Red Light, Green Light: Precise cell targeting with Co-LOCKR

Written by AE SPENS

Cancer cells are not monolithic. The individual cells that make up solid tumors or blood leukemias often show striking heterogeneity, or variation. The Achilles Heel of many cancer therapeutics is their inability to deal with heterogeneity. “A single antigen rarely distinguishes cancer cells from healthy tissue,” explains Dr. Marc Lajoie, a former Postdoc in the lab and Co-Founder and current CEO of Outpace Bio. “Rather, it is the unique combinations of antigens caused by aberrant gene expression patterns that distinguish cancer.” That is, healthy cells might display protein A or protein B on their surface whereas cancer cells display both. A therapy that targets these markers one at a time would kill both intended and unintended targets. But a therapy that only acts on cells displaying both proteins? Bingo. The labs of Cancer Consortium members Dr. David Baker, Dr. Stan Riddell, and Dr. Suzie Pun contributed to a new technology published in Science, called “Co-LOCKR”, that addresses this problem.

Co-LOCKR stands for Colocalization-dependent Latching Orthogonal Cage/Key pRoteins. The system was designed by Drs. Lajoie, Boyken, and Salter, from the Baker and Riddell Labs, to lock onto cells following Boolean AND, OR, and NOT operators. Engineered proteins referred to as the “Cage” and “Key” can be created to recognize specific and distinct cell surface markers. The synthetic proteins are then mixed with heterogeneous populations of cells and Co-LOCKR is recruited to cells that display the matching combinations of markers and avoid cells that lack either of the markers. Once the Cage and Key co-localize on a cell’s surface, they change shape to reveal a hidden segment of protein that can execute additional functions like fluorescing (glowing) or recruiting cytotoxic T-cells.

‍

‍

Original art by Ian Haydon shows the Cage (left, with beige helices) and Key (right, with blue helix) co-localizing on a cell expressing two targeted surface antigens. The proximity-based binding of this Cage and Key cause the Cage to unfold a functional domain that can recruit cytotoxic T-cells, generate fluorescence, and more.Original art by Ian Haydon

‍

Using Cages that fluoresce when active, the authors first showed that Co-LOCKR was only functional when bound to the cell surface rather than when floating untethered in solution. It was also capable of specifically labelling sub-populations of cells expressing distinct antigens in a heterogeneous pool. Following this proof of concept, the authors tested if Co-LOCKR could be designed to mark cells displaying two proteins but ignore those expressing a third. They accomplished this by designing a “decoy” Cage that lacked the functional domain for fluorescing but bound a third marker and could act as a sponge sequestering the Key. When they mixed the functional Cage, the Key, and the decoy Cage with cells displaying either two or three markers, they found that the functional domain activated on the cells expressing the first two antigens but not the cells expressing all three antigens. Finally, they re-engineered the functional Cage so that it would recruit cytotoxic T-cells when bound by the Key instead of merely glowing. Remarkably, they were able to target T-cells to cells based on recognition of surface markers following each of the same AND, OR, and NOT logical functions. This specificity of T-cell targeting could be a game-changer for CAR T cell therapies because nearby “healthy bystander cells expressing a subset of the same antigens would be spared,” said Dr. Lajoie.

Co-LOCKR isn’t ready for the clinic yet, but researchers are hopeful it could be soon. “There’s substantial work still to be done,” said Dr. Lajoie. “We’re working on next-gen versions of Co-LOCKR that exhibit improved properties in vivo.” As their approach relies on designing specific synthetic proteins, rather than tweaking or adapting pre-existing ones, they were able to create from first principles a fundamentally new biological function (logic gating) that offers a potential path to safer CAR T-cell therapies in the future. Developing technologies like Co-LOCKR is the bread and butter of the Baker Lab, and often leads to very productive collaborations with other labs in the CCG. “The fields of de novo protein design and cell therapy each separately are poised for major inflections, and the opportunity to bring them together in this Baker–Riddell collaboration highlights the incredible opportunity for synergy,” said Dr. Lajoie. “Also, from a personal standpoint, I’m grateful that this collaboration led to great friendships with Alex and Stan.”

This work was supported by the HHMI, the Open Philanthropy Project, the NSF, the DTRA, the Nordstrom Barrier IPD Directors Fund, the Washington Research Foundation and Translation Research Fund, the Audacious Project organized by TED, and the NIH. MJL was supported by a Washington Research Foundation Innovation Postdoctoral Fellowship and a Cancer Research Institute Irvington Fellowship from the Cancer Research Institute. SEB was supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface. AIS was supported by the FHCRC interdisciplinary training grant in cancer research and the Hearst Foundation. AO was supported by NIH NCI grants.

Cancer Consortium members Dr. David Baker, Dr. Stan Riddell, and Dr. Suzie Pun contributed to this work.

MJ Lajoie, SE Boyken, AI Salter, J Bruffey, A Rajan, RA Langan, A Olshefsky, V Muhunthan, MJ Bick, M Gewe, A Quijano-Rubio, J Johnson, G Lenz, A Nguyen, S Pun, CE Correnti, SR Riddell, and D Baker. 2020. Designed protein logic to target cells with precise combinations of surface antigens. Science. 369: 1637-1643.

‍