CD16A and NKP46-Based Therapy

• Gene Information: CD16A: Encoded by the FCGR3A gene on chromosome 1q23.1 and is a low-affinity activating Fcγ receptor for IgG. The V158F polymorphism modulates IgG binding affinity and clinical response to antibody therapies. NKP46: A transmembrane glycoprotein, located on chromosome 19q13.42. It belongs to the immunoglobulin superfamily and serves as a natural killer (NK) cell specific activating receptor that plays a critical role in NK-cell development, activation, and antitumor/antiviral surveillance throughout the innate immune system.

• Protein Expression: CD16A: Mainly expressed on NK cells, as well as on monocytes, macrophages, DCs and γδ T cell subsets. It associates with FcRγ/CD3ζ adaptor chains that contain ITAM motifs. NKP46: Expressed almost exclusively on natural killer (NK) cells, beginning at the early NK-cell developmental stage and persisting through mature NK cells.

• Signaling Pathway: CD16A: Upon binding IgG-opsonized targets, CD16A clustering triggers ITAM phosphorylation via Src-family kinases, activating downstream signaling. This leads to NK-cell activation, cytokine release (e.g., IFN-γ, TNF-α), and ADCC via perforin/granzyme-mediated cytotoxicity. CD16A signaling can be downregulated by ADAM17-mediated shedding. NKP46: A key activating receptor of the natural killer (NK) cell receptor complex. By associating with ITAM-containing adaptor molecules, it initiates signal transduction and activates downstream pathways including PI3K/AKT, MAPK/ERK, PLCγ, and NF-κB.

• Therapeutic Activation: CD16A and NKP46 targeting antibodies recruit and activate NK cells by simultaneously binding NKP46 and CD16A on NK cells, leading to targeted killing of different types of tumor cells depending on the TAA branch. Ongoing efforts aim to further enhance efficacy, durability, and safety through optimized Fc engineering.

CD16A

• The exons 1-5 of mouse Fcgr4 gene that encode signal peptide, extracellular domain, transmembrane domain, and cytoplasmic region are replaced by human FCGR3A(F158V) exons 1-6 in B-hCD16A/hNKP46 mice (CB-17 SCID).

• The promoter and 5’ UTR region of the mouse gene are replaced by human counterparts. The human FCGR3A expression is driven by human FCGR3A promoter, while mouse Fcgr4 gene transcription and translation will be disrupted.

NKP46

• The exons 1-7 of mouse NKp46 gene that encode the extracellular domain were replaced by human NKP46 exons 1-7 in B-hCD16A/hNKP46 mice (CB-17 SCID).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Splenocytes were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Splenocytes were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Blood cells were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Blood cells were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Bone marrow cells were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human CD16A expression analysis in multiple lymphocyte subtypes. Bone marrow cells were collected from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). CD16A expression on various types of immune cells was analyzed by flow cytometry using species-specific anti-mouse CD16.2 antibody (Biolegend, 149503) and anti-human CD16A antibody (BioLegend, 302008).

Mouse and human NKP46 expression analysis in multiple lymphocyte subtypes. Cells were collected across different tissues from wild-type CB-17 SCID mice (+/+) and homozygous B-hCD16A/hNKP46 mice (CB-17 SCID) (H/H) (female, 6-week-old, n = 3). NKP46 expression was analyzed with anti-mouse NKP46 antibody (eBioscience™, 17-3351-82) and anti-human NKP46 antibody (Biolegend, 331916) by flow cytometry.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and bone marrow cells were isolated from female CB-17 SCID and B-hCD16A/hNKP46 mice (CB-17 SCID) (female, 7-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.