Suppression of Normal and Malignant Kit Signaling by a Bispecific Antibody Linking Kit with CD300a

Abstract

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.

The Biology and Disease Relevance of CD300a, an Inhibitory Receptor for Phosphatidylserine and Phosphatidylethanolamine

“…Intriguingly, upon cross-linking of the receptor with mAbs, only SHIP-1 associated with CD300a in the same cell type (19). Furthermore, immunoprecipitation of CD300a from human mast cells that were treated with Kit-CD300a, a bispecific Ab fragment linking Kit with CD300a, induced its tyrosine phosphorylation and the recruitment of SHIP but not SHP-1 (53). Triggering of CD300a with mAbs induced weak phosphorylation of SHIP-1 on human basophils, but the phosphorylation status of SHP-1 and SHP-2 was not tested (21).…”

“…Some investigators demonstrated that coligation of CD300a with FceRI is essential for the CD300a-mediated inhibitory signal (52). Instead, others showed that there is no requirement for the coligation of CD300a with the activating receptor for CD300a to inhibit the FceRI-mediated activation signal, despite the fact that coligation increased the inhibitory effect (19,53). Interestingly, the effect on mast cell survival did not change significantly when CD300a was coligated with Kit (19).…”

“…Hence, its engagement by agonist mAbs decreased NK cell-mediated cytotoxicity and inhibited IgE-dependent Ca 2+ mobilization and mediator release from mast cells and SCF-mediated mast cell activation, differentiation, and survival, as well as IgEinduced basophil degranulation (9,14,19,21,25,31,53,56). The mAb-mediated cross-linking of CD300a also reduces FcgRIIa-triggered reactive oxygen species production and Ca 2+ flux in neutrophils (18), suppresses eosinophil survival, migration, and inflammatory mediator production triggered by eotaxin, IL-15, and GM-CSF (23), and LPS-and CpG-induced IL-8 secretion by myelomonocytic cell lines (55).…”

“…The expression of CD300a on cell types, such as mast cells, eosinophils, and basophils, that have an important role in the initiation, regulation, and effector phases of allergic responses, along with its ability to downregulate their activity in response to diverse stimuli, led to the design of bispecific Ab fragments targeting CD300a, along with other receptors, with the goal of downregulating the function of these cells during disease conditions in mouse models. Bispecific Ab fragments specific for CD300a and c-Kit abrogated mast cell degranulation induced by SCF during cutaneous anaphylaxis (53). Another bispecific Ab fragment linking CD300a to IgE bound to FceRI, and, therefore, specific for FceRI-expressing cells (i.e., basophils and mast cells), was able to abolish allergic and inflammatory responses in OVA-induced acute experimental asthma and IgEdependent passive cutaneous anaphylaxis (56).…”

CD300a is expressed on human basophils and seems to inhibit IgE/FcεRI‐dependent anaphylactic degranulation

“…With respect to mast cells, it has been shown that cross-linking of CD300a with the high affinity receptor for IgE (FceRI) down-regulates IgE-mediated mast cell degranulation and cytokine production (17,18), whereas linking to c-Kit abrogates c-Kit-mediated cell differentiation, survival, and activation (19). Furthermore, CD300a shares 80% structural homology with CD300c (8,9), whose function remains to be elucidated.…”