Description

• CD16A (FcγRIIIA) is a low-affinity IgG Fc receptor primarily expressed on the surface of natural killer (NK) cells, macrophages, monocytes, and mast cells. It recognizes and binds to the Fc region of antibodies (e.g., IgG), triggering NK cells to release cytotoxic granules (e.g., granzymes, perforin) and pro-inflammatory cytokines (e.g., IFN-γ, TNF-α), thereby killing target cells. It also enhances NK cell-target cell tight contact to improve killing efficiency. Dysregulation of CD16A function is closely linked to diseases such as cancer and autoimmune disorders. Current drug development targeting CD16A focuses on enhancing ADCC (antibody-dependent cellular cytotoxicity) effects and activating NK cells through targeted therapies.
• B-hCD3E/hCD16A plus mice was obtained by B-hCD3E mice(110008) cross breeding with B-hCD16A plus mice(112446). The exons 2-6 of mouse Cd3e gene that encode the extracellular domain is replaced by human CD3E exons 2-7 in B-hCD3E/hCD16A plus mice. The exons 1-5 of mouse Fcgr4 gene that encode signal peptide, extracellular domain and transmembrane domain are replaced by human FCGR3A counterparts with V158 variant in B-hCD3E/hCD16A plus mice.
• Human CD3E was exclusively detectable in T cells of homozygous B-hCD3E/hCD16A plus mice, but not in wild-type C57BL/6JNifdc mice.
• Human CD16A was detected in NK cells, macrophages and monocytes, but not in granulocytes. We predicate that CD16A has similar expression pattern between human and B-hCD3E/hCD16A plus mice.
• B-hCD3E/hCD16A plus mice provide a powerful preclinical model for in vivo evaluation of CD16A-targeted mAbs, BiKEs, TriKEs, NK cell engager, etc.

Targeting strategy

Gene targeting strategy for B-hCD3E/hCD16A plus mice.

The exons 2-6 of mouse Cd3e gene that encode the extracellular domain is replaced by human CD3E exons 2-7 in B-hCD3E/hCD16A plus mice.

The exons 1-5 of mouse Fcgr4 gene that encode signal peptide, extracellular domain and transmembrane domain is replaced by human FCGR3A counterparts with V158 variant in B-hCD3E/hCD16A plus mice. The genomic region of mouse Fcgr4 gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are replaced by human counterparts.

Protein expression analysis

Strain specific human CD3E expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hCD16A mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hCD16A mice (H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse CD3E antibody (BioLegend, 100312) and anti-human CD3E antibody (BD, 562426) by flow cytometry. Human CD3E was exclusively detectable in T cells of homozygous B-hCD3E/hCD16A plus mice, but not in wild-type C57BL/6JNifdc mice. Mouse CD3E was detectable in T cells of wild-type C57BL/6JNifdc mice.

Protein expression analysis in NK cells

Strain specific human CD16A expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hCD16A mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hCD16A mice (H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse Fcgr4 antibody (Biolegend, 149503) and anti-human CD16A antibody (Biolegend, 302008) by flow cytometry. Human CD16A was exclusively detectable in NK cells of homozygous B-hCD3E/hCD16A plus mice, but not in wild-type C57BL/6JNifdc mice. Mouse Fcgr4 was not detectable in NK cells of wild-type C57BL/6JNifdc mice.

Protein expression analysis in granulocytes

Strain specific human CD16A expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hCD16A mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hCD16A mice (H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse Fcgr4 antibody (Biolegend, 149503) and anti-human CD16A antibody (Biolegend, 302008) by flow cytometry. Human CD16A was not detectable in granulocytes of homozygous B-hCD3E/hCD16A plus mice. Mouse Fcgr4 was detectable in granulocytes of wild-type C57BL/6JNifdc mice.

Protein expression analysis in macrophages

Strain specific human CD16A expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hCD16A mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hCD16A mice (H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse Fcgr4 antibody (Biolegend, 149503) and anti-human CD16A antibody (Biolegend, 302008) by flow cytometry. Human CD16A was detectable in macrophages of homozygous B-hCD3E/hCD16A plus mice with a low level. Mouse Fcgr4 was detectable in macrophages of wild-type C57BL/6JNifdc mice.

Protein expression analysis in monocytes

Strain specific human CD16A expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hCD16A plus mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hCD16A mice (H/H) (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse Fcgr4 antibody (Biolegend, 149503) and anti-human CD16A antibody (Biolegend, 302008) by flow cytometry. Human CD16A was detectable in monocytes of homozygous B-hCD3E/hCD16A plus mice with a low level. Mouse Fcgr4 was not detectable in monocytes of wild-type C57BL/6JNifdc mice.