De novo-designed transmembrane domains tune engineered receptor functions
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Curated by eLife
Evaluation Summary:
This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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- Synthetic Biology (eLife)
Abstract
De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.
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Evaluation Summary:
This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, …
Evaluation Summary:
This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. During the design process it was important to explicitly reward sequence diversity as low diversity sequences (i.e. many leucines) produced the lowest energies when evaluated on the target backbone, but showed little specificity for a single conformation when docking simulations were performed with the designs. After experimentally evaluating a couple rounds of designs, the investigators settled …
Reviewer #1 (Public Review):
This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. During the design process it was important to explicitly reward sequence diversity as low diversity sequences (i.e. many leucines) produced the lowest energies when evaluated on the target backbone, but showed little specificity for a single conformation when docking simulations were performed with the designs. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.
In vitro experiments showed a clear correlation between oligomerization state of the CAR and cytokine secretion when CAR T cells were exposed to HER2+ cancer cells. The higher order oligomers also were more effective at slowing tumor growth in mice injected with HER2 tumor cells. These results confirm previous observations that dimeric CARs (via disulfide formation) are more effective than monomeric CARs. One exciting finding was that the designed CAR tetramer was as effective at suppressing tumor growth in mice as a standard CAR construct used in the field (transmembrane domain derived from CD28), but the tetramer CAR stimulated less cytokine release than the CD28TM CAR in vitro. The CAR T therapies currently used in the clinic frequently stimulate dangerous levels of cytokines, if a CAR T cell can be created that is as effective as current treatments but overall cytokine release is lowered, this could be an improvement over current treatment options. One caveat about the efficacy data presented in this paper is that CAR T cells were administered to the mice only a single day after injection of the cancer cells. More rigorous tests of efficacy will be needed to determine if the tetrameric CAR is on par with standard constructs used in therapy.
One thing that struck me is that the protein design process contained no consideration for how activation signals are transmitted from the extracellular domain (ECD) of the CAR to the intracellular activation domains. Does this suggest that specific conformational changes are not a part of the activation process? It would be great if the authors could comment on this. Do the results say anything about how binding to the ECD does or does not activate signaling?
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Reviewer #2 (Public Review):
The authors designed and computationally refined a set of novel self-associating TM helices which were shown to form dimers and trimers by X-ray crystallography. The dimer (Car2) and trimer (Car3) as well as a modelled tetramer (Car4) were then fused to extracellular antigen-recognition and intracellular signalling domains. They were then compared to a monomer (Car1) and the original CD28 TMD in their ability to kill cancer cells in vitro and in vivo and to release various cytokines. Also, the authors show that a specific amino acid motif within the wild-type CD28 TMD of the CD28 CAR mediates interaction with the endogenous CD28 and that this is responsible for cytokine release, an undesired side reaction associated with cancer cell killing. Since their CARs with de novo designed TMDs show much reduced …
Reviewer #2 (Public Review):
The authors designed and computationally refined a set of novel self-associating TM helices which were shown to form dimers and trimers by X-ray crystallography. The dimer (Car2) and trimer (Car3) as well as a modelled tetramer (Car4) were then fused to extracellular antigen-recognition and intracellular signalling domains. They were then compared to a monomer (Car1) and the original CD28 TMD in their ability to kill cancer cells in vitro and in vivo and to release various cytokines. Also, the authors show that a specific amino acid motif within the wild-type CD28 TMD of the CD28 CAR mediates interaction with the endogenous CD28 and that this is responsible for cytokine release, an undesired side reaction associated with cancer cell killing. Since their CARs with de novo designed TMDs show much reduced cytokine release, this confirms their notion that non-natural TMDs would isolate CARs from endogeneous CD28. The novel functional aspect about the designer CARs presented here is thus that cytokine secretion is less troubling when compared with the original CD28 CAR28. Most interestingly, they find that the antitumor activity of the constructs tested in an engineered mouse tumour model as well as induced cytokine release scales with the oligomeric state.
In sum, the work is not only very elegant from a membrane protein engineering point of view. Rather, it represents a fine example of how protein design can be translated into medical applications. -
Reviewer #3 (Public Review):
The paper by Elazar et al describes a highly significant and rigorously performed study that addresses the influence of TMD-mediated chimeric antigen receptor (CAR) oligomerization on the antitumor activity and cytokine release of CAR-T cells.
The transmembrane helices (TMHs) of most single-pass transmembrane receptors have long been considered as mere membrane anchors, and hence their function in receptor signaling has often been neglected. In recent years, however, several studies have found that TMH oligomerization can play an active and often essential role in the signaling mechanism of receptors including growth factor receptors, death receptors, and immune receptors. In this study, the authors have demonstrated that designed TMH oligomerization, which mediates CAR oligomerization, can have profound …
Reviewer #3 (Public Review):
The paper by Elazar et al describes a highly significant and rigorously performed study that addresses the influence of TMD-mediated chimeric antigen receptor (CAR) oligomerization on the antitumor activity and cytokine release of CAR-T cells.
The transmembrane helices (TMHs) of most single-pass transmembrane receptors have long been considered as mere membrane anchors, and hence their function in receptor signaling has often been neglected. In recent years, however, several studies have found that TMH oligomerization can play an active and often essential role in the signaling mechanism of receptors including growth factor receptors, death receptors, and immune receptors. In this study, the authors have demonstrated that designed TMH oligomerization, which mediates CAR oligomerization, can have profound impact on the activity and cytokine release of CAR-T cells. Their findings have illuminated another dimension for CAR-T/NK engineering and optimization.
Specifically, the authors first performed de novo design of completely new TMHs that can form stable parallel dimer, trimer, or tetramers, as validated by SDS-PAGE and crystallography. Then, by replacing the TMH of the most used CARs, which is the TMH of CD28, with the designed TMHs, they found that the antitumor activity of the CAR-T cells in mice scaled linearly with CAR oligomeric state encoded by the designed TMH. More strikingly, their in vitro assays showed that the CARs with the designed TMHs all induced 2-10-fold less cytokine release than the CAR with CD28 TMH, raising the suspicion that the CD28 TMH may pair CAR with endogenous T cell CD28 leading to higher level of CD28 signaling independent of the CARs. Indeed, they have shown that a set of mutations in the CD28 TM of the regular CAR that is likely to disrupt TMH homodimerization led to 2-6-fold reduction in CAR-T cell cytokine release. Collectively, their results suggest that there is ample room for improving the CAR-T antitumor activity / cytokine release ratio by optimizing the CAR TM sequence, and it remains to be seen if this approach can be used to achieve better outcomes in higher animals.
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