Bispecific Antibodies Targeting CD3 and FLT3

Introduction of CD3

CD3 is a protein complex functioning as a co-receptor for the T cell antigen receptor (TCR), playing a crucial role in T cell activation and signal transduction. Comprising four subunits—CD3γ, CD3δ, CD3ε, and CD3ζ—it forms three pairs of dimers: CD3γε, CD3δε, and CD3ζζ. These subunits associate with TCRαβ or TCRγδ to create the TCR complex on the surface of αβ or γδ T cells. CD3, part of the immunoglobulin superfamily, contains a single extracellular immunoglobulin domain. The intracellular tails of CD3 subunits feature conserved sequences known as immunoreceptor tyrosine-based activation motifs (ITAMs), crucial for TCR signaling. Initially expressed in the cytoplasm of developing T cells, CD3 later translocates to the membrane upon maturation. Upon antigen binding to TCR, phosphorylation of CD3ζ subunits at their ITAMs initiates downstream signaling pathways. CD3 serves as a common marker for T cells and is used for their identification via flow cytometry. Ongoing studies explore anti-CD3 antibodies as potential immunosuppressive agents for treating type 1 diabetes and other autoimmune diseases.

Introduction of FLT3

FLT3, a receptor tyrosine kinase, is frequently mutated or overexpressed in acute myeloid leukemia (AML) and other hematologic malignancies. Also known as CD135 or FLK-2, FLT3 exhibits low and restricted expression levels on normal hematopoietic stem and progenitor cells (HSPCs) and myeloid dendritic cells. Binding to its ligand, FLT3 ligand (FL), regulates cell survival and proliferation. In AML patients, FLT3 mutations, particularly the internal tandem duplication (ITD) in the juxtamembrane domain and point mutations like D835 in the activation loop, are associated with poor prognosis. ITD disrupts the autoinhibitory mechanism, resulting in constitutive FLT3 activation, while D835 mutation impairs kinase activity regulation. These mutations activate downstream signaling pathways, including RAS/RAF/MEK/ERK and PI3K/AKT/mTOR, contributing to leukemia initiation and progression. Despite the development of small molecule inhibitors and neutralizing antibodies targeting FLT3, resistance due to secondary mutations remains a significant challenge.

Fig.1 The FLT3 Tyrosine Kinase Comprising Five Domains (Zhao JC, 2022)

Signaling Pathways Involved in Bispecific Antibodies Targeting CD3 and FLT3

Bispecific antibodies (BsAbs) represent a groundbreaking advancement in cancer immunotherapy, engineered to simultaneously recognize two distinct antigens or epitopes. Within cancer immunotherapy, BsAbs play a pivotal role in activating T cells, fostering the formation of an immunological synapse to eliminate tumor cells—a strategy termed T cell-induced cytotoxicity (TACT). Notably, BsAbs targeting CD3 and FLT3 emerge as a novel category of TACT agents, leveraging T cell immune prowess to eradicate leukemia cells expressing FLT3. These BsAbs function by binding to CD3 on T cells and FLT3 on leukemia cells, establishing a bridge that brings them into close proximity. This interaction triggers the release of cytokines and cytolytic molecules, such as perforin and granzyme, from T cells, culminating in apoptosis of leukemia cells. Moreover, BsAbs exert influence over the survival and proliferation of leukemia cells by modulating downstream signaling pathways. For instance, BsAbs demonstrate the capability to inhibit the tyrosine kinase activity of FLT3, thereby impeding the signaling pathways activated by FLT3, including RAS/RAF/MEK/ERK and PI3K/AKT/mTOR.

Clinic Status of Bispecific Antibodies Targeting CD3 and FLT3

While no Bispecific antibodies targeting CD3 and FLT3 have received marketing approval, several are actively undergoing clinical trials, primarily focusing on the treatment of the treatment of AML and B-ALL.One promising candidate is CLN-049, an IgG heavy chain/single-chain variable fragment (scFv) fusion BsAb. It binds to the membrane proximal extracellular domain of FLT3 protein tyrosine kinase, enabling it to target leukemic blasts irrespective of FLT3 mutational status. CLN-049 has demonstrated favorable safety and efficacy in vitro and in vivo models, leading to its current evaluation in a phase I clinical trial (NCT04799466). This trial assesses its safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with relapsed/refractory AML and B-ALL.

Fig.2 Design of CLN-049

Another contender in clinical development is AMG427, a bispecific T cell engager molecule designed to target fms-like tyrosine kinase 3 (FLT3). Developed by Amgen in collaboration with Kyowa Kirin, AMG427 entered a phase 1 clinical trial in 2018. This trial aims to evaluate its safety, tolerability, pharmacokinetics, and preliminary efficacy in patients with relapsed or refractory AML.

Table 1. Comparison of Bispecific Antibodies Targeting CD3 and FLT3