High-throughput antibody discovery targeting cell surface receptors for signaling blocking in cancer

The use of monoclonal antibody-based therapeutics to target and treat cancer is considered to be one of the most successful therapeutic strategies within the oncology field to date. Both unconjugated and conjugated antibodies are currently approved by the FDA for cancer therapy, capitalizing on methods of action spanning from direct inhibition of cell survival pathways to the delivery of cytotoxic drugs. Specific cell surface antigen recognition is paramount to the success of these drugs, necessitating high-resolution, early-stage characterization of lead antibody drug candidates prior to preclinical development.

Major advancements in high-throughput flow cytometry have enabled a more thorough analysis of on-cell antigen specificity and function earlier in the drug discovery process, allowing for more exquisite antibody discovery within the oncology field. In this webinar, Tracey Mullen, COO of Abveris, reviews an antibody discovery campaign targeting a cell surface receptor implicated in cancer. Methods for immunization, antibody discovery, and subsequent functional characterization are also discussed.

Watch this webinar to receive a detailed overview of high-throughput antibody characterization in antibody discovery workflows and to learn the importance of leveraging high-content screening as early in the discovery process as possible to improve the likelihood of campaign success.

Read on for highlights from the live Q&A session or register to watch the webinar at any time that suits you>>

Q: In our workflow, we often screen by ELISA first and then flow, to reduce costs and workload. How do the results of that strategy compare to those when you do all the screening on the cells?

TM: In general, we always recommend performing primary and secondary fusion screening in the most relevant format for your downstream application. If your end-use requires on-cell binding, the antibodies should be assayed first—and as early as possible—for on-cell binding to increase the chance of success. The biggest issue we occasionally run into when screening via ELISA first and flow later is the potential mismatch between the recombinant protein and the cell surface protein, which can result in attrition between ELISA and flow assays. Depending on how representative your recombinant protein is of the on-cell protein, this may or may not be an issue. If you're able to validate the recombinant protein ahead of time and deem it biologically and functionally relevant, this approach shouldn't be a concern. But if there is an observed difference between the two, this will play out in your antibody triage and you'll likely see significantly fewer flow positives than ELISA positives.

Q: Do you have experience with cell or DNA immunizations for cell surface targets when the recombinant protein isn't available?

TM: Yes, to both. We have experience with both whole-cell and genetic vaccination. If you are unable to generate a recombinant protein due to the complexity of the cell surface protein that you're looking to target, we can incorporate those types of immunization strategies into that campaign. We combine these with proprietary adjuvant strategies that we work with, and we also have a host of proprietary methods for DNA immunizations for different types of targets that we leverage as well.

Q: What if we don't have a recombinant protein and still want to affinity rank?

TM: For most programs in which a partner requests affinity ranking for a panel of antibodies against a cell surface receptor, we will typically run on-cell apparent KD assessments in which we titrate the antibody concentrations and generate EC50 values for each interrogated antibody candidate. The iQue software makes this particularly easy: once you input the concentration series, it will generate a dose-response curve and automatically fit that curve to calculate the EC50 value for you. That enables us to very rapidly and easily rank the candidates by affinity.

Q: How do you confirm antibody specificity in your system?

TM: It depends on the system. If we're using our single B-cell screening platform, we will screen for specificity upfront on the Beacon instrument, with multiple back-to-back assays, depending on how deeply we're looking to characterize those individual antibodies. We can look at things like species cross-reactivity, specificity to different proteins, off-target binding or lack thereof, and/or binding to closely related family members. We can also perform cell surface binding analyses as well.

If we're using a hybridoma-based approach, our tactic is slightly different. We have to prioritize two reagents to screen during our primary fusion screening stage, so we’ll decide what our two most important specificity requirements are and screen for these first. Then, as we start to whittle down the number of potential candidates, we’ll expand out the number of screening reagents we use at the secondary screen stage. We'll screen for specificity not just with ELISA and flow cytometry, but also incorporate the Octet or Carterra platforms for additional specificity screening.

Q: Have you run a side-by-side epitope binning analysis of your DiversimAb mouse alongside a wild-type mouse as a function of immunization strategy? If so, do you see a loss of diversity with longer immunizations?

TM: We do have a case study in which we compared the epitopic diversity coming out of DiversimAb mice with that of wild-type BALB/c mice, in which we also incorporated multiple cohorts of DiversimAb immunization strategies—accelerated and extended schedules. In general, we found that the BALB/c epitopes were covered by the DiversimAb animals, with some additional epitopes targeted by the DiversimAb animals exclusively. More interestingly, we didn't observe a loss of diversity when comparing the antibodies generated by animals undergoing accelerated versus the extended immunization schedules. Of course, that's just a single recombinant protein study. We would need to do many more comparison studies to know for sure whether these two immunization strategies consistently deliver comparable diversity in our DiversimAb mice, but this was certainly a promising case study.

Q: Have you tested different mouse species in your experiments for higher efficiency?

TM: Yes, we often run multiple cohorts of immunizations incorporating different strains. It’s challenging to predict which strain will be more promising, so we prefer to generate broad diversity to start and then triage from there.

Q: How long, how many injections, and how many cells are needed for cell immunization?

TM: It depends on whether we're following an accelerated immunization schedule or extended, but in general, we tend to inject around five million cells per mouse per dose. The duration, again, will depend on the actual immunization strategy followed; if accelerated, the duration is three weeks and we'll inject every two to three days for a total of eight injections. For our extended immunization strategy, we inject once weekly for a total of five weeks before titer tests, and then potentially up to eight if required.

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