Navigating the Unexpected 

After some further work to validate expression of the target on the cell line, the client managed to find one commercial detection antibody which gave the expected answer – a positive result on the overexpression cell line. With these cells provided to Isogenica, the project appeared to be back on track. After all, most antibody discovery scientists have encountered bad detection mAbs at some time or another.

However, testing the lead panel of VHH antibodies against these cells revealed an unexpected result: none bound to the target. This was an unexpected result, especially now that we had greater confidence in the cell line. Even more perplexing, a similar parallel project against a different target showed no such issues and was proceeding smoothly.

In response, both parties jointly re-evaluated the data so far; perhaps this was just bad luck, and we needed to expand the numbers. We provided a repeat of the selection campaign at no extra charge and identified a total of 220 unique VHH clones that specifically bound the target in ELISA. But still, none of them showed binding to the overexpression cell line.

While it’s normal in antibody discovery projects to experience a drop in hit numbers when transitioning from recombinant proteins tested in vitro to cell-assays, this scenario was very unusual. Together we all took another close look at the data.

Rethinking the Strategy:  Precision and Adaptability in Action

Rather than halting the project, Isogenica adapted its approach to address the problem logically: 

• Challenging Assumptions: Which antibody was right? The first one, which worked in ELISA but not on the overexpression line, or the second, which confirmed the target’s presence on the cell surface?

•  Expanded Selection Strategies: The selection process was optimized to increase the likelihood of identifying a broad range of antibodies. 
• Alternative screening: Apart from the overexpression cell line, what other cell lines were available

After looking again at the original cell line QC data, there were a few clues suggesting that the target on the cells was significantly different to the recombinant protein. It turned out that the partner had had difficulties making the cell line, and that was the only antibody they had tried which showed a positive result. These findings suggested that the issue lay in the behavior of the target protein on the cell surface, not the clones themselves. To address this, the client requested nearly 300 purified clones for further validation in additional engineered and natural cell lines they held in-house. 

Results: The Power of Collaboration 

After shipping the panel of 290 purified VHHs, the partner tested their binding specificity to multiple cell lines, with many in the panel demonstrated successful binding. This lead panel of VHH antibodies showed notable diversity in affinity and sequence, providing several options for future development. 

Building on this success, the client selected a lead panel, which Isogenica further characterized for biophysical properties and binding kinetics, with an example BLI trace shown in Figure 2.

Lessons Learned: Commitment and Communication

This case underscores the critical role of technical rigor and open communication in overcoming unforeseen challenges. Central to the project’s success was open discussion and data critique, a commitment to asking the right questions, and a shared focus on finding solutions.

At Isogenica, Ownership and Integrity is one of our core values. This means transparency if things don’t go to plan, and doing everything we can to get the desired outcome from your antibody discovery campaign. Our scientists follow your project all the way, so when the cell binding results come in, they remember those early QC experiments and can offer creative solutions. Even in the most complex situations, collaboration paves the way to success.