​Science Zhao Xiang reveals the precise regulation of the activity of T cell receptors using the reverse lock key for tumor immunotherapy

The T cell receptor (TCR) recognizes its ligand peptide-major histocompatibility complex (pMHC), and this protein interaction determines the antigen specificity and activation of T cells strength
.

In tumor immunotherapy, TCRs that recognize tumor-specific antigen pMHC can be used for cell therapy, but the activation response level of endogenous TCR to tumor antigen pMHC is often low
.

In order to improve the activation strength of TCR, the traditional method is to use the technology of affinity maturation to increase the activation level of TCR by increasing the affinity (KD) of TCR binding to antigen pMHC by tens of thousands of times
.

The method of affinity maturation has been proven effective in numerous clinical trials of cell therapy, but there are also some problems, such as excessive KD may cause TCR to lose the specificity of antigen recognition: TCR not only recognizes specific tumor antigens, but also starts Recognition of antigens in healthy tissue, i.
e.
off-target toxicity
.

KD is considered to be an important biochemical indicator that determines the activation level of TCR, but researchers have also found that KD is not necessarily positively correlated with the activation level of TCR in many special cases, indicating that there are other factors that determine the activation of TCR, such as inverse locking ( catch bond)
.

How to obtain TCR with low KD and high activation strength independent of KD increase may be an important method to solve the off-target toxicity of TCR in cell therapy
.

On April 8, 2022, the laboratory of K.
Christopher Garcia from Stanford University published a research result entitled Tuning T cell receptor sensitivity through catch bond engineering in Science as a Research Article
.

This study revealed that the activation of TCR is accompanied by the formation of the reverse lock bond between TCR and pMHC, and the activation level of TCR is positively correlated with the peak bond lifetime of the reverse lock bond; Lock key to obtain low KD, high activation level TCR, applying this inverse lock key engineering technology to melanoma antigen-specific TCR to obtain high activation level TCR mutants, and the new mutants are no longer available in previous clinical trials Reported off-target toxicity that attacks the patient's heart due to affinity maturation
.

Catch bond is a non-covalent interaction caused by external force, which describes an "abnormal" phenomenon in the interaction between some proteins, that is, under the influence of shear tension of the order of pN, protein The bond lifetime between proteins increases with the increase in the strength of the applied pulling force.
Instead of dissociating between proteins, they are locked into a bound state by an external force
.

The opposite of inverse locking is the slip bond, in which the lifetime of the bond between proteins decreases as the strength of the applied pulling force increases
.

The inverse lock and slip bonds together form a pair of molecular switches that regulate the interaction of some proteins in physiological activities, such as the adhesion of E.
coli to the urethra, the adhesion of leukocytes to endothelial cells near the infected area, the TCR-pMHC interaction, and Notch signaling.
Pathway activation, actin-myosin interaction and filament depolymerization,
etc.

K.
Christopher Garcia's laboratory in the past research, found that an HIV-derived antigenic peptide (HIV), after forming a complex with B35 MHC (B35-HIV), B35-HIV and two TCRs (TCR589 and TCR55) With similar binding affinity KD; B35-HIV can activate TCR589, but cannot activate TCR55, indicating that KD cannot explain why TCR55 can bind its specific ligand, but cannot turn on TCR signaling
.

Biomembrane force probe (BFP) experiments found that B35-HIV can form an inverse lock bond with TCR589, but form a slip bond with TCR55, indicating that the inverse lock bond is not only necessary for TCR activation, but also TCR may be activated by Reverse the transformation and formation of the lock key to obtain TCR mutants with limited KD changes but significantly increased activation strength
.

The authors first hypothesized that by mutating some residues in the complementarity determining regions (CDRs) of TCRs to charged or polar amino acids, new inverse lock bonds could be generated in the TCR-pMHC interaction
.

These selected residues are generally at least 0.
4 nm away from the pMHC, thus preventing over-enrichment of affinity matured mutants
.

The TCR library was displayed on T cell lines by lentiviral transduction.
After antigen stimulation, the T cells displaying the TCR library were stained with CD69 antibody to mark the activation intensity of different TCR mutants, and pMHC tetramer staining was used to mark different TCR mutations.
The affinity of the body to bind to pMHC
.

Next, the T cell library was sorted.
In order to obtain TCR mutants with low KD and high activation levels due to the enrichment of inverse locking, the sorting target regions were high levels of CD69 antibody staining and low pMHC tetramer staining.
level
.

The authors firstly obtained two different TCR55 mutants by designing a TCR55 library and performing functional selection: one mutant only has a mutation of alanine at residue 98 of the TCR55 chain to histidine, and the other One mutant had only one mutation of alanine to aspartate at residue 50 of the TCR55 chain
.

Both single amino acid mutants were activated by B35-HIV and maintained the KD for binding to B35-HIV at the level of wild-type TCR55
.

BFP experiments found that the mechanism was that the slip bonds were transformed into inverse lock bonds in the mutants
.

In addition, the authors found that residue 98 of the TCR55 chain and residue 50 of the TCR55 chain are hotspot residues for inverse lock engineering, since these two residues also allow the formation of a variety of other polar or charged amino acids mutated to form Reverses the key and is activated by B35-HIV
.

Further research found that the activation level of different TCR55 mutants was not correlated with KD, but positively correlated with the peak bond lifetime during the formation of reverse locked bonds, indicating that the activation level of TCR can be precisely regulated by adjusting the peak bond lifetime
.

The authors used a Jurkat T cell signaling pathway reporter system to demonstrate that the reverse-lock-engineered TCR55 mutant can normally activate downstream signaling pathways such as ERK, p38, and NFAT
.

In addition, the author also used a "smart bead" platform technology BATTLES (Biomechanically-Assisted T-cell Triggering for Large-scale Exogenous-pMHC Screening), B35-HIV under physiological concentration conditions was plated on the smart beads, and the smart beads passed the At different temperatures, changing the size and size of the T cells attached to it exerted an external force, thereby simulating the formation of inverse bonds and activating T cells, demonstrating that the engineered TCR55 mutants can also be activated by pMHC at physiological concentrations
.

In order to verify whether inverse key engineering can be applied to tumor antigen-specific TCR without off-target toxicity, the authors selected a well-known cell therapy failure case: after affinity maturation of the specific wild-type TCR of the melanoma antigen MAGE-A3, A mutant A3A TCR with hundreds of times higher affinity and greatly enhanced activation strength was obtained
.

The A3A TCR was responsible for the deaths of two patients in clinical trials due to the discovery of massive T-cell infiltration in the heart and cardiac damage
.

The study found that the A3A TCR produced off-target toxicity, not only recognizing the tumor antigen MAGE-A3, but also recognizing the short peptide derived from the protein TITIN expressed in the heart, which caused the attack of the A3A TCR on the heart
.

The authors designed a library based on wild-type TCR and performed functional screening to obtain 13 different TCR mutants that could be activated by MAGE-A3, and none of these mutants could be activated by TITIN
.

The KD of these 13 mutants for binding to MAGE-A3 was 10-50 times lower than that of A3A, but the KD for binding to TITIN was too low to be detected, indicating that one of the mechanisms by which off-target toxicity was removed is that these TCR mutants no longer Combined with TITIN
.

The authors selected two of the TCR mutants (94a-14 and 20a-18) for further study, and found that both can form inverse lock bonds with MAGE-A3, but only form slip bonds with TITIN, indicating that off-target toxicity is removed.
Another mechanism is the inverse lock key/slip key conversion
.

The activation intensity of these two TCR mutants was still positively correlated with peak bond lifetime, and in primary T cells, they still exhibited tumor cell-killing ability almost close to that of the A3A TCR
.

To further rule out the off-target toxicity of other unknown proteins introduced by inverse key-binding engineering, the authors constructed a peptide-HLA-A1 library by yeast display technology, screened any short peptides that could bind to TCR mutants, and performed bioinformatics to predict which themselves Short protein peptides may activate these TCR mutants
.

The screening results found that for affinity matured A3A TCR, not only MAGE-A3 and TITIN were predicted to be high-ranked short peptides that could activate A3A TCR, but the authors also found MAGE-A6 and FAT2, two other possible off-target toxic protein short peptides
.

However, for the inverse-key engineered TCR mutants, only the tumor antigen MAGE-A3 was highly predicted to be a short peptide that could activate the TCR mutant, and none of the other predicted short peptides could be activated, and TITIN no longer appeared in the predicted list.
in
.

In summary, the authors further elucidate the importance of inverse lock bond formation for TCR activation by in-depth study of the mechanism of TCR activation, and found that the level of TCR activation can be precisely regulated by regulating the peak of bond lifetime
.

Based on this, the authors further proved that the inverse lock key is a biophysical indicator that can be engineered.
By enriching the inverse lock key, the activation level of the antigen pMHC for TCR can be greatly improved without greatly increasing the binding affinity
.

This finding can be exploited in the engineering of tumor-specific TCRs and further in adoptive cell therapy and tumor immunotherapy
.

Dr.
Zhao Xiang from Stanford University is the first author of the research paper, and Professor K.
Christopher Garcia is the corresponding author
.

Prof.
Brian Evavold and Dr.
Elizabeth Kolawole from University of Utah, Prof.
Ronald Germain and Dr.
Waipan Chan from NIH, Prof.
Polly Fordyce, Dr.
Feng Yinian, and Dr.
Yang Xinbo from Stanford University have made important contributions
.

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