Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins

<p>Abstract</p> <p>Background</p> <p>Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either V_αor V_β, with the well characterized murine TCR, 2C.</p> <p>Results</p> <p>Several TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading.</p> <p>Conclusions</p> <p>We show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs <it>in vitro </it>and <it>in vivo</it>. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.</p

Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

<p>Abstract</p> <p>Background</p> <p>Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs.</p> <p>Results</p> <p>The effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules.</p> <p>Conclusion</p> <p>The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.</p

A Practical Approach to T-Cell Receptor Cloning and Expression

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5′-RACE amplification. We here present an improved 5′-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality

T Cell Recognition of the Dominant I-Ak–Restricted Hen Egg Lysozyme Epitope: Critical Role for Asparagine Deamidation

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL48–62) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-Ak in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide–MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity

A human endothelial cell-based recycling assay for screening of FcRn targeted molecules.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation. Nat Commun 2018 Feb 12; 9(1):621

Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage

Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Methodology/Principal Findings: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS6 or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Conclusions/Significance: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited