Biophysical differences in IgG1 Fc-based therapeutics relate to their cellular handling, interaction with FcRn and plasma half-life.

Antibody-based therapeutics (ABTs) are used to treat a range of diseases. Most ABTs are either full-length IgG1 antibodies or fusions between for instance antigen (Ag)-binding receptor domains and the IgG1 Fc fragment. Interestingly, their plasma half-life varies considerably, which may relate to how they engage the neonatal Fc receptor (FcRn). As such, there is a need for an in-depth understanding of how different features of ABTs affect FcRn-binding and transport behavior. Here, we report on how FcRn-engagement of the IgG1 Fc fragment compare to clinically relevant IgGs and receptor domain Fc fusions, binding to VEGF or TNF-α. The results reveal FcRn-dependent intracellular accumulation of the Fc, which is in line with shorter plasma half-life than that of full-length IgG1 in human FcRn-expressing mice. Receptor domain fusion to the Fc increases its half-life, but not to the extent of IgG1. This is mirrored by a reduced cellular recycling capacity of the Fc-fusions. In addition, binding of cognate Ag to ABTs show that complexes of similar size undergo cellular transport at different rates, which could be explained by the biophysical properties of each ABT. Thus, the study provides knowledge that should guide tailoring of ABTs regarding optimal cellular sorting and plasma half-life.

A TRAV26-1-encoded recognition motif focuses the biased T cell response in celiac disease.

The semi-public T-cell response towards the gluten epitope DQ2.5-glia-α2 uses a prototypic TCR encoded by the germline segments TRAV26-1 and TRBV7-2. Through mutagenesis experiments, we show that a TRAV26-1encoded recognition motif contacts the MHC ß-chain and the TCR CDR3ß loop underpinning this conserved T-cell response restricted to the prototypic TCRs.

Targeting the MHC Ligandome by Use of TCR-Like Antibodies.

Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide-major histocompatibility molecule (pMHC) complexes are often referred to as "TCR-like" mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.

Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease.

BACKGROUND & AIMS: Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. METHODS: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). RESULTS: Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. CONCLUSIONS: A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.

Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture.

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system.

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

A TCRα framework-centered codon shapes a biased T cell repertoire through direct MHC and CDR3ß interactions.

Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3ß loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3ß loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3ß. This allowed prediction of expanded DQ2.5-glia-α2-reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues.

TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions.