Agent-Based Modeling of T Cell Receptor Cooperativity

Immunological synapse (IS) formation is a key event during antigen recognition by T cells. Recent experimental evidence suggests that the affinity between T cell receptors (TCRs) and antigen is actively modulated during the early steps of TCR signaling. In this work, we used an agent-based model to study possible mechanisms for affinity modulation during IS formation. We show that, without any specific active mechanism, the observed affinity between receptors and ligands evolves over time and depends on the density of ligands of the antigen peptide presented by major histocompatibility complexes (pMHC) and TCR molecules. A comparison between the presence or absence of TCR–pMHC centrally directed flow due to F-actin coupling suggests that centripetal transport is a potential mechanism for affinity modulation. The model further suggests that the time point of affinity measurement during immune synapse formation is critical. Finally, a mathematical model of F-actin foci formation incorporated in the agent-based model shows that TCR affinity can potentially be actively modulated by positive/negative feedback of the F-actin foci on the TCR-pMHC association rate kon.

Agent-Based Modeling of T Cell Receptor Cooperativity

Immunological synapse (IS) formation is a key event during antigen recognition by T cells. Recent experimental evidence suggests that the affinity between T cell receptors (TCRs) and antigen is actively modulated during the early steps of TCR signaling. In this work, we used an agent-based model to study possible mechanisms for affinity modulation during IS formation. We show that, without any specific active mechanism, the observed affinity between receptors and ligands evolves over time, and depends on the density of ligand pMHC (antigen peptide presented by major histocompatibility complexes) and TCR molecules. Comparison between the presence or absence of TCR-pMHC centrally directed flow due to F-actin coupling suggest centripetal transport is a potential mechanism for the affinity modulation. The model further suggests that the time point of affinity measurement during immune synapse formation is critical. Finally, a mathematical model of F-actin foci formation incorporated in the agent-based model, shows that TCR affinity can potentially be actively modulated by a positive/negative feedback of F-actin foci on the TCR-pMHC association rate kon.

Integrin Affinity Modulation Critically Regulates Atherogenic Endothelial Activation in vitro and in vivo

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5β1 integrins in endothelial cells in vitro, and α5β1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5β1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread, but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5β1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5β1 integrins in talin1 L325R cells partially restores fibronectin deposition, whereas NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

Antibody Structure and Function: The Basis for Engineering Therapeutics

Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.

The CC′ loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC′ loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC′ loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC′ loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC′ loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC′ loop in affinity modulation in the TIGIT/Nectin pathway. The CC′ loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

The CC′ loop of IgV domain containing immune checkpoint receptors: From a bystander to an active determinant of receptor:ligand binding

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks such as resistance, poor solid tumor penetration and so on, calls for the rational design of next generation of therapeutics. Soluble isoforms of negative regulators of immune checkpoints are expressed naturally and are shown to regulate the immune response, suggesting the soluble version of these molecules and affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To get a better insight on hotspot regions for modification, we have analysed structures of available immune receptor:ligand complexes containing IgV domains. Interestingly, this analysis reveals that the CC′ loop of IgV domain, a loop which is distinct from CDRs which are generally utilized by antibodies to recognize antigens, plays a pivotal role in affinity modulation. Here, we present several examples of cognate partner specific conformational variation observed in CC′ loop of several checkpoint receptor:ligand complexes. In addition,in silicoswapping of CC′ loop targeting TIGIT:Nectin-2/PVR pathway corroborated well with biophysically determined affinity values for these complexes. Thus, CC′ loop appears to be a hotspot for affinity modification without affecting the specificity to their cognate receptors, an important requirement to avoid unintended interaction of these modified molecules with undesired targets.