The role of Lck inhibition on lymphocyte function
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Date
2025-01-15
Authors
Τυριτίδης, Ιωάννης
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Abstract
The Lymphocyte-specific protein tyrosine kinase Lck, a member of the evolutionarily conserved family of Src kinases (SFKs), is predominantly expressed in T cell and is responsible for the differentiation, proliferation, and activation of T cells, because of its vital role in the initiation of T cell receptor (TCR) signaling following engagement with an antigen. Based on its essential role in these cells, Lck is implicated in aberrant T-cell responses such as autoimmunity, leukemias, allograft rejections and Graft Versus Host Disease as well as the occurrence of toxicity accompanying immunotherapy using Chimeric Antigen Receptor (CAR)- T cells.
As a result, Lck has become a highly attractive target for the development of small molecule inhibitors. However, designing these inhibitors has been exceptionally challenging, as currently available compounds exhibit very low target specificity. The primary obstacle is the remarkable homology shared among SFK members within their catalytic centers, which makes the development of selective inhibitors exceedingly difficult. Recent studies from our laboratory and others have identified an N-terminal regulatory domain (LcKSH4) located outside the catalytic center, as mandatory for Lck anchoring on plasma membrane and its activity. Importantly, LckSH4 displays negligible homology with other members of the SFK family or any other kinases, making it a perfect target for selective Lck inhibition.
The goal of the current study is the efficient and highly selective inhibition of Lck by blocking the LckSH4 domain, which in turn will prohibit anchoring of the kinase at the plasma membrane and decrease its enzymatic activity. The LckSH4 blocking will be accomplished using nanobodies (anti-LckSH4 Nbs). Nbs are single-domain antibody fragments derived from heavy chain-only antibodies (HcAbs) which are naturally found in camelids. Nbs consist of one single variable heavy chain (VHH) region, which is the site of epitope binding. In recent years Nbs have been characterized as the “rising stars” of the antibody world, occupying a central position in cutting-edge applied medical research, particularly in the field of diagnosis and treatment of diseases. Due to their unique structural features and advantageous properties, Nbs are expected to quickly replace conventional monoclonal antibodies as therapeutic and diagnostic tools.
30 different anti-LckSH4 Nbs were tested for their ability to bind Lck intracellularly. Initial data revealed that the Nbs predominantly localized to intracellular compartments different from those of Lck, preventing effective binding. To address this, we introduced various N-terminal modifications to the Nbs with the aim to enforce their colocalization with Lck, thereby increasing the likelihood of successful Lck binding.
To select the top Nb Lck-binder, we assessed whether the N-terminal modifications successfully directed the Nbs to the same intracellular compartments as Lck, as well as their ability to bind Lck in different cell lines (HEK293 and Jurkat). Additionally, we evaluated the capacity of these Nbs to interfere with Lck activation and block Lck-mediated TCR signaling.
While the majority of N-terminal modifications successfully directed the Nbs to the same compartments as Lck, only one modification—the addition of the LckSH4 domain to NbGT, termed Lck-NbGT—resulted in significant Lck binding. The other modifications exhibited minimal or no binding. Importantly, Lck-NbGT was specific for Lck, as it failed to bind chimeric forms of Lck in which the LckSH4 domain had been replaced by those of other SFK members, and it did not interfere with signaling in B cell lines, in which another SFK member-Lyn is expressed. Although, our initial working hypothesis that Nb-mediated masking of LckSH4 will abolish Lck enzymatic activity, could not been proved, we identified a specific and efficient Lck binder, which we then employed for the selective knock down of the kinase.
Specifically, we repurposed the usage of Lck-NbGT as a mediator for Lck degradation using the proteasome system. Lck-NbGT was fused with the ubiquitin E3 ligase RNF4, with the purpose to bind and lead Lck to ubiquitin proteasome-mediated destruction. This fusion showed promising results since endogenous Lck expression levels were significantly reduced. Additionally, collective data from confocal microscopy and co-immunoprecipitation experiments revealed that NbGT can be used as a non-invasive, specific and high resolution Lck probe, able to pinpoint native Lck with high accuracy. Based on these data, we propose the broader usage of Nbs as a new biochemical tool to determine the intracellular localization of target molecules with superior resolution than the one provided by imaging techniques.
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Keywords
Lck, T cells, Nanobodies, Src family kinases