The Thermodynamic Mechanism of Peptide-MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM.

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306-319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by "sensing" flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

Dissipative particle dynamics: Systematic parametrization using water-octanol partition coefficients.

We present a systematic, top-down, thermodynamic parametrization scheme for dissipative particle dynamics (DPD) using water-octanol partition coefficients, supplemented by water-octanol phase equilibria and pure liquid phase density data. We demonstrate the feasibility of computing the required partition coefficients in DPD using brute-force simulation, within an adaptive semi-automatic staged optimization scheme. We test the methodology by fitting to experimental partition coefficient data for twenty one small molecules in five classes comprising alcohols and poly-alcohols, amines, ethers and simple aromatics, and alkanes (i.e., hexane). Finally, we illustrate the transferability of a subset of the determined parameters by calculating the critical micelle concentrations and mean aggregation numbers of selected alkyl ethoxylate surfactants, in good agreement with reported experimental values.

HLA-DM: arbiter conformationis.

The recognition by CD4(+) T cells of peptides bound to class II MHC (MHCII) molecules expressed on the surface of antigen-presenting cells is a key step in the initiation of an adaptive immune response. Presentation of peptides is the outcome of an intracellular selection process occurring in dedicated endosomal compartments involving, among others, an MHCII-like molecule named HLA-DM (DM). The impact of DM on the epitope selection machinery has been known for more than 15 years. However, the mechanism by which DM skews the presented repertoire in favour of kinetically stable complexes has remained elusive. Here, a review of the most recent observations in the field is presented, pointing to the possibility that DM decides the survival of a peptide-MHCII complex (pMHCII) on the basis of its conformational flexibility, which is a function of the 'tightness' of interaction between the peptide and the MHCII at a specific region of the binding site.

Cross-reactivity of T cells and its role in the immune system.

T-cell receptors recognize peptides presented by the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APC). The ability of the T-cell receptor (TCR) to recognize more than one peptide-MHC structure defines cross-reactivity. Cross-reactivity is a documented phenomenon of the immune system whose importance is still under investigation. There are a number of rational arguments for cross-reactivity. These include the discrepancy between the theoretical high number of pathogen-derived peptides and the lower diversity of the T-cell repertoire, the need for recognition of escape variants, and the intrinsic low affinity of this receptor-ligand pair. However, quantifying the phenomenon has been difficult, and its immunological importance remains unknown. In this review, we examined the cases for and against an important role for cross reactivity. We argue that it may be an essential feature of the immune system from the point of view of biological robustness.

Naive T cell repertoire skewing in HLA-A2 individuals by a specialized rearrangement mechanism results in public memory clonotypes.

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2-restricted CD8 memory T cell responses to influenza M1(58-66). We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M1(58-66) epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

Cutting edge: HLA-DM-mediated peptide exchange functions normally on MHC class II-peptide complexes that have been weakened by elimination of a conserved hydrogen bond.

The mechanism by which HLA-DM (DM) promotes exchange of peptides bound to HLA-DR (DR) is still unclear. We have shown that peptide interaction with DR1 can be considered a folding process as evidenced by cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative, which could be a function of the breaking of a critical interaction. The hydrogen bond (H-bond) between beta-chain His(81) and the peptide backbone at the -1 position is a candidate for such a target. In this study, we analyze the exchange of peptides bound to a DR1 mutant in which formation of this H-bond is impaired. We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond contributes to the overall energetics of the complex and its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable complex.

Peptide-MHC class II complex stability governs CD4 T cell clonal selection.

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity, but the mechanisms regulating the responding TCR repertoire remain poorly defined. In this study, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that, although all peptides elicited similar T cell response size on immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high-affinity clonotypes expressing restricted public TCR, whereas peptides with longer half-lives broadened CD4 T cell response by recruiting lower-affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high-affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher-affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.

A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics.

Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.

Comparability study of monocyte derived dendritic cells, primary monocytes, and THP1 cells for innate immune responses.

Immunogenicity is one major challenge to the successful development of biotherapeutics because it could adversely affect PK/PD, safety, and efficacy. Preclinical immunogenicity risk assessment strategies and assays have been developed and implemented to screen and optimize discovery molecules. Internalization by antigen presenting cells (APC) and innate immune activation are initial prerequisite steps in eliciting immune responses to biotherapeutics. Dendritic cells (DC)- and monocyte-based assays are employed to interrogate such risks, and their value has been well documented in the literature. However, these assays have limited throughput, exhibit higher variability, and entail lengthy and complex procedures as they are based on primary cells such as peripheral blood mononuclear cells (PBMC) from individual donors. Herein, we investigated THP1 cells as surrogate cells to study APC internalization and innate immune activation. Comparability studies showed that THP1 cells could resemble innate immune responses of monocyte-derived DC and primary CD14+ monocytes using a panel of therapeutic antibodies. In addition, an automated high throughput THP1 internalization assay was qualified to enable risk assessment at pre­lead stages. The results demonstrated that THP1 cells can be utilized to assess immunogenicity risk in a high throughput manner.

Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment.

Treatment-emergent antidrug antibodies (TE-ADA) pose a major challenge to the development of biotherapeutics. The antidrug antibody responses are highly orchestrated and involve many types of immune cells and biological processes. Biological drug internalization and processing by antigen-presenting cells (APCs) are two initial and critical steps in the cascade of events leading to T cell-dependent ADA production. The assays thus far described in literature to evaluate immunogenicity potential/risk as a function of APC activity mainly focus on internalization of labeled drug candidates in vitro. Herein, we describe a high-throughput Förster Resonance Energy Transfer (FRET)-based assay for assessing both internalization and processing using CD14+ monocyte-derived dendritic cells (DCs) as APCs. Antigen-binding fragment F(ab')2 against IgG fragment crystallizable gamma (Fcγ) was labeled with the activatable FRET pair TAMRA-QSY7 as a universal probe for antibodies and proteins with a fragment crystallizable (Fc) domain. The assay was qualified using six mAbs of known clinical immunogenicity and one IgG1 isotype antibody using Design of Experiment (DoE). Correlation analysis of internalization and clinical immunogenicity data showed that this FRET-based internalization assay was able to detect clinically immunogenic antibodies. This method provides a tool for analyzing/screening the immunogenicity risk of biological candidates by assessing one of the critical components of the ADA formation process within the broader context of an immunogenicity risk assessment strategy.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Micelle Formation in Alkyl Sulfate Surfactants Using Dissipative Particle Dynamics.

We use dissipative particle dynamics (DPD) to study micelle formation in alkyl sulfate surfactants, with alkyl chain lengths ranging from 6 to 12 carbon atoms. We extend our recent DPD force field [ J. Chem. Phys. 2017 , 147 , 094503 ] to include a charged sulfate chemical group and aqueous sodium ions. With this model, we achieve good agreement with the experimentally reported critical micelle concentrations (CMCs) and can match the trend in mean aggregation numbers versus alkyl chain length. We determine the CMC by fitting a charged pseudophase model to the dependence of the free surfactant on the total surfactant concentration above the CMC and compare it with a direct operational definition of the CMC as the point at which half of the surfactant is classed as micellar and half as monomers and submicellar aggregates. We find that the latter provides the best agreement with experimental results. Finally, with the same model, we are able to observe the sphere-to-rod morphological transition for sodium dodecyl sulfate (SDS) micelles and determine that it corresponds to SDS concentrations in the region of 300-500 mM.

Enhanced quantification of serum immunoglobulin G from a non-model wildlife species, the Steller sea lion (Eumetopias jubatus), using a protein A ELISA.

Immunoglobulins (Ig) are proteins that preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring Ig in non-model wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide reliable results especially when lacking species-specific reagents. We modified and optimized existing ELISA methodology utilizing the binding properties of Staphylococcus-derived Protein A (PrtA) to immunoglobulin G (IgG). We enhanced the assay for quantifying IgG in Steller sea lion (SSL) serum using critical quality control measures including dilution linearity, spike and percent recoveries, and internal controls. Of the modifications made, heat treatment of SSL serum enhanced accuracy and precision of IgG measurements by improving linearity and percent recovery in parallel dilutions and serum spikes. Purified canine IgG standard was not affected by heat inactivation. These results support that confounding serum proteins interfere with binding of PrtA with IgG demonstrating the need for heat treatment of serum to optimize IgG quantification using the PrtA-ELISA. Further, essential validation measures ensure proper assay performance. Consequently, the improved PrtA-ELISA provides species-independent IgG detection with validation criteria to enhance accuracy and precision for addressing future immunological questions in non-model wildlife in clinical, ecological, and conservation contexts.

Nfix Induces a Switch in Sox6 Transcriptional Activity to Regulate MyHC-I Expression in Fetal Muscle.

Sox6 belongs to the Sox gene family and plays a pivotal role in fiber type differentiation, suppressing transcription of slow-fiber-specific genes during fetal development. Here, we show that Sox6 plays opposite roles in MyHC-I regulation, acting as a positive and negative regulator of MyHC-I expression during embryonic and fetal myogenesis, respectively. During embryonic myogenesis, Sox6 positively regulates MyHC-I via transcriptional activation of Mef2C, whereas during fetal myogenesis, Sox6 requires and cooperates with the transcription factor Nfix in repressing MyHC-I expression. Mechanistically, Nfix is necessary for Sox6 binding to the MyHC-I promoter and thus for Sox6 repressive function, revealing a key role for Nfix in driving Sox6 activity. This feature is evolutionarily conserved, since the orthologs Nfixa and Sox6 contribute to repression of the slow-twitch phenotype in zebrafish embryos. These data demonstrate functional cooperation between Sox6 and Nfix in regulating MyHC-I expression during prenatal muscle development.

Thermodynamics of Peptide-MHC Class II Interactions: Not all Complexes are Created Equal.

The adaptive immune response begins when CD4+ T cells recognize antigenic peptides bound to class II molecules of the Major Histocompatibility Complex (MHCII). The interaction between peptides and MHCII has been historically interpreted as a rigid docking event. However, this model has been challenged by the evidence that conformational flexibility plays an important role in peptide-MHCII complex formation. Thermodynamic analysis of the binding reaction suggests a model of complexation in which the physical-chemical nature of the peptide determines the variability in flexibility of the substates in the peptide-MHC conformational ensemble. This review discusses our understanding of the correlation between thermodynamics of peptide binding and structural features of the resulting complex as well as their impact on HLA-DM activity and on our ability to predict MHCII-restricted epitopes.

For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection.

The adaptive immune response starts when CD4+ T cells recognize peptide antigens presented by class II molecules of the Major Histocompatibility Complex (MHCII). Two outstanding features of MHCII molecules are their polymorphism and the ability of each allele to bind a large panoply of peptides. The ability of each MHCII molecule to interact with a limited, though broad, range of amino acid sequences, or "permissive specificity" of binding, is the result of structural flexibility. This flexibility has been identified through biochemical and biophysical studies, and molecular dynamic simulations have modeled the conformational rearrangements that the peptide and the MHCII undergo during interaction. Moreover, there is evidence that the structural flexibility of the peptide/MHCII complex correlates with the activity of the "peptide-editing" molecule DM. In light of the impact that these recent findings have on our ability to predict MHCII epitopes, a review of the structural and thermodynamic determinants of peptide binding to MHCII is proposed.

Enthalpy-entropy compensation and cooperativity as thermodynamic epiphenomena of structural flexibility in ligand-receptor interactions.

Ligand binding is a thermodynamically cooperative process in many biochemical systems characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to cooperativity of ligation needs to be elucidated. Here, we perform kinetic and thermodynamic analyses on a panel of cycle-mutated peptides, derived from influenza H3 HA(306-319), interacting with wild type and a mutant HLA-DR. We observe that, within a certain range of peptide affinity, this system shows isothermal entropy-enthalpy compensation (iEEC). The incremental increases in conformational entropy measured as disruptive mutations are added in the ligand or receptor are more than sufficient in magnitude to account for the experimentally observed lack of free-energy decrease cooperativity. Beyond this affinity range, compensation is not observed, and therefore, the ability of the residual interactions to form a stable complex decreases in an exponential fashion. Taken together, our results indicate that cooperativity and iEEC constitute the thermodynamic epiphenomena of the structural fluctuation that accompanies ligand-receptor complex formation in flexible systems. Therefore, ligand binding affinity prediction needs to consider how each source of binding energy contributes synergistically to the folding and kinetic stability of the complex in a process based on the trade-off between structural tightening and restraint of conformational mobility.

A Peptide/MHCII conformer generated in the presence of exchange peptide is substrate for HLA-DM editing.

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that DM-mediated peptide release from HLA-DR (DR) is dependent on the presence of exchange peptide. However, DM also promotes a small amount of peptide release in the absence of exchange peptide. Here we show that SDS-PAGE separates purified peptide/DR1 complexes (pDR1) into two conformers whose ratio is peptide K(d)-dependent. In the absence of exchange peptide, DM only releases peptide from the slower migrating conformer. Addition of exchange peptide converts the DM-resistant conformer to the slower migrating conformer, which is DM labile. Thus, exchange peptide generates a conformer of pDR1 which constitutes the intermediate for peptide exchange and the substrate for DM activity. The resolution of the intermediate favors the highest affinity peptide. However, once folded into the DM-resistant conformer, even low affinity peptides can be presented in the absence of free peptide, broadening the repertoire available for presentation.