Cryptic Epitopes Research Articles

Designing occluded-open Env-based immunogens for HIV-1.

The highly variable and heavily glycosylated surface of the HIV-1 Envelope (Env) trimer present formidable challenges for eliciting broad and potent responses to the virus. While the overwhelming majority of broadly neutralizing antibodies described to date target the pre-fusion closed form of Env, a few neutralizing antibodies, including the broadly neutralizing antibody b12, target an open state of the trimer known as occluded-open. In the occluded-open state, glycans are splayed outwards and conserved residues that are inaccessible on the closed state are revealed. Here, I present a strategy for stabilizing this otherwise transient state of the trimer with the goal of eliciting antibodies that target the unexplored landscape of cryptic epitopes present on the occluded-open state of HIV-Env.

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies.

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency. IMPORTANCE An effective HIV-1 vaccination strategy will need to stimulate rare precursor B cells of multiple bNAb lineages and affinity mature them along desired pathways. Here, we searched for V2-apex germ line-targeting Envs among a large set of diverse primate lentiviruses and identified minimally modified versions of one chimpanzee SIV Env that bound several human V2-apex bNAb precursors and stimulated one of these in a V2-apex bNAb precursor-expressing knock-in mouse. We also generated chimeric simian-chimpanzee immunodeficiency viruses and showed that they elicit low-titer V2-directed heterologous plasma breadth in six of seven infected rhesus macaques. Characterization of this antibody response identified a new class of weakly cross-reactive neutralizing antibodies that target the V2-apex, but only in occluded-open Env trimers. The existence of this cryptic epitope, which in some Env backgrounds is immunodominant, needs to be considered in immunogen design.

Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

In vitro characterization of a novel Arg102 mutation in the ADAMTS13 metalloprotease domain

BackgroundCongenital thrombotic thrombocytopenic purpura is caused by defects in the ADAMTS13 gene. ADAMTS13 is normally preactivated by conformational changes of the Metalloprotease (M) domain. Studying a novel congenital thrombotic thrombocytopenic purpura p.R102S mutation in the M domain, which results in undetectable ADAMTS13 activity in the patient, could help to explain the patients’ phenotype and to elucidate the currently unclear mechanism of allosteric preactivation. ObjectivesTo investigate the in vitro effect of p.R102S mutation on ADAMTS13 secretion, activity, and allosteric preactivation. MethodsMolecular modeling was used to study the effect of the mutation on the stability of ADAMTS13. Recombinant mutant ADAMTS13 was generated by transient and stable transfection of, respectively, CHO K1 and HEK293-T cells. ADAMTS13 antigen was measured in enzyme-linked immunosorbent assay. ADAMTS13 activity was measured in a FRETS-VWF73 assay. Allosteric preactivation was assessed in FRETS-VWF73 assay, using monoclonal antibody (mAb) 17G2 that normally induces a ∼2-fold increase in activity, and in enzyme-linked immunosorbent assay using mAb 6A6 recognizing a cryptic epitope in the M domain that becomes exposed after binding of 17G2. Resultsp.R102S mutation destabilizes the interactions between the M and Disintegrin-like (D) domain. p.R102S mutant secretion was impaired (35% of wild type) and activity was severely reduced (12% of wild type). p.R102S mutant could still be activated and the cryptic epitope of 6A6 was still fully exposed by 17G2 addition. Conclusionp.R102S mutation destabilizes the M–D domain interactions, causing impaired ADAMTS13 secretion and activity, which explains the patients’ phenotype. Allosteric preactivation of ADAMTS13 remains conserved in the presence of the p.R102S mutation.

Nanobody-peptide-conjugate (NPC) for passive immunotherapy against SARS-CoV-2 variants of concern (VoC): a prospective pan-coronavirus therapeutics.

The COVID-19 crisis, incited by the zoonotic SARS-CoV-2 virus, has quickly escalated into a catastrophic public health issue and a grave threat to humankind owing to the advent of mutant viruses. Multiple pharmaceutical therapies or biologics envision stopping the virus from spreading further; however, WHO has voiced concerns about the variants of concern (VoCs) inability to respond. Nanobodies are a new class of antibody mimics with binding affinity and specificity similar to classical mAbs, as well as the privileges of a small molecular weight, ease of entry into solid tissues, and binding cryptic epitopes of the antigen. Herein, we investigated multiple putative anti-SARS-CoV-2 nanobodies targeting the Receptor binding domain of the WHO-listed SARS-CoV-2 variants of concern using a comprehensivecomputational immunoinformatics methodology. With affinity maturation via alanine scanning mutagenesis, we remodeled an ultrapotent nanobody with substantial breadth and potency, exhibiting pico-molar binding affinities against all the VoCs. An antiviral peptide with specificity for ACE-2 receptors was affixed to make it multispecific and discourage viral entry. Collectively, we constructed a broad-spectrum therapeutic biparatopic nanobody-peptide conjugate (NPC) extending coverage to SARS-CoV-2 VoCs RBDs. We PEGylated the biparatopic construct with 20kD maleimide-terminated PEG (MAL-(PEG)n-OMe) to improve its clinical efficacy limiting rapid renal clearance, and performed in silico cloning to facilitate future experimental studies. Our findings suggest that combining biparatopic nanobody conjugate with standard treatment may be a promising bivariate tool for combating viral entry during COVID-19 illness.

Nilabh Shastri – Towards understanding classical and non-classical MHC-I antigen processing and presentation

Major histocompatibility complex (MHC-I) peptide antigen processing and presentation has experienced a revived interest in the context of immuno oncology, immune surveillance escape by pathogen mutations and technical advances that accelerate vaccine design. This sheds new light on the discoveries made by Nilabh Shastri and colleagues that includes the characterisation of cryptic MHC-I peptide antigen epitopes derived from untranslated regions and the N-terminal trimming of peptide antigen precursors by the aminopeptidase ERAAP (ERAP1/2 / ARTS1/LRAP) in the endoplasmic reticulum (ER) prior to the complete assembly of MHC-I complexes and their subsequent exposure to the cell surface. These scientific findings have important implications for developing novel therapeutic approaches in immunotherapy and modern vaccine design.

Antigenic mapping reveals sites of vulnerability on α-HCoV spike protein

Understanding the antigenic signatures of all human coronaviruses (HCoVs) Spike (S) proteins is imperative for pan-HCoV epitopes identification and broadly effective vaccine development. To depict the currently elusive antigenic signatures of α-HCoVs S proteins, we isolated a panel of antibodies against the HCoV-229E S protein and characterized their epitopes and neutralizing potential. We found that the N-terminal domain of HCoV-229E S protein is antigenically dominant wherein an antigenic supersite is present and appears conserved in HCoV-NL63, which holds potential to serve as a pan-α-HCoVs epitope. In the receptor binding domain, a neutralizing epitope is captured in the end distal to the receptor binding site, reminiscent of the locations of the SARS-CoV-2 RBD cryptic epitopes. We also identified a neutralizing antibody that recognizes the connector domain, thus representing the first S2-directed neutralizing antibody against α-HCoVs. The unraveled HCoVs S proteins antigenic similarities and variances among genera highlight the challenges faced by pan-HCoV vaccine design while supporting the feasibility of broadly effective vaccine development against a subset of HCoVs.

Carboxyl-Terminal Decoy Epitopes in the Capsid Protein of Porcine Circovirus Type 2 Are Immunogenicity-Enhancers That Elicit Predominantly Specific Antibodies in Non-Vaccinated Pigs.

In the context of the carboxyl-terminus (C-terminus) of the capsid protein of porcine circovirus type 2a (PCV2a) and PCV2a vaccines, this study aimed to explore its unrevealing cryptic epitope and its relation to PCV2-infected herd immunity. To discover the C-terminus of the capsid protein of PCV2a, monoclonal antibodies (mAbs) were generated in this work. Two mAbs bound the two minimal linear epitopes (229PPLKP233 and 228DPPLNP233 (or 229PPLNP233)), which were located at the C-terminus of the capsid proteins of PCV2a and PCV2b, respectively. One mAb bound to the minimal linear epitope (220QFREFNLK227, peptide P82), but it neither bound the virus-like particle (VLP) of PCV2a nor produced positive staining in PCV2a-infected cells by immunofluorescence assay. Further, the residues 220-227 were not accessible on the surface of the VLP on the three-dimensional model, but the residues 228-231 extend toward the VLP exterior. Immunoassays were conducted in this study to screen anti-viral peptide-specific IgGs, which could differentiate vaccinated pigs from non-vaccinated ones. The data show two 220QFREFNLKDPPLKP233-containing peptides had a significantly higher binding reactivity with sera from PCV2-infected pigs in the control group than with sera from the VLP-vaccine group, particularly seen in sera from swine aged 15 weeks to 24 weeks. However, the peptide P82 had not this phenomenon in that test. This study confirmed that C-terminal epitopes play an important role in PCV2-induced decoy of swine humoral immunity.

A bovine antibody possessing an ultralong complementarity-determining region CDRH3 targets a highly conserved epitope in sarbecovirus spike proteins

Broadly neutralizing antibodies have huge potential as novel antiviral therapeutics due to their ability to recognize highly conserved epitopes that are seldom mutated in viral variants. A subset of bovine antibodies possess an ultralong complementarity-determining region (CDR)H3 that is highly adept at recognizing such conserved epitopes, but their reactivity against Sarbecovirus Spike proteins has not been explored previously. Here, we use a SARS-naïve library to isolate a broadly reactive bovine CDRH3 that binds the receptor-binding domain of SARS-CoV, SARS-CoV-2, and all SARS-CoV-2 variants. We show further that it neutralizes viruses pseudo-typed with SARS-CoV Spike, but this is not by competition with angiotensin-converting enzyme 2 (ACE2) binding. Instead, using differential hydrogen-deuterium exchange mass spectrometry, we demonstrate that it recognizes the major site of vulnerability of Sarbecoviruses. This glycan-shielded cryptic epitope becomes available only transiently via interdomain movements of the Spike protein such that antibody binding triggers destruction of the prefusion complex. This proof of principle study demonstrates the power of invitro expressed bovine antibodies with ultralong CDRH3s for the isolation of novel, broadly reactive tools to combat emerging pathogens and to identify key epitopes for vaccine development.

Epitope of antiphospholipid antibodies retrieved from peptide microarray based on R39‐R43 of β2‐glycoprotein I

BackgroundAntiphospholipid antibody (aPL) syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies and thromboembolic or pregnancy complications. Although cryptic epitope R39‐R43 belonging to beta‐2‐glycoprotein 1 (β2GP1) has been identified as the main antigenic determinant for aPLs, we have recently demonstrated that the epitope is a motif determined by the polarity, rather than by the sequence or charge of amino acids. ObjectiveIn the present study, we wanted to identify the association of residues needed to obtain the highest aPL affinity. MethodsBased on the epitope R39‐R43 and our identified motif, we generated a printed peptide microarray of 676 different peptides. These peptides have been then screened for their ability to interact with the plasmas from 11 well‐characterized APS patients and confirmed by surface plasma resonance assay. Results and ConclusionsWe identified a peptide that selectively bound immunoglobulin G (IgG) derived from APS patients with 100 times more affinity than β2GP1, Domain I, or epitope R39‐R43. This peptide is able to inhibit the activity of IgG derived from APS patients in vitro. We have also generated a monoclonal IgG antibody against this peptide. Using both peptide and monoclonal antibody, we have been able to develop a fully standardized indirect colorimetric immunoassay with highly sensitivity. The identification of the optimized peptide offers a new standardized and accurate tool for diagnostics of APS. Furthermore, having increased affinity for aPL, this peptide could represent a useful tool as prevention strategy for APS and an alternative to the use of anticoagulants.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Omicron SARS-CoV-2 mutations stabilize spike up-RBD conformation and lead to a non-RBM-binding monoclonal antibody escape

Omicron SARS-CoV-2 is rapidly spreading worldwide. To delineate the impact of emerging mutations on spike’s properties, we performed systematic structural analyses on apo Omicron spike and its complexes with human ACE2 or S309 neutralizing antibody (NAb) by cryo-EM. The Omicron spike preferentially adopts the one-RBD-up conformation both before and after ACE2 binding, which is in sharp contrast to the orchestrated conformational changes to create more up-RBDs upon ACE2 binding as observed in the prototype and other four variants of concern (VOCs). Furthermore, we found that S371L, S373P and S375F substitutions enhance the stability of the one-RBD-up conformation to prevent exposing more up-RBDs triggered by ACE2 binding. The increased stability of the one-RBD-up conformation restricts the accessibility of S304 NAb, which targets a cryptic epitope in the closed conformation, thus facilitating the immune evasion by Omicron. These results expand our understanding of Omicron spike’s conformation, receptor binding and antibody evasion mechanism.

Molecular dynamics studies reveal structural and functional features of the SARS-CoV-2 spike protein.

The SARS‐CoV‐2 virus is responsible for the COVID‐19 pandemic the world experience since 2019. The protein responsible for the first steps of cell invasion, the spike protein, has probably received the most attention in light of its central role during infection. Computational approaches are among the tools employed by the scientific community in the enormous effort to study this new affliction. One of these methods, namely molecular dynamics (MD), has been used to characterize the function of the spike protein at the atomic level and unveil its structural features from a dynamic perspective. In this review, we focus on these main findings, including spike protein flexibility, rare S protein conformational changes, cryptic epitopes, the role of glycans, drug repurposing, and the effect of spike protein variants.

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies.

The coronavirus spike (S) glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus S proteins. This class of mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and beta-coronaviruses, including animal coronavirus WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope, which is hidden in prefusion stabilized S, and becomes exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.

IMM-BCP-01, a patient-derived anti-SARS-CoV-2 antibody cocktail, is active across variants of concern including Omicron BA.1 and BA.2.

Monoclonal antibodies are an efficacious therapy against SARS-CoV-2. However, rapid viral mutagenesis, led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta, Omicron BA.1 and BA.2. Here, we describe an antibody cocktail IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD, altered the conformation of the Spike Trimer, promoting release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo, and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrated that the three antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.

High frequency of anti-DSG 2 antibodies in post COVID-19 serum samples

BackgroundThere is growing recognition that COVID-19 does cause cardiac sequelae. The underlying mechanisms involved are still poorly understood to date. Viral infections, including COVID-19, have been hypothesized to contribute to autoimmunity, by exposing previously hidden cryptic epitopes on damaged cells to an activated immune system. Given the high incidence of cardiac involvement seen in COVID-19, our aim was to determine the frequency of anti-DSG2 antibodies in a population of post COVID-19 patients. Methods and results300 convalescent serum samples were obtained from a group of post COVID-19 infected patients from October 2020 to February 2021. 154 samples were drawn 6 months post-COVID-19 infection and 146 samples were drawn 9 months post COVID infection. 17 samples were obtained from the same patient at the 6- and 9- month mark. An electrochemiluminescent-based immunoassay utilizing the extracellular domain of DSG2 for antibody capture was used. The mean signal intensity of anti-DSG2 antibodies in the post COVID-19 samples was significantly higher than that of a healthy control population (19 ± 83.2 in the post-COVID-19 sample vs. 2.1 ± 7.2 (p < 0. 0001) in the negative control healthy population). Of note, 29.3% of the post COVID-19 infection samples demonstrated a signal higher than the 90th percentile of the control population and 8.7% were higher than the median found in ARVC patients. The signal intensity between the 6-month and 9-month samples did not differ significantly. ConclusionsWe report for the first time that recovered COVID-19 patients demonstrate significantly higher and sustained levels of anti-DSG2 autoantibodies as compared to a healthy control population, comparable to that of a diagnosed ARVC group.

Counter changes with changelessness: cope with SARS-CoV-2 immune evasion by targeting cryptic epitopes

Counter changes with changelessness: cope with SARS-CoV-2 immune evasion by targeting cryptic epitopes

P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS

Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies. Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients. Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples. Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10-5 or 10-6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies. Image:Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy.

Citrullination modulates MHC class II antigen processing and presentation by revealing cryptic epitopes

Abstract Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and can facilitate loss of tolerance to self-antigens. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines; however, the initiating events in RA remain poorly understood. We investigated the hypothesis that citrullination exposes cryptic epitopes by modifying protein structure and proteolysis in a manner sufficient to activate a previously ignorant repertoire of self-reactive T cells. We first utilized in vitro proteolytic mapping to define the effect of citrullination on four well-defined RA autoantigens-. We then used a natural antigen processing assay to elucidate the molecular mechanisms of citrullination-modulated processing and evaluated autoantigen-specific T cell responses against the unique peptide repertoire. We show that citrullination alters MHC class II processing and presentation of RA autoantigens, resulting in the destruction of dominant epitopes and the generation and presentation of cryptic epitopes, composed primarily of native peptide sequences. The citrullination-dependent repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than the native repertoire and control T cells. The generation of this unique repertoire is achieved through altered protease affinity and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA. Supported by a grant from the Rheumatology Research Foundation and by the Luke Evnin and Deann Wright Fellowship

Understanding the Role of Cancer Glycosylation on Antibody Binding to MUC16

Protein glycosylation is a common post‐translation modification where carbohydrates are anchored to a protein. Glycans play critical roles in mediating cell‐cell interactions, protein‐receptor signaling, and protein folding during translation. One of the hallmarks of cancer is aberrant glycosylation, where shortened glycans specific to cancer are observed. Tumorigenesis, tumor progression, and migration correlate with abnormal glycosylation. Overexpression of the membrane‐bound glycoproteins, MUC16, has been linked to poor prognosis and metastasis in pancreatic and ovarian cancer patients. A common feature of tumor‐associated MUC16 is altered glycosylation, revealing cryptic epitopes where antibodies can bind. These tumor‐specific antibodies can form the basis of new cancer immunotherapies. This project aims to establish an expression system using genetically modified Chinese Hamster Ovary (CHO) cell lines to produce cancer‐associated MUC16 with truncated O‐glycans. A CRISPR/Cas9 modified CHO cell line (CHO‐S□cosmc/mcherry+) was obtained, which has had the gene mcherry inserted into the cosmc locus, a mutation that is hypothesized to produce truncated O‐linked glycans. Here we report the purification and Enzyme‐Linked Immunosorbent Assay (ELISA) analysis of recombinant MUC16 produced in these cell lines. MUC16 in CHO (ExpiCHOwildtype and CHO‐S□cosmc/mcherry+)cell lines were transiently transfected and purified through the Nickel affinity chromatography. The purified protein was analyzed through SDS‐PAGE. Quantitative analysis of MUC16 binding to three different therapeutic antibodies was performed using ELISA. The results revealed that MUC16 produced in CHO‐Smcherry+displayed a higher binding affinity towards the antibodies than ExpiCHOwildtype. The recombinant proteins produced will be further used for examining the role of cancer‐associated glycans binding to therapeutic antibodies.