Bispecific Antibodies Targeting CD3 and CD16A

Introduction to CD3

CD3 serves as a coreceptor of the T cell receptor (TCR), forming a complex of four polypeptide chains (γ, δ, ε, and ζ) that associate with the TCR α and β chains, collectively forming the TCR-CD3 complex. The CD3 chains, encoded by genes on chromosome 11, possess a single extracellular immunoglobulin-like domain, a transmembrane region with charged residues, and an intracellular tail with immunoreceptor tyrosine-based activation motifs (ITAMs). Expressed on all mature T cells, CD3 plays an essential role in T cell activation, differentiation, and survival. Additionally, CD3 is involved in various immune-mediated diseases, such as autoimmune disorders, allergies, graft-versus-host disease (GVHD), and cancer.

Introduction to CD16A

CD16A, also known as FcγRIIIA or low-affinity receptor IIIa for the Fc fragment of IgG, is a member of the immunoglobulin superfamily. It consists of two extracellular Ig-like domains, a transmembrane region with a charged residue, and a short cytoplasmic tail without ITAMs. Encoded by a gene on chromosome 1, CD16A is expressed on natural killer (NK) cells, macrophages, mast cells, and monocytes. CD16A can bind to IgG1 and IgG3 subclasses with low affinity, mediating antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, cytokine production, and cell activation. Additionally, CD16A is implicated in various diseases, such as infections, autoimmune disorders, inflammation, and cancer.

Signaling Pathways Involved in Bispecific Antibodies Targeting CD3 and CD16A

The signaling pathways involved in BsAbs targeting CD3 and CD16A are mainly derived from the activation of T cells and NK cells by their respective receptors. Upon binding to a BsAb that recognizes a TAA on tumor cells, CD3 undergoes conformational changes and recruits several protein tyrosine kinases, such as Lck, Fyn, ZAP-70, and Syk. These kinases phosphorylate multiple tyrosine residues on the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 subunits, creating docking sites for downstream signaling molecules. The activation of phospholipase C-γ (PLC-γ) leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG activates protein kinase C-θ (PKC-θ), which in turn activates the nuclear factor-κB (NF-κB) pathway via the IKK complex. IP3 induces the release of calcium from the endoplasmic reticulum (ER), which triggers the opening of calcium channels on the plasma membrane and the influx of extracellular calcium. The increased intracellular calcium concentration activates calcineurin, which dephosphorylates nuclear factor of activated T cells (NFAT), allowing its translocation to the nucleus. NF-κB and NFAT cooperate with activator protein-1 (AP-1), which is activated by the mitogen-activated protein kinase (MAPK) pathway via Ras/Raf/MEK/ERK cascade. These transcription factors regulate the expression of genes involved in T cell activation, proliferation, differentiation, survival, and effector functions.

Similarly, CD16A also initiates ITAM-based signaling upon binding to a BsAb that recognizes a TAA on tumor cells. However, unlike T cells that express their own kinases, NK cells rely on the transmembrane adaptor protein DNAX-activation protein 12 (DAP12) to provide ITAMs for CD16A signaling. DAP12 associates with CD16A via electrostatic interactions between their transmembrane domains. The phosphorylation of DAP12 ITAMs by Src-family kinases recruits Syk, which activates PLC-γ and leads to DAG and IP3 production. DAG activates PKC-δ, which activates NF-κB via the CARMA1/Bcl10/MALT1 complex. IP3 induces calcium mobilization and calcineurin activation, which activates NFAT. In addition to PLC-γ, Syk also activates the MAPK pathway via Vav1/Ras/Raf/MEK/ERK cascade. The MAPK pathway activates AP-1 and also regulates the expression of microRNAs that modulate NK cell functions. NF-κB, NFAT, and AP-1 induce the expression of genes involved in NK cell activation, cytotoxicity, cytokine production, and survival.

References

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