5 min read
By Joshua Lowitz on 01/12/2021
Editor’s note: Welcome to 2021, and to our first in a year-long series of posts that will explore the essentials in antibody discovery. We’re starting off with a frequently under-appreciated technique for getting to “yes” or “no” faster — target analysis. Next up, in early February, we’ll be talking about fusions. Thanks for reading, and we invite your ideas on what you consider to be “the essentials” of antibody research success.
The First Step in Increasing the Odds of Hitting Your Target
“Measure twice, cut once” is one of the most practical and logical pieces of advice we learn growing up. And while the folks who first published this maxim back in the Middle Ages obviously weren’t referencing life sciences, that phrase can — and should — be applied to virtually every aspect of the process underlying scientific research.
Take target analysis in antibody discovery, for instance.
How Skipping Ahead Risks Putting You Behind
Too often, there’s a tendency in antibody discovery campaigns to jump directly into immunization with an available antigen, under the assumption that as long as there is an immune response that the right antibodies will be “in there somewhere.” This would be like cooking dinner without checking for all the ingredients in your fridge first. To paraphrase the immortal Ferris Bueller, drug discovery “ . . . moves pretty fast. If you don’t stop and look around once in a while, you could miss it.”
- Immunogens can take many forms, and there may be particular biological functions that need to be identified first and addressed through the antigen format.
- There are many adjuvants to choose from for immunization — each with its own advantages (and, in turn, potential disadvantages) in concert with specific antigens.
- Protocols for immunization are varied, and some may be more appropriate than others depending on the target and desired antibody application.
- Depending on the biology and structure of the target, a particular species (e.g., mouse, rat, etc.), strain (i.e., MHC haplotype) or otherwise engineered animal model (like a transgenic or knockout) may be particularly advantageous for immunization.
- Finally, you may be looking for the resulting antibody to have a specific application or function that could be affected by your choice of assays. (So, for example, an antibody that works in a flow assay may not be appropriate if you are hoping to run a Western Blot as well.)
You’ll see there’s a theme here: Looking ahead (or, better still, thinking and planning ahead) at the onset of antibody discovery isn’t so much a “nice-to-have” luxury as it is a “need-to-have” prerequisite. Proceeding directly to immunization with even the most highly reputable protein as a foundation might not be the best course.
A Proven Way Forward
If target analysis isn’t typically on the “lab bench” in your campaigns today, there’s an easy way to make it part of your future repertoire. It starts with asking a handful of key questions that help establish a clear and reliable pathway to success:
- What immunogens make the most sense? Some examples:
- Proteins: Is purified or recombinant protein available? Is the available protein bioactive and appropriately formulated? Has it been tested for endotoxin? If available from multiple sources, what criteria should you use when choosing one?
- Peptides: Is the target difficult to produce recombinantly or purify? Is it highly conserved, or are there any conformational issues that could exist? In many cases, a synthetic peptide fragment, if appropriately designed, can be the best immunogen.
- Cells: For some membrane protein targets, immunizing with cells is the only way to present a native, properly folded antigen. In most cases, engineering a syngeneic mouse or rat cell line over-expressing the target is required to avoid background response.
- DNA: Is the DNA vector appropriate for immunization? Without the appropriate enhancers, promoters and codon optimization, it may not generate a robust response.
- Are there conformational aspects to the immunogen that will be disrupted by certain adjuvants? For example, some adjuvants might denature the protein in a different way so that certain epitopes are linearized or exposed; anticipating those variables can help you decide which adjuvant is most appropriate.
- How is the immunogen presented (think “route, frequency, timing and dosing”)?
- What’s the principal biology of the target you’re exploring? What’s the proverbial “bigger picture”?
An “Everything-To-Gain” Proposition?
Answering key target analysis questions doesn’t necessarily require hitting the “pause” button on your research. In fact, in the end, this approach can ultimately both improve the efficiency, expedite downstream processes and enhance the cost-effectiveness of your overall discovery efforts.
Target analysis can represent a natural “arm” of your research and immunization can, if necessary, happen in parallel with it. The goal is to increase your odds of a successful “hit.” Put another way, the operative question is “How do we enhance our odds of prompting the right response in the most direct way?” rather than “How do we enhance our odds of prompting any response in the most direct way?”
So, you naturally might ask, does target analysis lead to success 100 percent of the time? Unfortunately not. Even when you start with that as your first step, there’s no guarantee that you’ll always get a hit. But if you don’t do it and something goes awry in the middle of the campaign, it’s almost certain that you’ll have to start all over (and there’s certainly no way of getting back the month or two lost while waiting on the results).
The bottom line? You have nothing to lose and everything to gain through target analysis.
Putting Goals First
Because we believe so strongly in the value of target analysis, we typically offer it as a complementary first step for our clients. It’s a natural part of our process at Antibody Solutions (not to mention a reflection of our commitment to full transparency in the scientific process and how we collaborate with your team). We can’t prepare an accurate work order, after all, without a plan for how we intend to pursue your target.
Fit-for-purpose antibodies rarely pop up out of the blue (or culture plate, in this case). It’s thoughtful, precise science that yields responses and delivers results.
Pairing Up The Art And Science Of Target Analysis
There’s an art, too, that accompanies the science of target analysis. Nuance — the kind of insights only revealed through firsthand experience — matters.
We touch on some of those firsthand insights in our scientific poster, “Generation of Antibodies to Difficult Membrane Protein Targets,” which explains some of the key dynamics that influence how an antibody is generated. You also can gain an understanding of how we incorporate the target analysis process to streamline our work on our clients’ behalf and improve their odds of earlier success.
And since we prefer dialogues to monologues, we’d welcome your perspectives on how target analysis plays a role in your current research as well as the potential value it could offer in your future work.
Written by Joshua Lowitz
Josh is the Manager of Project Management of Antibody Solutions. Since joining the company in 2010, Josh has served multiple roles at the company and is currently providing scientific support and guidance to the Project Management team as well as helping clients achieve their antibody discovery and research needs.