Although anti‐α4 integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil α4 integrins or indirectly on another cell type. The role of α4 integrins on the accumulation of 111In‐labelled eosinophils in allergic and non‐allergic inflammation in guinea‐pig skin was therefore investigated. Intradermal injection of antigen in sensitized skin sites induced accumulation of 111In‐eosinophils that was reduced up to 70% by two anti‐α4 integrin mAbs. In contrast, accumulation of 111In‐eosinophils to intradermal chemoattractants was unaffected by the same mAbs. Accumulation of 111In‐eosinophils in allergic and non‐allergic conditions was partly inhibited by a low dose of an anti‐β2 integrin mAb. In combination with anti‐α4 integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. Pretreating skin sites with antiserum or contaminating LPS did not reveal an α4 integrin dependent pathway for chemoattractant‐induced 111In‐eosinophil accumulation. These data suggest that α4 integrins are involved in the response to antigen in sensitized skin sites. Pretreating 111In‐eosinophil with α4 integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. These data support the concept that targeting α4 integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

Discovery and evaluation of potent, cysteine-based α4β1 integrin antagonists

Abstract Acyclic, disulphide derivatives of cysteine have been identified as moderately potent antagonists of α4β1-mediated leukocyte cell adhesion to VCAM. This communication describes how they were discovered from a simple l -cystine derivative and using the structure–activity data of C*DThioPC* related cyclic peptides.