IntroductionAntibody maturation in the lymphoid follicle produces antibodies with improved binding affinity. This process requires iterative rounds of mutation and B cell expansion, supported by T cells that recognize epitopes presented on the B cell’s MHC-II. In this comprehensive antibody repertoire analysis, we find that established regulatory T cell epitopes (Tregitopes) decline in maturing antibody sequences as somatic hypermutation (SHM) increases, but potential T effector epitopes do not decline. A previous analysis of B cell receptor (BCR)-derived HLA-DR epitopes present in memory antibody repertoires from seven healthy human donors revealed a decrease in donor-specific epitope content with SHM. Moreover, T cell epitope depletion was associated with class-switching and long-term secretion of antibody into serum. Significant depletion of high-affinity germline-encoded epitopes in high SHM sequences was also observed, but the predicted phenotype of T cells responding to the BCR-derived epitopes (regulatory vs. effector) was not previously evaluated.MethodsIn this follow-on study, we screened a different set of four donor repertoires to investigate the dynamics of donor-specific HLA-DR T cell epitopes and three subsets of T cell epitope content: previously validated T cell epitopes recognized by thymus-derived Tregs (Tregitopes), potentially tolerated T cell epitopes, and potential effector T cell epitopes.ResultsOur results show that Tregitope content reduction is correlated with SHM, suggesting that Tregitopes are removed during maturation. Moreover, T cell epitopes that are likely to be tolerated or tolerogenic were also removed with SHM progression. In contrast, potential T effector epitope content increased with SHM. Tregitope depletion occurred in multiple V-gene pair combinations and was the most frequent T cell epitope change. Furthermore, Tregitope content in IgA and IgG sequences was lower and had greater negative correlation with SHM than IgM, indicating that Tregitope removal is likely associated with class-switching. Tregitope depletion was also associated with maturation to plasmablasts. In vitro, representative Tregitopes inhibited CD4+ T cell proliferation. Mutations introduced by SHM altered Tregitope HLA-DR binding affinities.DiscussionThe correlation of Tregitope depletion with increasing SHM implies that the activity of thymus-derived Treg cells in immune responses to antibodies is diminished with SHM, maturation, and isotype switching, supporting the generation of anti-idiotype responses.

Therapeutic drug monitoring (TDM) for dose modification of biologics has the potential to improve patient outcomes. The US Food and Drug Administration (FDA) and the American Association of Pharmaceutical Scientists (AAPS) hosted the first US-based public workshop on TDM of biologics with contributions from a broad array of interested parties including healthcare providers, clinical pharmacologists, test developers, bioanalysis and immunogenicity scientists, health economics and outcomes research (HEOR) experts and regulators. The key insight was that despite a body of evidence to support TDM in certain therapeutic areas, there remain substantial challenges to widespread clinical implementation. There is a lack of consensus regarding the integration of TDM in clinical guidelines, and a lack of consensus on the cost-effectiveness of TDM; both factors contribute to the difficulty that healthcare providers face in obtaining reimbursement for TDM (both coverage of testing itself, and coverage of potential dosing modifications). The HEOR experts outlined alternative routes to obtaining reimbursement and suggested advocating for changes in coverage policies to promote TDM use in the clinic. Reaching alignment across policy makers, patients and advocacy groups, payers, and healthcare providers, on specific treatment settings where TDM will be clearly beneficial, was identified as an important step to advancing TDM implementation for the benefit of patients.

Editorial: Innovative approaches to immune tolerance and regulation with gene, cellular, protein, and microbiome based therapeutics

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.

IntroductionThe efficacy of enzyme replacement therapy (ERT) with alglucosidase alfa for infantile-onset Pompe disease (IOPD) is limited in some patients due to the development of high and sustained antibody titers (HSAT; ≥12,800).MethodsWe carried out detailed immunophenotyping of IOPD patients (n=40), including analysis of circulating cell populations by flow cytometry and plasma cytokines by multiplex array, to determine whether patients with HSAT have unique immunological characteristics compared to those with low titers (LT; <12,800).ResultsCompared to patients with LT, patients who develop HSAT were skewed toward a type 2 immune profile, with an increased frequency of Th2 cells that was positively correlated with levels of Th2 (IL-4, IL-5, IL-13) and pro-inflammatory (IL-6, TNF-α, MIP-1α, MIP-1β) cytokines. B cells were increased in HSAT patients with a decreased fraction of unswitched memory B cells. Plasma GM-CSF concentrations were lower on average in HSAT patients, while CXCL11 was elevated. Finally, using principal components analysis, we derived an HSAT Signature Score that successfully stratified patients according to their antibody titers.DiscussionThe immune profiles revealed in this study not only identify potential biomarkers of patients that developed HSAT but also provide insights into the pathophysiology of HSAT that will ultimately lead to improved immunotherapy strategies.

COPYRIGHT © 2023 Rosenberg, Tourdot and Markusic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 22 August 2023 DOI 10.3389/fimmu.2023.1219854

Context: Chronic myeloid leukemia (CML) is characterized by a relapsing evolution, increased disease burden and poor prognosis in the advanced phases. Objective: The assessment of diagnosis and management options of CML in the Republic of Moldova. Design: We performed the case-control study of 134 CML patients, who were treated and followed up at the comprehensive cancer center – Institute of Oncology between 2007–2022. The real-time PCR was used with the aim to determine the expression of BCR-ABL p210 and p190 transcripts while proceeding CML diagnosis. Setting: The study was related to the outpatient and hospitalized care. Participants: The patients' age ranged between 14-81 years. The median age was 51.4 ± 2.13 years. There were 78 (58.2%) males and 56 (41.8%) females. Interventions: Tyrosine kinase inhibitors (TKIs) were supplied by The Max Foundation via Max Access Solutions (MAS). Imatinib, nilotinib, and ponatinib were used as a front-line therapy in newly diagnosed CML patients and in the cases of resistance to non-TKIs treatment. Main Outcome Measures: Complete response (CR) rate estimated the short-term results of chemotherapy, and the overall survival (OS) –long-term results. Results: CML was diagnosed in chronic phase in 122 (91,04 ± 2.32%) patients. The rate of Ph-positive bone marrow (BM) cells ranged between 20 – 100%. In 72.7% of cases Ph-chromosome was detected in over 70% of the BM cells. BCR-ABL p210 transcript range was 21.84–100%. In 69.8% of cases BCR-ABL transcripts were identified in over 65% of the peripheral blood cells. Under the TKIs therapy the hematologic CR was achieved in 92.8% of cases, and the molecular CR – in 28.5%. One- and 5-year OS of patients treated with TKIs was 98.5% and 91%. IFN a-2b was used with partial response in cases of resistance to conventional chemotherapy (CC) and TKIs. In 79% of patients treated initially with CC, only the minor cytogenetic response was obtained. Conclusions: MAS program may be considered an efficient and transparent management option to ensure CML patients with potentially curable TKIs medication in the emerging regions regardless the age, gender, and social categories. The Max Foundation delivers TKIs medication to CML patients on a regular basis, accelerating health equity in low-and middle-income countries.

BackgroundA major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice. MethodsAn AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human alpha-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood. ResultsIn naive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19(+)B220(+) B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration. ConclusionsOur data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naive mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.

Table S1. Tumor sample and patient information; Table S2a. 9 upregulated MT-tRNAs; Table. S2b. 13 upregulated snoRNAs; Table. S2c. 19 downregulated snoRNAs; Table S3: CRC tumor fingerprint based on T/N PRKM ratio (1); Figure S1. RPKMs of snord12 cluster (a) and snord114 cluster (b) were plotted in 35 CRC pairs; Fig. S2. (a) 3D PCA of 22 MT-tRNAs (RPKM) in 35 CRC pairs; Fig. S3. Nine regulated MT-tRNAs (a), 13 upregulated (b) and 19 downregulated snoRNAs (c) were plotted by their RPKMs in 35 CRC pairs; Fig. S4. (a) Top twenty sRNAs with smallest p-values (paired t-test) in all 35 CRCs. (b) All examined 14 tumors had higher expression of ZNFX1-AS1 (host gene of snord12 cluster). (c) Expression correlation between snord12B and ZNFX1-AS1 in 14 tumors; Fig. S5. snord1114 cluster (a) and MEG3 (b) were plotted by their RPKM in 14 CRC pairs; Supplementary information (S1) Fidelity of RNA-Seq analysis and identification of house keeping genes; Supplementary information S2. Correlation between MT-tRNAs and miRNAs.

The identification and removal of host cell proteins (HCPs) from biologic products is a critical step in drug development. Despite recent improvements to purification processes, biologics such as monoclonal antibodies, enzyme replacement therapies, and vaccines that are manufactured in a range of cell lines and purified using diverse processes may contain HCP impurities, making it necessary for developers to identify and quantify impurities during process development for each drug product. HCPs that contain sequences that are less conserved with human homologs may be more immunogenic than those that are more conserved. We have developed a computational tool, ISPRI-HCP, that estimates the immunogenic potential of HCP sequences by evaluating and quantifying T cell epitope density and relative conservation with similar T cell epitopes in the human proteome. Here we describe several case studies that support the use of this method for classifying candidate HCP impurities according to their immunogenicity risk.

The in silico prediction of T cell epitopes within any peptide or biologic drug candidate serves as an important first step for assessing immunogenicity. T cell epitopes bind human leukocyte antigen (HLA) by a well-characterized interaction of amino acid side chains and pockets in the HLA molecule binding groove. Immunoinformatics tools, such as the EpiMatrix algorithm, have been developed to screen natural amino acid sequences for peptides that will bind HLA. In addition to commonly occurring in synthetic peptide impurities, unnatural amino acids (UAA) are also often incorporated into novel peptide therapeutics to improve properties of the drug product. To date, the HLA binding properties of peptides containing UAA are not accurately estimated by most algorithms. Both scenarios warrant the need for enhanced predictive tools. The authors developed an in silico method for modeling the impact of a given UAA on a peptide's likelihood of binding to HLA and, by extension, its immunogenic potential. In silico assessment of immunogenic potential allows for risk-based selection of best candidate peptides in further confirmatory in vitro, ex vivo and in vivo assays, thereby reducing the overall cost of immunogenicity evaluation. Examples demonstrating in silico immunogenicity prediction for product impurities that are commonly found in formulations of the generic peptides teriparatide and semaglutide are provided. Next, this article discusses how HLA binding studies can be used to estimate the binding potentials of commonly encountered UAA and "correct" in silico estimates of binding based on their naturally occurring counterparts. As demonstrated here, these in vitro binding studies are usually performed with known ligands which have been modified to contain UAA in HLA anchor positions. An example using D-amino acids in relative binding position 1 (P1) of the PADRE peptide is presented. As more HLA binding data become available, new predictive models allowing for the direct estimation of HLA binding for peptides containing UAA can be established.

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.

PurposeTo assess the magnitude of benefit to early treatment initiation, enabled by newborn screening or prenatal diagnosis, in patients with cross-reactive immunological material (CRIM)-negative infantile Pompe disease (IPD), treated with enzyme replacement therapy (ERT) and prophylactic immune tolerance induction (ITI) with rituximab, methotrexate, and intravenous immunoglobulin (IVIG).MethodsA total of 41 CRIM-negative IPD patients were evaluated. Among patients who were treated with ERT + ITI (n = 30), those who were invasive ventilator–free at baseline and had ≥6 months of follow-up were stratified based on age at treatment initiation: (1) early (≤4 weeks), (2) intermediate (>4 and ≤15 weeks), and (3) late (>15 weeks). A historical cohort of 11 CRIM-negative patients with IPD treated with ERT monotherapy served as an additional comparator group.ResultsTwenty patients were included; five, seven, and eight in early, intermediate, and late treatment groups, respectively. Genotypes were similar across the three groups. Early-treated patients showed significant improvements in left ventricular mass index, motor and pulmonary outcomes, as well as biomarkers creatine kinase and urinary glucose tetrasaccharide, compared with those treated later.ConclusionOur preliminary data suggest that early treatment with ERT + ITI can transform the long-term CRIM-negative IPD phenotype, which represents the most severe end of the Pompe disease spectrum.

In the midst of resurging COVID-19 cases, the second NIH/FDA virtual COVID-19 and Cytokines symposium was held on 1 December 2020, focusing on longitudinal studies of COVID-19 immunity, including long-term consequences, potential associations with autoimmunity and the multisystem inflammatory syndrome in children (MIS-C).

Background: Ivosidenib (Servier Pharmaceuticals LLC) and enasidenib (Celgene) are inhibitors of mutant IDH1 and IDH2, respectively, approved for the treatment of relapsed/refractory (R/R) AML with an IDH1 or IDH2 mutation (ivosidenib is also approved for front-line use in patients ≥ 75 years or with comorbidities). While these drugs were approved based on durable complete remission (CR) + CR with partial hematologic recovery (CRh) rate, only 23-43% of patients responded. Thus, there is a need for a biomarker to better predict response. We previously presented a genomic model of response to enasidenib using linear discriminate analysis (Ghazanchyan et al., ASH 2018). In the two-part study presented here, we improved this modeling by adding clinical data, using machine learning-based approaches to model response to both enasidenib and ivosidenib, and generated an independent validation cohort using clinical samples.

To evaluate the expression of immune checkpoint genes, their concordance with expression of IFNγ, and to identify potential novel ICP related genes (ICPRG) in colorectal cancer (CRC), the biological connectivity of six well documented (“classical”) ICPs (CTLA4, PD1, PDL1, Tim3, IDO1, and LAG3) with IFNγ and its co-expressed genes was examined by NGS in 79 CRC/healthy colon tissue pairs. Identification of novel IFNγ- induced molecules with potential ICP activity was also sought. In our study, the six classical ICPs were statistically upregulated and correlated with IFNγ, CD8A, CD8B, CD4, and 180 additional immunologically related genes in IFNγ positive (FPKM > 1) tumors. By ICP co-expression analysis, we also identified three IFNγ-induced genes [(IFNγ-inducible lysosomal thiol reductase (IFI30), guanylate binding protein1 (GBP1), and guanylate binding protein 4 (GBP4)] as potential novel ICPRGs. These three genes were upregulated in tumor compared to normal tissues in IFNγ positive tumors, co-expressed with CD8A and had relatively high abundance (average FPKM = 362, 51, and 25, respectively), compared to the abundance of the 5 well-defined ICPs (Tim3, LAG3, PDL1, CTLA4, PD1; average FPKM = 10, 9, 6, 6, and 2, respectively), although IDO1 is expressed at comparably high levels (FPKM = 39). We extended our evaluation by querying the TCGA database which revealed the commonality of IFNγ dependent expression of the three potential ICPRGs in 638 CRCs, 103 skin cutaneous melanomas (SKCM), 1105 breast cancers (BC), 184 esophageal cancers (ESC), 416 stomach cancers (STC), and 501 lung squamous carcinomas (LUSC). In terms of prognosis, based on Pathology Atlas data, correlation of GBP1 and GBP4, but not IFI30, with 5-year survival rate was favorable in CRC, BC, SKCM, and STC. Thus, further studies defining the role of IFI30, GBP1, and GBP4 in CRC are warranted.

The first case of human transmission of SARS-CoV-2 was reported in China in December 2019. A few months later, this viral infection had spread worldwide and became a pandemic. The disease caused by SARS-CoV-2, termed COVID-19, is multifactorial and associated with both specific antiviral as well as inflammatory responses, the extent of which may determine why some individuals are asymptomatic while others develop serious complications. Here we review possible life-threating immune events that can occur during disease progression to uncover key factors behind COVID-19 severity and provide suggestions for interventions with repurposed drugs in well-controlled and randomized clinical trials. These drugs include therapeutics with potential to inhibit SARS-CoV-2 entry into host cells such as serine protease inhibitors of the cellular protease TMPS2 and drugs targeting the renin-angiotensin system; antivirals with potential to block SARS-CoV-2 replication or factors that could boost the antiviral response; monoclonal antibodies targeting pro-inflammatory cytokines that drive the hyperinflammatory response during COVID-19 progression toward the severe stage and therapeutics that could ameliorate the function of the lungs. Furthermore, in order to help make more informed decisions on the timing of the intervention with the drugs listed in this review, we have grouped these therapeutics according to the stage of COVID-19 progression that we considered most appropriate for their mechanism of action.

1 Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States, 2 Translational Medicine and Early Development, Sanofi Research and Development, Sanofi, Framingham, MA, United States, 3 Université de ParisSaclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé, SIMoS, Gif-sur-Yvette, France, 4 Centre for Rheumatology, Division of Medicine, University College London, London, United Kingdom, 5 Laboratory of Immunology, Division of Biotechnology Product Review and Research 3, Office of Biotechnology Products, Center for Drugs Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States

“The role of cytokines in COVID-19” online symposium was presented on 18 June 2020 by the NIH/FDA Immunology and Cytokine Interest Groups and was purposed to discuss our rapidly changing understanding of COVID-19-related cytokine responses in different stages of infection, including the etiologies, downstream consequences and possible mitigation strategies. The recording is available at https://nci.rev.vbrick.com/sharevideo/03106730-66cc-47ba-870b-f6e6274a998a .

Immune modulation with rituximab, methotrexate, and intravenous immunoglobulin (IVIG) has shown great success in inducing immune tolerance in a large cohort of enzyme replacement therapy (ERT)-naïve infantile Pompe disease patients. Antibody-dependent cellular cytotoxicity, the principal mechanism by which rituximab depletes B-cells, requires CD20 binding by Fab'2 of rituximab on B-cells and the concomitant binding of its Fc region to Fc receptors on effector cells or to complement. To protect patients against microbial infections when using rituximab, IVIG was added to the immunomodulation regimen used in Pompe disease. Administration of IVIG can saturate neonatal Fc receptors (FcRn), which recycle endogenous as well as administered polyclonal/monoclonal antibodies via the binding of the Fc moiety to FcRn. As such, the administration of IVIG prior to rituximab, a chimeric mouse-human monoclonal antibody, may sharply reduce the half-life of rituximab and in turn, its efficacy. Based on this understanding, it is vital to understand the optimal timing of IVIG administration in relation to rituximab administration for the purposes of inducing immune tolerance.