Multiformat T‐Cell‐Engaging Bispecific Antibodies Targeting Human Breast Cancers

Abstract

We generated four different formats of bispecific antibodies (bsAbs) consisting of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency, or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…4,12 However, the activity of T-cell redirecting bsAbs is dependent on multiple interrelated factors, and the identification of the desired target product profiles remains a difficult task; past experience had shown that engineered bsAbs do not always result in enhanced anti-tumor activity or improved therapeutic windows. 13,14 Factors to consider when optimizing the design and dosing regimen of T-cell redirecting bsAbs include the protein scaffold, binding affinities to receptors on effector cells (e.g., CD3) and target cells (e.g., CD19), selection of binding epitopes, the drug concentrations, receptor expression levels on effector and target cells, and the dynamic interplay between T-cell redirecting bsAbs and other forms of treatment. A holistic approach must consider all of the above factors, as well as the in vivo pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of the bsAbs.…”

Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection

“…Thus, high affinity or bivalent CD3 binding, at clinically-relevant concentrations, did not induce non-target—specific T cell activation, which requires cell-to-cell crosslinking as opposed to bivalent cis antibody engagement of CD3. Although others have observed that bivalent binding to CD3 does not increase T cell cytotoxic potency [51], current [21*, 52, 53] and future T cell redirecting strategies are sure to clinically evaluate whether high affinity CD3 binding results in optimal T cell redirection.…”

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer

“…MS analysis confirmed that the PNE fusions were not subject to post‐translational modification or proteolysis (Figure S4 and Table S2). The binding affinity of all of the switches to SKBR3 (Her2 3+, clinical immunohistochemistry score), MDA‐MB453 (Her2 2+), MDA‐MB231, MDA‐MB435 (Her2 1+), and MDA‐MB468 (Her2 0) cancer cells was assessed by flow cytometry. As shown in Figure S5, FITC‐ and PNE‐based switches bound to Her2‐expressing cancer cells to a similar extent as wild‐type 4D5 Fab (Table S3 and S4).…”

“…To determine the optimal switch/sCAR combination for targeting Her2‐expressing cells, we measured sCAR‐T cell activation with monovalent (HS75X and LS202X for FITC; HCNT and LCCT for PNE) and bivalent (LG68X/HS75X and LS202X/HK136X for FITC; NTBV and CTBV for PNE) switches against breast cancer cells with varying levels of Her2 expression (Figures S9 and S10) . In general, switches with FITC or PNE placed distal to the antigen‐binding domain provided the greatest activation.…”