Diversity and evolution of computationally predicted T cell epitopes against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory infection. Despite more than 60 years of research, there is no licensed vaccine. While B cell response is a major focus for vaccine design, the T cell epitope profile of RSV is also important for vaccine development. Here, we computationally predicted putative T cell epitopes in the Fusion protein (F) and Glycoprotein (G) of RSV wild circulating strains by predicting Major Histocompatibility Complex (MHC) class I and class II binding affinity. We limited our inferences to conserved epitopes in both F and G proteins that have been experimentally validated. We applied multidimensional scaling (MDS) to construct T cell epitope landscapes to investigate the diversity and evolution of T cell profiles across different RSV strains. We find the RSV strains are clustered into three RSV-A groups and two RSV-B groups on this T epitope landscape. These clusters represent divergent RSV strains with potentially different immunogenic profiles. In addition, our results show a greater proportion of F protein T cell epitope content conservation among recent epidemic strains, whereas the G protein T cell epitope content was decreased. Importantly, our results suggest that RSV-A and RSV-B have different patterns of epitope drift and replacement and that RSV-B vaccines may need more frequent updates. Our study provides a novel framework to study RSV T cell epitope evolution. Understanding the patterns of T cell epitope conservation and change may be valuable for vaccine design and assessment.

Development of IFNß-1a versions with reduced immunogenicity and full in vitro biological activity for the treatment of multiple sclerosis.

IFNß (recombinant interferon Beta) has been widely used for the treatment of Multiple sclerosis for the last four decades. Despite the human origin of the IFNß sequence, IFNß is immunogenic, and unwanted immune responses in IFNß-treated patients may compromise its efficacy and safety in the clinic. In this study, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of IFNß-1a. Two de-immunized versions of IFNß-1a were produced in CHO cells and designated as IFNß-1a(VAR1) and IFNß-1a(VAR2). First, the secondary and tertiary protein structures were analyzed by circular dichroism spectroscopy. Then, the variants were also tested for functionality. While IFNß-1a(VAR2) showed similar in vitro antiviral activity to the original protein, IFNß-1a(VAR1) exhibited 40% more biological potency. Finally, in vivo assays using HLA-DR transgenic mice revealed that the de-immunized variants showed a markedly reduced immunogenicity when compared to the originator.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

Identification of a potent regulatory T cell epitope in factor V that modulates CD4+ and CD8+ memory T cell responses.

Identification of T cell epitopes that are recognized by Tregs may elucidate the relative contributions of thymic Tregs and induced Tregs to control of autoimmune diseases and allergy. One such T regulatory cell epitope or 'Tregitope', derived from blood Factor V, is described here. Tregs responding to Tregitope FV621 are potent suppressors of CD4+ T effector responses to Tetanus Toxoid in an in vitro bystander suppression assay, strongly inhibit proliferation of effector CD8+ T cells, down-modulate CD86 and HLA DR on antigen-presenting cells, and enhance expression of granzyme B in Tregs. Tregitope FV621 also suppresses anti-OVA immune responses in vivo. The immunomodulatory effect of Tregitope FV621 is enhanced when conjugated to albumin, suggesting that the short half-life of Tregitope peptides can be prolonged. The in silico tools used to prospectively identify the FV Tregitope described here, when combined with in vitro /in vivo validating assays, may facilitate future Tregitope discoveries.

Coxiella burnetii Epitope-Specific T-Cell Responses in Patients with Chronic Q Fever.

Infection with Coxiella burnetii, the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes.

Mass spectrometry-assisted identification of ADAMTS13-derived peptides presented on HLA-DR and HLA-DQ.

Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an in silico analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.

De-immunized and Functional Therapeutic (DeFT) versions of a long lasting recombinant alpha interferon for antiviral therapy.

Interferon α (IFN-α) exerts potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. However, repeated IFN-α administration induces neutralizing antibodies (NAb) against the therapeutic in a significant number of patients. Associations between IFN-α immunogenicity and loss of efficacy have been described. So as to improve the in vivo biological efficacy of IFN-α, a long lasting hyperglycosylated protein (4N-IFN) derived from IFN-α2b wild type (WT-IFN) was developed. However, in silico analysis performed using established in silico methods revealed that 4N-IFN had more T cell epitopes than WT-IFN. In order to develop a safer and more efficient IFN therapy, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of 4N-IFN. Using the OptiMatrix in silico tool in ISPRI, the 4N-IFN sequence was modified to reduce HLA binding potential of specific T cell epitopes. Following verification of predictions by HLA binding assays, eight modifications were selected and integrated in three variants: 4N-IFN(VAR1), (VAR2) and (VAR3). Two of the three variants (VAR1 and VAR3) retained anti-viral function and demonstrated reduced T-cell immunogenicity in terms of T-cell proliferation and Th1 and Th2 cytokine levels, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). It was previously demonstrated that N-glycosylation improved IFN-α pharmacokinetic properties. Here, we further reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling by modifying the T cell epitope content of a protein (de-immunizing). Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-α2b variants, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for improved IFN-α therapy of HCV and HBV.

Development and validation of an epitope prediction tool for swine (PigMatrix) based on the pocket profile method.

BACKGROUND: T cell epitope prediction tools and associated vaccine design algorithms have accelerated the development of vaccines for humans. Predictive tools for swine and other food animals are not as well developed, primarily because the data required to develop the tools are lacking. Here, we overcome a lack of T cell epitope data to construct swine epitope predictors by systematically leveraging available human information. Applying the "pocket profile method", we use sequence and structural similarities in the binding pockets of human and swine major histocompatibility complex proteins to infer Swine Leukocyte Antigen (SLA) peptide binding preferences. We developed epitope-prediction matrices (PigMatrices), for three SLA class I alleles (SLA-1*0401, 2*0401 and 3*0401) and one class II allele (SLA-DRB1*0201), based on the binding preferences of the best-matched Human Leukocyte Antigen (HLA) pocket for each SLA pocket. The contact residues involved in the binding pockets were defined for class I based on crystal structures of either SLA (SLA-specific contacts, Ssc) or HLA supertype alleles (HLA contacts, Hc); for class II, only Hc was possible. Different substitution matrices were evaluated (PAM and BLOSUM) for scoring pocket similarity and identifying the best human match. The accuracy of the PigMatrices was compared to available online swine epitope prediction tools such as PickPocket and NetMHCpan. RESULTS: PigMatrices that used Ssc to define the pocket sequences and PAM30 to score pocket similarity demonstrated the best predictive performance and were able to accurately separate binders from random peptides. For SLA-1*0401 and 2*0401, PigMatrix achieved area under the receiver operating characteristic curves (AUC) of 0.78 and 0.73, respectively, which were equivalent or better than PickPocket (0.76 and 0.54) and NetMHCpan version 2.4 (0.41 and 0.51) and version 2.8 (0.72 and 0.71). In addition, we developed the first predictive SLA class II matrix, obtaining an AUC of 0.73 for existing SLA-DRB1*0201 epitopes. Notably, PigMatrix achieved this level of predictive power without training on SLA binding data. CONCLUSION: Overall, the pocket profile method combined with binding preferences from HLA binding data shows significant promise for developing T cell epitope prediction tools for pigs. When combined with existing vaccine design algorithms, PigMatrix will be useful for developing genome-derived vaccines for a range of pig pathogens for which no effective vaccines currently exist (e.g. porcine reproductive and respiratory syndrome, influenza and porcine epidemic diarrhea).

HCV epitope, homologous to multiple human protein sequences, induces a regulatory T cell response in infected patients.

BACKGROUND & AIMS: Spontaneous resolution of hepatitis C virus (HCV) infection depends upon a broad T cell response to multiple viral epitopes. However, most patients fail to clear infections spontaneously and develop chronic disease. The elevated number and function of CD3(+)CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg)) in HCV-infected patients suggest a role of Treg cells in impaired viral clearance. The factors contributing to increased Treg cell activity in chronic hepatitis C cases remain to be delineated. METHODS: Immunoinformatics tools were used to predict promiscuous, highly-conserved HLA-DRB1-restricted immunogenic consensus sequences (ICS), each composed of multiple T cell epitopes. These sequences were synthesized and added to cultures of peripheral blood mononuclear cells (PBMCs), derived from patients who resolved HCV infection spontaneously, patients with persistent infection, and non-infected individuals. The cells were collected and following 5days incubation, quantified and characterized by flow cytometry. RESULTS: One immunogenic consensus sequence (ICS), HCV_G1_p7_794, induced a marked increase in Treg cells in PBMC cultures derived from infected patients, but not in patients who spontaneously cleared HCV or in non-infected individuals. An analogous human peptide (p7_794), on the other hand, induced a significant increase in Treg cells among PBMCs derived from both HCV-infected and non-infected individuals. JanusMatrix analyses determined that HCV_G1_p7_794 is comprised of Treg cell epitopes that exhibit extensive cross-reactivity with the human proteome. CONCLUSIONS: A virus-encoded peptide (HCV_G1_p7_794) with extensive human homology activates cross-reactive CD3(+)CD4(+)CD25(+)FoxP3(+) natural Treg cells, which potentially contributes to immunosuppression and to the development of chronic hepatitis C.

C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells.

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T-cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 knockout mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T-cell recognition of C3d T-helper epitopes processed and presented by major histocompatibility complex class II on the B-cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T-cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple human leukocyte antigen (HLA)-DR alleles with high affinity, and induce interferon-γ responses in healthy donor peripheral blood mononuclear cells. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. In addition, we show that the T-cell phenotype of C3d-specific reactive T cells is CD4(+)CD45RO(+) memory T cells. Finally, mutation of a single T-cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4(+) T-helper cells in the context of HLA class II.

Vida Sana: a lifestyle intervention for uninsured, predominantly Spanish-speaking immigrants improves metabolic syndrome indicators.

Metabolic syndrome is an increasingly common condition that can contribute to the development of type 2 diabetes and cardiovascular disease. 35 % of adults living in the United States meet the criteria for having metabolic syndrome, with that number being even higher in populations with health disparities. We describe a 'healthy lifestyles' program implemented at a free clinic serving a predominantly Hispanic cohort of low-income, uninsured individuals living in Providence, Rhode Island. The "Vida Sana/Healthy Life" (Vida Sana) program uses low literacy, language-appropriate materials and trained peers to educate participants about healthy lifestyles in a setting that also provided opportunities for social engagement. 192 of 126 (65.6 %) participants in Vida Sana completed 6 out of 8 sessions of the Vida Sana program over a 12-month period. At the completion of the program, nearly 90 % of Vida Sana participants showed an increase in their health literacy, and at least 60 % of participants decreased each of the risk factors (blood sugar, cholesterol, body mass index or waist circumference) associated with metabolic syndrome.

ICoVax 2013: the 3rd ISV Pre-conference Computational Vaccinology Workshop.

Following last year's computational vaccinology workshop in Shanghai, China, the third ISV Pre-conference Computational Vaccinology Workshop (ICoVax 2013) was held in Barcelona, Spain. ICoVax 2013 provided an international platform for the attendees to showcase their research and discuss problems and solutions in the development and application of computational vaccinology and vaccine informatics tools. The first of the three full-length papers presented at ICoVax discussed the discovery of viral "camouflage" through cross-conservation of T-cell epitopes using a tool called JanusMatrix. This important paper reports that viruses may camouflage their presence in the human body by incorporating sequences in their proteins that are highly cross-conserved at the T-cell receptor surface with human genome proteins, a discovery that has wide ranging implications for the development of vaccines against viruses that use the camouflage method. The other papers described a database for storing experimentally verified data on DNA vaccines and compared therapeutic targets of western drugs to Chinese herbal medicines for cardiovascular diseases. The short poster presentations covered various uses of informatics tools for processing the DNA and microRNA of pathogens to improve vaccine coverage, efficacy and development. A live (on-line) demonstration of the vaccine design toolkit, iVax, presented by Frances Terry of EpiVax, illustrated how computational vaccinology could be used in the design of next generation vaccines.

Integrated assessment of predicted MHC binding and cross-conservation with self reveals patterns of viral camouflage.

BACKGROUND: Immune recognition of foreign proteins by T cells hinges on the formation of a ternary complex sandwiching a constituent peptide of the protein between a major histocompatibility complex (MHC) molecule and a T cell receptor (TCR). Viruses have evolved means of "camouflaging" themselves, avoiding immune recognition by reducing the MHC and/or TCR binding of their constituent peptides. Computer-driven T cell epitope mapping tools have been used to evaluate the degree to which particular viruses have used this means of avoiding immune response, but most such analyses focus on MHC-facing 'agretopes'. Here we set out a new means of evaluating the TCR faces of viral peptides in addition to their agretopes, integrating evaluations of both sides of the ternary complex in a single analysis. METHODS: This paper develops what we call the Janus Immunogenicity Score (JIS), bringing together a well-established method for predicting MHC binding, with a novel assessment of the potential for TCR binding based on similarity with self. Intuitively, both good MHC binding and poor self-similarity are required for high immunogenicity (i.e., a robust T effector response). RESULTS: Focusing on the class II antigen-processing pathway, we show that the JIS of T effector epitopes and null or regulatory epitopes deposited in a large database of epitopes (Immune Epitope Database) are significantly different. We then show that different types of viruses display significantly different patterns of scores over their constituent peptides, with viruses causing chronic infection (Epstein-Barr and cytomegalovirus) strongly shifted to lower scores relative to those causing acute infection (Ebola and Marburg). Similarly we find distinct patterns among influenza proteins in H1N1 (a strain against which human populations rapidly developed immunity) and H5N1 and H7N9 (highly pathogenic avian flu strains, with significantly greater case mortality rates). CONCLUSION: The Janus Immunogenicity Score, which integrates MHC binding and TCR cross-reactivity, provides a new tool for studying immunogenicity of pathogens and may improve the selection and optimization of antigenic elements for vaccine design.

CHOPPI: a web tool for the analysis of immunogenicity risk from host cell proteins in CHO-based protein production.

Despite high quality standards and continual process improvements in manufacturing, host cell protein (HCP) process impurities remain a substantial risk for biological products. Even at low levels, residual HCPs can induce a detrimental immune response compromising the safety and efficacy of a biologic. Consequently, advanced-stage clinical trials have been cancelled due to the identification of antibodies against HCPs. To enable earlier and rapid assessment of the risks in Chinese Hamster Ovary (CHO)-based protein production of residual CHO protein impurities (CHOPs), we have developed a web tool called CHOPPI, for CHO Protein Predicted Immunogenicity. CHOPPI integrates information regarding the possible presence of CHOPs (expression and secretion) with characterizations of their immunogenicity (T cell epitope count and density, and relative conservation with human counterparts). CHOPPI can generate a report for a specified CHO protein (e.g., identified from proteomics or immunoassays) or characterize an entire specified subset of the CHO genome (e.g., filtered based on confidence in transcription and similarity to human proteins). The ability to analyze potential CHOPs at a genomic scale provides a baseline to evaluate relative risk. We show here that CHOPPI can identify clear differences in immunogenicity risk among previously validated CHOPs, as well as identify additional "risky" CHO proteins that may be expressed during production and induce a detrimental immune response upon delivery. We conclude that CHOPPI is a powerful tool that provides a valuable computational complement to existing experimental approaches for CHOP risk assessment and can focus experimental efforts in the most important directions. Biotechnol. Bioeng. 2014;111: 2170-2182. © 2014 Wiley Periodicals, Inc.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

Neoadjuvant personalized cancer vaccines: the final frontier?

INTRODUCTION: Clinical trials of personalized cancer vaccines have shown that on-demand therapies that are manufactured for each patient, result in activated T cell responses against individual tumor neoantigens. However, their use has been traditionally restricted to adjuvant settings and late-stage cancer therapy. There is growing support for the implementation of PCV earlier in the cancer therapy timeline, for reasons that will be discussed in this review. AREAS COVERED: The efficacy of cancer vaccines may be to some extent dependent on treatment(s) given prior to vaccine administration. Tumors can undergo radical immunoediting following treatment with immunotherapies, such as checkpoint inhibitors, which may affect the presence of the very mutations targeted by cancer vaccines. This review will cover the topics of neoantigen cancer vaccines, tumor immunoediting, and therapy timing. EXPERT OPINION: Therapy timing remains a critical topic to address in optimizing the efficacy of personalized cancer vaccines. Most personalized cancer vaccines are being evaluated in late-stage cancer patients and after treatment with checkpoint inhibitors, but they may offer a greater benefit to the patient if administered in earlier clinical settings, such as the neoadjuvant setting, where patients are not facing T cell exhaustion and/or a further compromised immune system.

In silico methods for immunogenicity risk assessment and human homology screening for therapeutic antibodies.

In silico immunogenicity risk assessment has been an important step in the development path for many biologic therapeutics, including monoclonal antibodies. Even if the source of a given biologic is 'fully human', T cell epitopes that are contained in the sequences of the biologic may activate the immune system, enabling the development of anti-drug antibodies that can reduce drug efficacy and may contribute to adverse events. Computational tools that identify T cell epitopes from primary amino acid sequences have been used to assess the immunogenic potential of therapeutic candidates for several decades. To facilitate larger scale analyses and accelerate preclinical immunogenicity risk assessment, our group developed an integrated web-based platform called ISPRI, (Immunogenicity Screening and Protein Re-engineering Interface) that provides hands-on access through a secure web-based interface for scientists working in large and mid-sized biotech companies in the US, Europe, and Japan. This toolkit has evolved and now contains an array of algorithms that can be used individually and/or consecutively for immunogenicity assessment and protein engineering. Most analyses start with the advanced epitope mapping tool (EpiMatrix), then proceed to identify epitope clusters using ClustiMer, and then use a tool called JanusMatrix to define whether any of the T cell epitope clusters may generate a regulatory T cell response which may diminish or eliminate anti-drug antibody formation. Candidates can be compared to similar products on a normalized immunogenicity scale. Should modifications to the biologic sequence be an option, a tool for moderating putative immunogenicity by editing T cell epitopes out of the sequence is available (OptiMatrix). Although this perspective discusses the in-silico immunogenicity risk assessment for monoclonal antibodies, bi-specifics, multi-specifics, and antibody-drug conjugates, the analysis of additional therapeutic modalities such as enzyme replacement proteins, blood factor proteins, CAR-T, gene therapy products, and peptide drugs is also made available on the ISPRI platform.

ISPRI (Interactive Screening and Protein Reengineering Interface): Integrated, cloud-based, comprehensive toolkit for Immunogenicity Risk Assessment.EpiMatrix Immunogenicity Score: Combined T effector and Treg Epitope Content per unit protein.Tregitopes: Treg Epitopes found in IgG Framework that have been shown to modulate antigen-specific effector T cell responses.ClustiMer: Tool for identifying epitope rich polypeptides from within a given protein sequence.JanusMatrix: Tool for Predicting Tolerance, Putative Treg Epitopes, and Anti-self-immune responses.OptiMatrix: Tool for modifying T cell epitope sequences to reduce (or enhance) MHC binding.

Optimization of therapeutic antibodies for reduced self-association and non-specific binding via interpretable machine learning.

Antibody development, delivery, and efficacy are influenced by antibody-antigen affinity interactions, off-target interactions that reduce antibody bioavailability and pharmacokinetics, and repulsive self-interactions that increase the stability of concentrated antibody formulations and reduce their corresponding viscosity. Yet identifying antibody variants with optimal combinations of these three types of interactions is challenging. Here we show that interpretable machine-learning classifiers, leveraging antibody structural features descriptive of their variable regions and trained on experimental data for a panel of 80 clinical-stage monoclonal antibodies, can identify antibodies with optimal combinations of low off-target binding in a common physiological-solution condition and low self-association in a common antibody-formulation condition. For three clinical-stage antibodies with suboptimal combinations of off-target binding and self-association, the classifiers predicted variable-region mutations that optimized non-affinity interactions while maintaining high-affinity antibody-antigen interactions. Interpretable machine-learning models may facilitate the optimization of antibody candidates for therapeutic applications.

Individual and population-level variability in HLA-DR associated immunogenicity risk of biologics used for the treatment of rheumatoid arthritis.

Hypothesis: While conventional in silico immunogenicity risk assessments focus on measuring immunogenicity based on the potential of therapeutic proteins to be processed and presented by a global population-wide set of human leukocyte antigen (HLA) alleles to T cells, future refinements might adjust for HLA allele frequencies in different geographic regions or populations, as well for as individuals in those populations. Adjustment by HLA allele distribution may reveal risk patterns that are specific to population groups or individuals, which current methods that rely on global-population HLA prevalence may obscure. Key findings: This analysis uses HLA frequency-weighted binding predictions to define immunogenicity risk for global and sub-global populations. A comparison of assessments tuned for North American/European versus Japanese/Asian populations suggests that the potential for anti-therapeutic responses (anti-therapeutic antibodies or ATA) for several commonly prescribed Rheumatoid Arthritis (RA) therapeutic biologics may differ, significantly, between the Caucasian and Japanese populations. This appears to align with reports of differing product-related immunogenicity that is observed in different populations. Relevance to clinical practice: Further definition of population-level (regional) and individual patient-specific immunogenic risk profiles may enable prescription of the RA therapeutic with the highest probability of success to each patient, depending on their population of origin and/or their individual HLA background. Furthermore, HLA-specific immunogenicity outcomes data are limited, thus there is a need to expand HLA-association studies that examine the relationship between HLA haplotype and ATA in the clinic.