IL-2R binders queued for testing
Starting in summer 2021, we ran 6 rounds of IL-2R binder design puzzles. Now we’ve selected 115 promising Foldit designs to test for binding in the lab! In early 2022, we will conduct a FACS experiment to test if these protein designs successfully bind to the IL-2R target.
Targeting IL-2R for cancer treatment
IL-2R is a protein found on the surface of human immune cells, and is composed of three different protein chains (α, β, and γ). Due to its role in regulating the immune system, IL-2R is an important target in cancer immunotherapy. However, IL-2R drugs are associated with severe side effects that seem to arise from over-activation of the α chain.
The goal of the Foldit IL-2R puzzles is to design a protein binder for the α chain of IL-2R, to block over-activation by immunotherapy drugs. The designed protein could potentially be given to cancer patients alongside normal immunotherapy to reduce its side effects. (In a different approach, other researchers have recently designed a protein binder for IL-2R β/γ that avoids the α chain altogether; that protein is currently in Phase I clinical trials.)
Selecting Foldit solutions for testing
For this experiment, we did not factor in AlphaFold confidence when selecting designs to test, even though the AlphaFold tool was available in some of the IL-2R binder puzzles. However, most of the selected designs have AlphaFold confidence greater than 75%, so we think they have a good chance of folding.
2011731_c0001 Crossed Sticks
2011731_c0024 Mike Cassidy
2011731_c0038 Bruno Kestemont
2011852_c0001 Bletchley Park,BootsMcGraw
2011852_c0028 Mike Cassidy
2011852_c0029 Bruno Kestemont
2011852_c0032 Bruno Kestemont
2011852_c0044 silent gene
2011926_c0001 Crossed Sticks
2011926_c0003 toshiue,Bruno Kestemont,Phyx
2011926_c0004 Bletchley Park,spvincent
2011926_c0007 Bruno Kestemont
2011926_c0009 Mike Cassidy
2011926_c0011 Bletchley Park
2011926_c0061 Bletchley Park,spvincent
2011958_c0011 Crossed Sticks
2011958_c0049 silent gene
2012023_c0004 silent gene
2012023_c0007 Bruno Kestemont
2012023_c0035 Bruno Kestemont
2012023_c0038 Bruno Kestemont
2012150_c0032 Crossed Sticks
2012150_c0046 Bruno Kestemont
2012150_c0070 Bruno Kestemont
2012150_c0093 Bruno Kestemont
We start with the amino acid sequence of each protein design and reverse-translate it into a DNA sequence. This custom DNA is ordered from a specialized company, and the DNA is inserted into yeast so that the yeast cells can produce our proteins and display them on the cell surface. Finally, we use fluorescent tags and microfluidics technology to sort out the yeast cells that can bind to our target protein--in this case, the α chain of IL-2R. See this blog post for a full description of the experiment.
Diversifying the testing pool
To increase our chances of success and make the most of Foldit players’ work, we again used a grafting technique to expand the diversity of the 115 Foldit designs.
We combine the binding interfaces from Foldit solutions with a large library of stable scaffold proteins to make variations of the original Foldit designs. This boosts the number of proteins in our testing pool, and allows us to more thoroughly test the binding interfaces designed by Foldit players. Our grafting method is described in more detail in this previous blog post about binders for MERS-CoV spike.
Diversifying the testing pool helps to bank against cases where the interface looks good but the binder protein fails to fold correctly. If your binder protein misfolds, then the interface residues will not be correctly positioned to bind the target. It doesn’t matter how good your DDG or Contact Surface metrics look if your protein design doesn’t fold!
By grafting the interface residues of each Foldit design onto a diversity of protein scaffolds, we generate multiple designs with the same interface, but with (potentially) very different folding behavior. This maximizes the chances that at least one of these proteins will fold correctly and present the designed interface to bind the target as intended.
We used this grafting method to generate over 1700 variations of the original 115 Foldit designs. Together with a third set of experimental re-designs (described in a future blog post), we’ll be testing a total of 1997 IL-2R binder designs from the work of Foldit players. These will be tested at the Institute for Protein Design alongside 30,000 additional designs from IPD researchers.
A big thank you goes to all Foldit players who participated in our IL-2R binder puzzles! We are very excited to get some experimental data about how these Foldit solutions behave in the real world. Keep an eye out for experiment results in the early months of 2022. In the meantime, happy folding!( Posted by bkoep 60 500 | Thu, 12/16/2021 - 00:26 | 1 comment )