Article Text
PDF
Abstract
Background CD3 T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T-cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies, whereas suboptimal efficacy in the treatment of solid tumors and the risk of inducing cytokine release syndrome (CRS) continue to be major challenges. Several factors, such as format, affinity, and valency of the antibody, TAA copy number, antigen size and epitope can impact therapeutic efficacy and safety of a TCE. In this study, we developed a novel ‘2+1’ TCE platform, AnTenGager TM, which enables potent anti-tumor activity with reduced risk of CRS.
Methods Humanized CD3 antibodies was derived from mouse hybridoma, followed by affinity optimization utilizing Computer Aided Drug Design (CADD). Fab-scFv-Fab format was used as the backbone structure of AnTenGager TM. The anti-CD3 scFv sequences and the length of peptide linkers between VH/VL, scFv/CH1, and scFv/CH2 were screened and compared in a variety of preclinical assays. LALA mutations (L234A, L235A) were introduced to abolish the Fc receptor binding capability. In a series of in vitro and/or in vivo models, the efficacy and safety of AnTenGagers targeting multiple TAAs, such as Her2, GD2, CD20, LILRB4, and GPRC5D, were evaluated.
Results We generated a library of humanized parental CD3 antibodies with a broad spectrum of affinities, some of which cross react with monkey CD3. AnTenGagers exhibited limited binding to CD3-positive cells without TAA-crosslinking, suggesting reduced risk of CRS caused by systemic CD3 activation. For instance, four CD3 sequences with varying affinities were utilized to construct AnTenGagers with the same TAA-targeting arm. Compared to clinical benchmarks targeting the same TAA, all four AnTenGagers demonstrated substantially lower binding capacity to CD3+ cells without TAA-crosslinking, while inducing increased cytotoxicity against target-positive tumor cells (figure 1). In addition, they induced significantly less ex-vivo cytokine release by human PBMC than benchmarks. Notably, four AnTenGagers demonstrated enhanced in vivo efficacy compared to the benchmark, exhibiting complete response (CR) in PBMC humanized mouse tumor model. The serum concentration of proinflammatory cytokines was substantially lower in groups treated with AnTenGagers (figure 2).
Conclusions These results suggested that AnTenGager is promising platform to develop the next generation of TCEs with improved efficacy and safety profiles.
Ethics Approval Crownbio: 001243
AnTenGagers showed target-depend CD3 binding and cytotoxicity. Antengagers has reduced CD3 binding in the absent of TAA-crosslinking (left) and enhanced TAA-specific cytotoxicity compared with benchmarks (right).
Antengagers demonstrates in vivo efficacy and good safety profile in human PBMC reestablished MM tumor model. (A) Average tumor growth curves of PBMC humanized MM1S CDX model. Antengagers produced significant anti-tumor efficacy with TGI value of 102.46%, 100.55%, 102.46% and 102.46%, respectively compared with 57.82% of the 1+1 bivalent benchmark (BMK). (B) The concentration of cytokines in the serum collected 2 hours after the first dosing from mice shown in (A) Compared with BMK, ATG-021 induced reduced release of cytokines, such as IL-2, INF-γ, and TNF-α, indicating lower risk of cytokine release syndrome.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.