In the field of immunotherapy drug development, B7H3 (also known as CD276) has become a hot target due to its high expression in various solid tumors as an emerging immune checkpoint molecule. However, antibodies or CAR-T cells targeting B7H3 often face major hurdles in preclinical research: on-target/off-tumor toxicity caused by low-level expression in normal tissues, and antibody cross-reactivity/off-target binding. Kyinno Biotechnology has developed a 293T-B7H3-KO cell line using CRISPR gene editing technology, which is now a critical tool to tackle these challenges and provides “nuclear-level precision” to the MPSA-AB5000 off-target screening platform.
I. The Two Major “Roadblocks” in B7H3 Drug Development
B7H3 promotes tumor immune evasion by suppressing T-cell activity within the tumor microenvironment, but its low-level expression in normal tissues (such as placenta, testes, and brain endothelial cells) poses unique challenges for drug development:
On-target/off-tumor toxicity: B7H3-targeting bispecific antibodies (BsAbs) or ADCs may attack normal cells expressing low levels of B7H3, causing nonspecific toxicity. Some studies have shown that B7H3 bispecific therapy can lead to adverse events such as skin rashes, though the exact mechanisms remain unclear. Based on its expression in fibroblasts and some normal tissues, researchers speculate these toxicities may be linked to nonspecific T-cell activation, though direct evidence is still lacking.
Cross-reactivity risk: Because B7H3 shares structural homology with other members of the B7 family (such as B7H1 and B7H4), some antibodies may cross-bind, potentially leading to immune dysregulation.
II. Targeting the Bystander Effect: Supporting ADC Drug Development
In vitro cytotoxicity experiments using 293T-B7H3-KO and wild-type 293T-B7H3 cells create the “perfect control”:
Experimental design: ADC drugs are co-cultured with cells expressing different levels of B7H3. Cytotoxicity is measured via flow cytometry. Killing activity against wild-type cells reflects “specific targeting activity,” while killing of KO cells directly reveals the “bystander effect” (non–B7H3-dependent cytotoxicity).
Data example: Kyinno Biotechnology’s gene-edited cells have already supported a B7H3×PD-L1 bispecific antibody study. Results showed ~20% killing in the co-culture group, while KO cells and isotype controls had bystander effects below 5%.
Empowering the MPSA-AB5000 Platform: Making Off-Target Signals “Unhideable”
The MPSA-AB5000 platform’s core advantage is its ability to capture weak binding signals, while the 293T-B7H3-KO cell line acts as a “cleaner” to eliminate background noise.
Off-target screening logic: When testing B7H3 antibodies, wild-type cell signals represent “specific binding + nonspecific binding,” while KO cell signals represent only “nonspecific binding.” The difference between the two is the true targeting activity.
For example, B7H3 and B7H4 share 37% sequence homology, making cross-reactivity difficult to distinguish with traditional methods. However, combining KO cells with the MPSA-AB5000 platform reduces experimental background noise. In one case, a candidate antibody showed high background specificity in conventional tests, but KO cell verification revealed potential cross-reactivity risks and prevented overlooked hazards in further drug development.
III. Beyond B7H3: The “Platform Value” of the CRISPR-Edited Cell Library
The success of the 293T-B7H3-KO cell line is only one example from Kyinno Biotechnology’s CRISPR-edited cell library. To date, the library covers over 80 targets (including CD47, EGFR, etc.), supporting diverse in vitro testing scenarios and off-target screening evaluations for molecules such as ADCs.
Gene-edited negative control cell lines are considered the “gold standard” for specificity evaluation in immunotherapy drug development. Kyinno Biotechnology not only provides customized 293T-B7H3-KO cells but also integrates them with the MPSA-AB5000 platform to deliver a one-stop “off-target screening + functional validation” solution. This ensures that every targeted drug can clearly “see” potential risks before entering clinical trials.
