Development of Monoclonal Antibodies Specific to Either CD300A R111 or CD300A Q111 or Both

Introduction

The CD300 family molecules are encoded by 8 genes mapped on chromosome 17 in humans and 12 genes on chromosome 11 in mice. They belong to the immunoglobulin (Ig) gene superfamily and contain one Ig-like domain in the extracellular portion. They are preferentially expressed on myeloid cells and mediate either activating or inhibitory signals through associated adaptor proteins, such as DAP 12 or FcɛRIγ, and immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region, respectively.^1,2

CD300a and CD300A, one of the CD300 family members in mice and in humans, respectively, have a long cytoplasmic region containing ITIM, which provides a docking site for Src homology 2 (SH2)-containing cytoplasmic phosphatases, including SHP1, SHP2, and SHIP^1,3 and mediates an inhibitory signal in myeloid cells upon binding to the ligands phosphatidylserine and phosphatidylethanolamine (PE).^4,5 We previously reported that CD300a suppresses innate immune responses by mast cells, dendritic cells, and macrophages in mice.^6–8 Moreover, blockade of CD300a signaling ameliorated the pathogenesis of allergies, including passive systemic anaphylaxis and airway inflammation, sepsis, enteritis, tumor formation, and acute ischemic stroke in mouse models.^9–11 These results suggested that a neutralizing anti-CD300A monoclonal antibody (mAb) may be useful for the treatment of these diseases. However, CD300A has a single nucleotide polymorphism (SNP) (rs2272111), which is a nonsense mutation encoding glutamine (CD300A^Q111) instead of arginine (CD300A^R111) at residue 111 in the Ig-like domain of CD300A.

The CD300 family molecules show high homology with each other. Among them, CD300A shows the highest homology with CD300C. Thus, the anti-CD300A mAb should specifically recognize CD300A but not other CD300 family molecules. In addition, mAbs specific to either CD300A^Q111 or CD300A^R111, but not to other CD300 family molecules, would also be useful for the analysis of CD300A genotype by flow cytometry. Therefore, anti-CD300A mAbs that specifically recognize either CD300A^Q111 or CD300A^R111, but not CD300C, would be useful for the analysis of CD300A genotype by flow cytometry. In addition, anti-CD300A mAbs specific to both CD300A^Q111 and CD300A^R111, but not CD300C, are required for the blockade of CD300A-mediated signaling as an antibody drug candidate. In this study, we successfully generated antibodies specific to either CD300A^R111 or CD300A^Q111 or both but not to CD300C.

Materials and Methods

Results and Discussion

We found that the extracellular portions of CD300A^R111 and CD300A^Q111 showed 81% and 80% homologies with that of CD300C, respectively (Fig. 1A). We established the BW5147 transfectants stably expressing CD300A^R111, CD300A^Q111, or CD300C (CD300A^R/BW, CD300A^Q/BW, or CD300C/BW, respectively). We found that TX49 clone, which was previously generated in our laboratory, ⁴ recognizes all CD300A^R111, CD300A^Q111, and CD300C (Fig. 1B). To establish mAbs specific to either human CD300A^R111 or CD300A^Q111 or both, but not to CD300C, we generated chimeric fusion proteins of the extracellular portion of CD300A^R111 or CD300A^Q111 with the Fc portion of human IgG (CD300A^R111–Fc and CD300A^Q111–Fc, respectively). We immunized mice with either CD300A^R111-Fc, CD300A^Q111-Fc, or both and obtained hybridomas secreting antibodies against CD300A. We successfully obtained mAbs TX112 and TX113, TX114, and EX67, which specifically recognize both CD300A^R111 and CD300A^Q111, CD300A^R111 alone, and CD300A^Q111 alone, respectively. However, all these clones did not bind to CD300C (Fig. 1B).

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FIG. 1.

Generation of anti-CD300A mAbs. (A) Amino acid sequences of the extracellular portions of CD300A^R111, CD300A^Q111, and CD300C. The red box indicates SNP (rs227221). (B) Flow cytometric analysis of the specificities of TX49, TX112, TX113, TX114, and EX67 mAbs to BW5147 transfectants expressing CD300A^R111, CD300A^Q111, or CD300C and the parental BW5147 cells. Representative histograms are shown. mAbs, monoclonal antibodies; SNP, single nucleotide polymorphism.

Flow cytometry analysis using TX112 mAb, which recognizes both CD300A^R111 and CD300A^Q111, but not CD300C, demonstrated that CD300A was expressed on all the subpopulations of monocytes in the human peripheral blood. CD300a expression was also detected on a subpopulation of B cells and CD16-CD56^hi NK cell population (Fig. 2A–C), consistent with previous reports.^12–15

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FIG. 2.

Expression of CD300A in humans and Macaca fascicularis. (A–C) Flow cytometric analysis of CD300A expressions on human PBMCs (A), each monocyte subpopulation (B), and NK cells subpopulation (C) by using TX112 mAb. (D) The amino acid sequence of the extracellular portions of CD300A in humans and Macaca fascicularis. (E) Flow cytometric analysis of CD300A expression on PBMC of Macaca fascicularis using TX112 mAb.

The extracellular portion of CD300A of Macaca fascicularis shows 78% homology to that of human CD300A (Fig. 2D). We found that TX112 mAb cross-reacted to Macaca fascicularis CD300A (Fig. 2E).

A genetic linkage study demonstrated that the SNP in the Ig domain of CD300A (rs2272111), which revealed 20%–30% frequency of the population, may link to psoriasis susceptibility. ¹⁶ In this study, we successfully generated TX114 and EX67 mAbs that distinguish CD300A^R111 from CD300A^Q111 and vice versa. These mAbs may be useful for the analysis of CD300A genotype by flow cytometry. Moreover, TX112 and TX113 recognize both CD300A^R111 to CD300A^Q111, but not CD300C. These mAbs may be useful for the development of antibody drugs because these mAbs can bind CD300A of any genotypes of patients, although further functional analyses of these mAbs should be required.