In the field of biopharmaceutical R&D, the development of candidate molecules is always accompanied by significant scientific challenges and clinical translation risks. Among these, off-target binding has increasingly drawn attention due to its potential clinical impact. As highly specific biologics, antibody drugs’ targeting accuracy directly determines the therapeutic window. However, due to the molecular complexity of biological systems—including protein homology and conformational polymorphism—antibody molecules may cross-react with unintended targets, a phenomenon known as polyspecific binding. For example, the anti-PD1 antibody SHR-1210 unexpectedly caused severe capillary hemangiomas in a phase I clinical trial, with studies indicating this might be related to its off-target binding to VEGFR2. In another case, the anti-β-amyloid antibody ABT-736 caused severe toxicity in monkeys due to off-target binding to platelet factors, forcing the termination of its development. This demonstrates that early precise detection and assessment of off-target effects is a key factor determining the success of antibody drug development.
MPSA-AB5000 Platform: A Powerful Tool Against Off-Target Issues
Kyinno Biotechnology has launched the MPSA-AB5000 antibody off-target screening platform, providing a comprehensive and efficient solution for antibody specificity screening and antigen recognition specificity assessment of CAR-modified immune cells. The MPSA-AB5000 platform offers unique technical advantages. It uses a reporter cell system combined with a no-wash strategy, avoiding false negatives caused by extensive washing steps in traditional methods when detecting weakly binding biologics. Its sensitivity exceeds technologies such as FACS. Meanwhile, the system amplifies signals to produce strong readouts even for weak receptor-ligand interactions. For instance, even weak interactions with micromolar dissociation constants, such as PD1 binding to PDL1, can be clearly distinguished.
It is worth noting that Kyinno Biotechnology has a mature gene knockout (KO) cell line construction platform, providing strong support for experimental optimization of the MPSA-AB5000 off-target screening platform. We have established an industry-leading HEK-293T gene knockout cell library covering numerous key targets, including immune checkpoint molecules (such as B7H3), tumor-associated antigens (such as HER2, EGFR), and signaling pathway node proteins. 293T cells, as a human cell line that is easily transfectable and relevant, are commonly used in protein overexpression and binding activity screening studies. However, high endogenous expression of certain proteins (such as B7-H3) in this cell line may mask important target and off-target interactions. In off-target screening experiments, these 293T KO cell lines effectively eliminate interference from non-specific binding, optimize background values, and make experimental results more accurate and reliable. For example, when performing off-target screening for an antibody targeting a tumor-associated antigen, using 293T cells with the antigen gene knocked out as target cells allows clear distinction of antibody binding signals to other potential targets, avoiding misinterpretation due to non-specific binding, and providing precise data support for antibody drug development.
Successful Cases and Broad Applications
As the only domestic company providing membrane protein screening and assessment services for biologic drug binding specificity, Kyinno Biotechnology has fully validated the reliability and effectiveness of the MPSA-AB5000 platform in practice. Over the past year, we have successfully completed more than 100 antibody and CAR protein specificity evaluation projects, with multiple projects utilizing CRISPR gene-edited 293T-KO cell lines to efficiently support antibody specificity screening.
The MPSA-AB5000 membrane protein off-target screening platform has a wide range of applications. Beyond antibody specificity screening and CAR-modified immune cell antigen recognition specificity assessment, it also plays an important role in drug target discovery and validation. Assisted by CRISPR gene-edited cell models, it enables rapid and efficient screening, discovery, and off-target evaluation of antibodies for all targets, opening new paths for antibody drug development.
Case 1: WT HEK293T Cells and HEK293T-B7H3-KO Cells
Case 2: WT HEK293T Cells and HEK293T-EGFR-KO Cells
Case 3: 293T-EGFR-B7H3-KO Cells
