Background Several peptide-HLA targets for T cell receptor (TCR)-based immunotherapies are currently being evaluated in the field, however, many are limited by their overall low prevalence, low copy numbers or relevant expression in healthy tissues. A T cell target with nearly ideal properties has high, homogenous and prevalent expression across multiple cancers in the absence of significant safety/toxicity liabilities. Here, we describe the in-depth characterization of an HLA-A*02:01-presented peptide derived from the cancer germline antigen preferentially expressed antigen in melanoma (PRAME) that opens an avenue of new opportunities for patients with solid cancers which we aim to leverage by two distinct TCR-based therapeutic modalities, TCR-engineered T cells (ACTengine® IMA203) and TCR Bispecifics (TCER® IMA402). Methods PRAME target peptide presentation and mRNA expression in tumor and normal tissues was assessed by quantitative mass spectrometry (MS) and transcriptomics. In situ hybridization was used to analyze target homogeneity. We compare target prevalences based on IMADetect® qPCR testing of screening biopsies from clinical trial patients with prevalences based on a PRAME target tailored, MS-based mRNA expression threshold applied to a large RNA sequencing dataset. Results PRAME RNA expression is elevated across many tumor types and only minimal in some normal tissues except testis. As demonstrated by MS, RNA expression does not translate into relevant peptide presentation on normal tissues. Peptide copy numbers range from 100 to 1,000 peptide copies per cell (target density) in the majority of tumor tissues as measured by highly sensitive MS-based AbsQuant® technology. Histologic analysis of PRAME RNA in different solid tumors demonstrates homogenous expression of PRAME with a high frequency of positive tumors cells. PRAME shows a prevalence between 80-100% in uterine and ovarian carcinoma, sarcoma subtypes, cutaneous and uveal melanoma and high prevalence in many other solid cancer types, such as cholangiocarcinoma, lung, kidney, breast, head and neck, esophageal, bladder and hepatocellular carcinoma. Prevalence numbers obtained during patient screening in our clinical trials match predicted prevalences. Interim phase 1a data from the IMA203 TCR-T trial (cut-off Oct 05, 2021) showed clinical responses in head and neck carcinoma, synovial sarcoma, uveal melanoma, and cutaneous melanoma. Conclusions Here, we demonstrate comprehensive target characterization and validation data supporting the nearly ideal target properties of PRAME that can be exploited for the benefit of patients: PRAME is highly cancer-associated, homogenously expressed, presented at high target density, highly prevalent across many solid cancers and clinically validated, underlining its potential to reach a large cancer patient population. Trial Registration NCT03686124 Ethics Approval The study was approved by the institutional review board/ethics committee as required for each participating site.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

Background T cell engaging bispecifics have emerged as a promising therapeutic opportunity for patients with solid cancers. However, challenges related to target specificity and drug safety profiles remain and many efforts are being made to generate optimized molecules with improved pharmacodynamic characteristics while reducing T cell engager-associated toxicities. We have developed a pipeline of novel bispecific molecules comprising a T cell receptor (TCR) for giving access to intracellular tumor antigens presented as peptide-HLA molecules and a unique T cell recruiting antibody aiming at conferring a favorable safety profile. Methods We designed a novel TCR-incorporating bispecific format, called T cell engaging receptor (TCER®). TCER® molecules targeting different peptide-HLA antigens and using different recruiting moieties were generated and assessed for preclinical characteristics such as in vitro efficacy, in vitro safety and anti-tumor responses in tumor xenograft models. Results Based on comparative preclinical testing of different TCR bispecific formats and T cell recruiting antibodies, we have developed a next-generation bispecific (TCER®) consisting of a high-affinity TCR capable of targeting tumor-specific peptide antigens and a low-affinity T cell recruiter designed to maximize efficacy while minimizing toxicity. The TCER® format harbors an effector function-silenced Fc part for the extension of serum half-life and improved manufacturability. For the development of different TCER® candidates, TCRs with promising functional avidity and high target-specificity are identified from the human repertoire and matured via yeast surface display to enhance TCR stability and to increase TCR affinity towards the target-peptide by at least 1,000-fold while retaining the target-specific binding pattern. TCER® molecules built with the maturated TCRs show in vitro activity at picomolar concentrations against tumor cell lines presenting the target peptide at similar copy numbers as found on patient tumors. Further, the TCER® molecules demonstrate consistent tumor regression including complete remissions in tumor xenograft models in mice and thereby also uncovered an essential role for the type of T cell recruiting antibody. For our clinical lead TCER® candidates we confirmed a favorable in vitro safety profile with a broad therapeutic window between tumor and normal cell reactivity against more than 20 different human normal tissue cell types. Conclusions We have developed a next-generation, half-life extended TCR Bispecific format that in preclinical tests demonstrated higher potency than multiple other established formats. By incorporating an innovative T cell recruiter we aim to reduce the risk for toxicities, specifically CRS, in patients. For each TCER® candidate we generate a robust preclinical data package before entering clinical development.

Preferentially expressed antigen in melanoma (PRAME) is an intracellular protein that has been shown to have attractive target attributes that can be leveraged by T cell receptor (TCR)-based immunotherapy. PRAME is cancer-specific, homogenously expressed and presented at high target density. In contrast to most other cancer germline antigens, PRAME is expressed across a multitude of solid tumors as well as hematological malignancies including acute myeloid leukemia and diffuse large B-cell lymphoma. We have developed IMA402, a bispecific T cell engaging receptor (TCER®) molecule targeting an HLA-A*02-presented peptide derived from PRAME. TCER® molecules are fusion proteins consisting of a high-affinity TCR for peptide-HLA targeting and a humanized T cell-recruiting antibody coupled to an effector function-silenced IgG1 Fc domain that aims to extend half-life together with providing antibody-like stability and manufacturability characteristics. For IMA402, a TCR recognizing the selected PRAME pHLA molecule with high avidity and specificity was affinity maturated via yeast surface display resulting in more than 1,000-fold increased binding affinity. During maturation we applied an off-target toxicity screening against normal tissue peptides selected from the our immunopeptidome database to retain specificity and deselect cross-reactive candidate TCRs. The affinity maturated TCR was engineered into the bispecific TCER® scaffold and produced in Chinese hamster ovary cells. In preclinical assays, IMA402 required only picomolar concentrations to induce lysis of various tumor cells with PRAME target peptide levels in the physiological range as found in patients. In different in vivo xenograft mouse models, IMA402 induced consistent tumor regression including complete remissions. In these models, pharmacokinetic profiling determined a terminal half-life of several days. In vivo analyses of various T cell recruiting domains with different affinities confirmed the superiority of the low affinity T cell recruiting domain used in IMA402 when compared to analogous TCER® molecules with higher-affinity T cell recruiter domains. For the safety assessment, we measured TCER® reactivity against more than 20 different human normal tissue cell types. We could demonstrate that IMA402 did not induce killing of normal tissue cells at concentrations of at least 1,000-fold higher than required for tumor cell lysis, suggesting a broad therapeutic window. In patients, the design of our next-generation TCER® molecule harbouring a high-affinity TCR, a low-affinity T cell recruiter and a Fc domain aims at optimizing biodistribution and activation of T cells at the tumor site instead of the periphery. This might reduce occurrence of immune-related toxicities, such as cytokine release syndrome and allow reaching relevant doses in the tumor tissue. Taken together, our preclinical studies demonstrate that our novel, next-generation bispecific T cell engager IMA402 could be a promising therapeutic option for PRAME-positive patients. The phase 1 clinical trial designed as basket trial will start recruitment in 2023.

Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HX-MS). However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.

Cell survival after oxidative DNA damage requires signaling, repair and transcriptional events often enabled by nucleosome displacement, exchange or removal by chromatin remodeling enzymes. Here, we show that Chromodomain Helicase DNA-binding protein 6 (CHD6), distinct to other CHD enzymes, is stabilized during oxidative stress via reduced degradation. CHD6 relocates rapidly to DNA damage in a manner dependent upon oxidative lesions and a conserved N-terminal poly(ADP-ribose)-dependent recruitment motif, with later retention requiring the double chromodomain and central core. CHD6 ablation increases reactive oxygen species persistence and impairs anti-oxidant transcriptional responses, leading to elevated DNA breakage and poly(ADP-ribose) induction that cannot be rescued by catalytic or double chromodomain mutants. Despite no overt epigenetic or DNA repair abnormalities, CHD6 loss leads to impaired cell survival after chronic oxidative stress, abnormal chromatin relaxation, amplified DNA damage signaling and checkpoint hypersensitivity. We suggest that CHD6 is a key regulator of the oxidative DNA damage response.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross‐linking between hydrophilic lysine‐rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross‐linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post‐translational modifications and cross‐linked peptides by high‐resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross‐link types or remained unmodified. Furthermore, both types of cross‐linking domains, Lys‐Pro and Lys‐Ala domains, participate not only in bifunctional inter‐ but also in intra‐domain cross‐links. We elucidated the sequences of several desmosine‐containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross‐links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys‐Ala domains. In summary, these results provide new and detailed insights into the cross‐linking process, which takes place within and between human tropoelastin units in a stochastic manner.

Quantification in proteomics largely relies on the incorporation of stable isotopes, with protocols that either introduce the label through metabolic incorporation or chemical tagging. Most methods rely on the use of trypsin and/or LysC to generate labeled peptides. Although alternative proteases can enhance proteome coverage, generic quantitative methods that port over to such enzymes are lacking. Here we describe a quantification strategy amenable to most proteases, which involves propionylation of metabolically labeled lysine, using a "silent stable isotope labeling by amino acids in cell culture (SILAC)" strategy that reveals isotopic labels on second-stage mass spectrometry (MS2) fragmentation in a tandem mass tag (TMT)-like manner. We selectively propionylated lysine residues prior to digestion to generate pure ArgC-like digestion for trypsin and novel ArgN-like digestions for LysargiNase, by restricting digestion at lysine. The modification offers highly complementary sequence coverage, and even enhanced protein identification rates in certain situations (GluC digestion). Propionylated lysine residues were present in the majority of identified peptides generated from digests of cell lysates and led to the consistent release of an intense cyclic imine reporter ion at mass-to-charge ratio (m/z) 140 using higher-energy collisional dissociation. We grew A549 cells in media containing either l-1-13C-lysine or l-6-13C-lysine, to generate proteins that share the same accurate mass when paired. Peptides were indistinguishable on the first-stage mass spectrometry (MS1) level and, upon fragmentation, released reporter ions at m/z 140 and m/z 141, without otherwise affecting sequence ion mass. The quantification approach is independent of the number of peptide lysines and offers a new strategy for quantitative proteomics.

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

Williams–Beuren syndrome (WBS) is a congenital disorder, which involves the heterozygous deletion of the elastin gene and other genes on chromosome 7. Clinical symptoms that are associated with hemizygosity of the essential extracellular matrix protein elastin include premature aging of the skin and supravalvular aortic stenosis. However, only little is known about the molecular basis of structural abnormalities in the connective tissue of WBS patients. Therefore, for the first time this study aimed to systematically characterize and compare the structure and amount of elastin present in skin and aortic tissue from WBS patients and healthy individuals. Elastin fibers were isolated from tissue biopsies, and it was found that skin of WBS patients contains significantly less elastin compared to skin of healthy individuals. Scanning electron microscopy and mass spectrometric measurements combined with bioinformatics data analysis were used to investigate the molecular‐level structure of elastin. Scanning electron microscopy revealed clear differences between WBS and healthy elastin. With respect to the molecular‐level structure, it was found that the proline hydroxylation degree differed between WBS and healthy elastin, while the tropoelastin isoform appeared to be the same. In terms of cross‐linking, no differences in the content of the tetrafunctional cross‐links desmosine and isodesmosine were found between WBS and healthy elastin. However, principal component analysis revealed differences between enzymatic digests of elastin from healthy probands and WBS patients, which indicates differing susceptibility toward enzymatic cleavage. Overall, the study contributes to a better understanding of the correlation between genotypic and elastin‐related phenotypic features of WBS patients. © 2016 Wiley Periodicals, Inc.

Elastin is a vital protein of the extracellular matrix of jawed vertebrates and provides elasticity to numerous tissues. It is secreted in the form of its soluble precursor tropoelastin, which is subsequently cross-linked in the course of the elastic fiber assembly. The process involves the formation of the two tetrafunctional amino acids desmosine (DES) and isodesmosine (IDES), which are unique to elastin. The resulting high degree of cross-linking confers remarkable properties, including mechanical integrity, insolubility, and long-term stability to the protein. These characteristics hinder the structural elucidation of mature elastin. However, MS(2) data of linear and cross-linked peptides released by proteolysis can provide indirect insights into the structure of elastin. In this study, we performed energy-resolved collision-induced dissociation experiments of DES, IDES, their derivatives, and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type CxHyN, suggesting that the pyridinium core of DES/IDES remains intact even at relatively high collision energies. The finding of these specific product ions enabled the development of a similarity-based scoring algorithm that was successfully applied on LC-MS/MS data of bovine elastin digests for the identification of DES-/IDES-cross-linked peptides. This approach facilitates the straightforward investigation of native cross-links in elastin.

This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS2 data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin.

Background: Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.Methods: Small peptides containing reactive allysine residues based on sequences or cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.Results: MS2 data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.Conclusions: The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.General significance: The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin. (C) 2013 Elsevier B.V. All rights reserved.