Parallel Measurements of Hundreds of TCR/pMHC Affinities Using The Carterra Surface Plasmon Resonance Technology

Key Highlights

• Carterra’s high-throughput surface plasmon resonance (HT-SPR) technology enables parallel measurements of hundreds of TCR/pMHC affinities at 37 °C within hours.
• Affinities measured with Carterra LSA® or LSA^XT are equal to those obtained with a Biacore T200.
• Carterra HT-SPR can easily be integrated into pipelines that aim to understand and exploit T-cell receptors,
  including clinical applications.

Introduction

T cells are essential in directing adaptive immune responses against infected or cancerous cells while avoiding harm to normal cells. This selective targeting is facilitated by the T-cell receptor (TCR), which recognizes peptide antigens presented by major histocompatibility complexes (pMHCs) on the surface of nearly all cells in the body (1). Unlike conventional surface receptors, the TCR is highly crossreactive, capable of recognizing hundreds to potentially millions of different pMHC molecules (2). This extensive crossreactivity is believed to be crucial for enabling a limited pool of T cells to recognize the vast array of potential non-self pMHCs.

There is a growing focus on harnessing TCRs to redirect immune responses toward various pMHC targets, particularly in cancer therapy (3, 4). This includes the development of soluble TCRs, such as the ImmTAX molecule tebentafusp, which is approved for the treatment of uveal melanoma, and adoptive cell therapies using TCR-T cells, like the clinically approved Afami-cel for synovial sarcoma.

A critical aspect of developing therapeutic TCRs in both academic and industrial settings involves characterising the on-target and offtarget pMHC binding properties of candidate TCRs. Central to this process is the accurate measurement of TCR/pMHC affinities. Two significant challenges hinder large-scale affinity measurements: the production of pMHC complexes and the measurement of affinities. Consequently, only a limited number of affinity measurements are typically performed for any given TCR.

Traditionally, producing large quantities of pMHCs has relied on standard protein production protocols, which are time-consuming and not easily scalable. Generating a single pMHC complex can take up to five days, making it impractical to produce hundreds of these molecules simultaneously. However, recent advancements have introduced more efficient  methods, such as the use of empty MHC molecules (5, 6), or MHCs with a placeholder peptide that is tethered to MHC via a protease-cleavable linker (7). These MHCs can be rapidly loaded with exogenous peptides within hours, enabling high-throughput peptide exchange, thus allowing the high-throughput parallel production of pMHC molecules.

The ability to conduct large-scale affinity measurements has been significantly enhanced by Carterra’s high-throughput surface plasmon resonance (HT-SPR) technology. This platform is now widely used to study various molecular interactions, including large-scale antibody-antigen binding studies. By using highly-sensitive SPR technology, its instrument is particularly well-suited for examining the typically weak interactions between TCRs and their many pMHC ligands.

In this white paper, we describe a workflow leveraging Carterra instruments to measure the affinities of two TCRs against a positional-scanning library of 163 pMHCs, resulting in a total of 326 TCR/pMHC affinity measurements within 16 hours. A subset of the data presented here have been published as part of a wider project on TCR cross-reactivity (8).