A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.

Multivalent pIX phage display selects for distinct and improved antibody properties.

Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration. This approach was combined with the use of an engineered helper phage known to improve antibody pIX display and retrieval. By direct comparison with pIII display, we demonstrate that antibody display using this pIX system translates into substantially improved retrieval of desired specificities with favorable biophysical properties in de novo selection. We show that the effect was due to less E. coli host toxicity during phage propagation conferred by the lack of a signal sequence. This pIX combinatorial display platform provides a generic alternative route for obtaining good binders with high stability and may thus find broad applicability.

Phage Display Engineered T Cell Receptors as Tools for the Study of Tumor Peptide-MHC Interactions.

Cancer immunotherapy has finally come of age, demonstrated by recent progress in strategies that engage the endogenous adaptive immune response in tumor killing. Occasionally, significant and durable tumor regression has been achieved. A giant leap forward was the demonstration that the pre-existing polyclonal T cell repertoire could be re-directed by use of cloned T cell receptors (TCRs), to obtain a defined tumor-specific pool of T cells. However, the procedure must be performed with caution to avoid deleterious cross-reactivity. Here, the use of engineered soluble TCRs may represent a safer, yet powerful, alternative. There is also a need for deeper understanding of the processes that underlie antigen presentation in disease and homeostasis, how tumor-specific peptides are generated, and how epitope spreading evolves during tumor development. Due to its plasticity, the pivotal interaction where a TCR engages a peptide/MHC (pMHC) also requires closer attention. For this purpose, phage display as a tool to evolve cloned TCRs represents an attractive avenue to generate suitable reagents allowing the study of defined pMHC presentation, TCR engagement, as well as for the discovery of novel therapeutic leads. Here, we highlight important aspects of the current status in this field.

Chimeric anti-CD14 IGG2/4 Hybrid antibodies for therapeutic intervention in pig and human models of inflammation.

CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases.

Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display.

We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random mutation scTCR phage libraries were prepared in E. coli over-expressing the periplasmic chaperone FkpA, and such over-expression during library preparation proved crucial for successful downstream selection. The thermostabilized scTCR(mut) variants selected were produced in high yields and isolated as monomers. Thus, the purified scTCRs could be studied with regard to specificity and equilibrium binding kinetics to pMHC using surface plasmon resonance (SPR). The results demonstrate a difference in affinity for pMHCs that display germ line or tumor-specific peptides which explains the tumor-specific reactivity of the TCR. This FkpA-assisted thermostabilization strategy extends the utility of recombinant TCRs and furthermore, may be of general use for efficient evolution of proteins.

Posttranslational modification of gluten shapes TCR usage in celiac disease.

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vß6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vß6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3ß loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.

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

BACKGROUND: 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. RESULTS: 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 Valphabeta domain orientation was far superior to the Vbetaalpha domain orientation regarding monomeric yield of functionally folded molecules. CONCLUSION: 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.