Bispecific Antibodies Targeting CD3 and GPRC5D

Introduction of CD3

Introducing CD3 (Cluster of Differentiation 3), a pivotal T cell co-receptor comprised of four distinct subunits—CD3γ, CD3δ, CD3ε, and CD3ζ. This member of the immunoglobulin superfamily is situated on human chromosome 11. CD3 plays a crucial role in T cell antigen receptor (TCR) function, influencing TCR cell surface expression and signal transduction. Initially expressed in the cytoplasm during T cell development, CD3 subsequently relocates to the cell membrane. Activation of TCR occurs when CD3 recognizes the antigen peptide-major histocompatibility complex (pMHC), leading to the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) on the CD3ζ subunit. This process triggers downstream signaling pathways, including Ras-MAPK, PI3K-Akt, and NF-κB, impacting T cell proliferation, differentiation, activation, and effector functions. CD3 subunit defects result in severe combined immunodeficiency (SCID), characterized by T cell deficiency. Monoclonal antibodies targeting CD3, such as antithymocyte antibody (ATG), have gained approval for treating allograft rejection and show promise in addressing autoimmune diseases.

Introduction of GPRC5D

GPRC5D (G protein-coupled receptor class C group 5 member D) is an orphan G protein-coupled receptor found on human chromosome 12. This receptor, part of the 7-transmembrane protein family, remains elusive in terms of its endogenous ligand. With a 377-amino acid protein encoding, GPRC5D demonstrates wide expression in malignant plasma cells, hair follicles, and lungs, while remaining low or absent in normal tissues. Despite an unclear biological function, studies suggest GPRC5D may play a role in regulating cell cycle, apoptosis, and autophagy, potentially influencing the Ras-MAPK signaling pathway. Notably, GPRC5D is specifically overexpressed in multiple myeloma (MM) cells, making it a promising therapeutic target for MM. Its superior specificity compared to BCMA, coupled with independent expression, allows for flexible single and dual targeting of therapeutic agents.

Signaling Pathways Involved in Bispecific Antibodies Targeting CD3 and GPRC5D

The signaling pathways associated with bispecific antibodies targeting CD3 and GPRC5D can be categorized into three main groups: TCR-CD3 complex-activated pathways, GPRC5D-activated pathways, and bispecific antibody-induced pathways. TCR-CD3 complex-activated signaling pathways are initiated by bispecific antibody-mediated cross-linking of the TCR-CD3 complex in the presence of pMHC on MM cells. This activation leads to phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) on the CD3ζ subunit, recruiting and activating the ZAP70 kinase. ZAP70 subsequently phosphorylates downstream substrates like LAT and SLP76, forming a signalosome that activates Ras-MAPK, PI3K-Akt, and NF-κB. These signaling molecules play pivotal roles in regulating T cell proliferation, differentiation, activation, and effector functions, including cytokine production and cytotoxic granule release.

The GPRC5D-activated signaling pathways are triggered by the bispecific antibody binding to GPRC5D on MM cells. Although the exact mechanism of GPRC5D signaling remains unclear due to the unidentified endogenous ligand, studies suggest coupling to various G proteins, such as Gαs, Gαq, and Gαi. Downstream effectors like Ras-MAPK, PKA, PKC, and others may modulate MM cell growth, survival, and drug resistance by regulating cell cycle, apoptosis, and autophagy.

The bispecific antibody-induced signaling pathways are initiated by the bispecific antibody interacting with both CD3 on T cells and GPRC5D on MM cells. This interaction enhances the affinity and avidity of the bispecific antibody for both targets, resulting in increased T cell activation and MM cell killing. Additionally, the interaction may induce cytokine secretion by T cells, such as IL-2 and IFN-γ, amplifying T cell immune response and memory function through activation of STAT5 and JAK-STAT pathways.

Fig.1 Action Mechanism of BsAbs targeting CD3 and GPRC5D (Pillarisetti K, 2020)

Clinic Status of Bispecific Antibodies Targeting CD3 and GPRC5D

JNJ-64407564, also known as Talquetamab, stands out as a promising bispecific antibody designed to target CD3 and GPRC5D for the treatment of relapsed or refractory multiple myeloma. Currently advancing through a Phase II clinical trial, this novel therapeutic is under scrutiny to assess its safety and efficacy. Based on the most recent phase I clinical trial outcomes, Talquetamab exhibits excellent tolerability and demonstrates significant anti-tumor activity. Notably, Talquetamab holds the distinction of being the first drug to specifically target GPRC5D—an orphan receptor found in abundance in malignant plasma cells. GPRC5D's association with prognosis and drug resistance in multiple myeloma adds a critical dimension to the drug's significance. Talquetamab operates by inducing T cell-mediated killing of GPRC5D-positive multiple myeloma cells. This is achieved through the recruitment and activation of T cells, leading to inhibition of tumor formation and growth.

Fig.2 Talquetamab, a Bispecific Antibody for Multiple Myeloma (Chari A, 2022)

Table 1. BsAbs Targeting CD3 and GPRC5D for MM Therapy