Impact of Decalcification, Cold Ischemia, and Deglycosylation on Performance of Programmed Cell Death Ligand-1 Antibodies With Different Binding Epitopes: Comparison of 7 Clones.

Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions.

Tissue Age Affects Antigenicity and Scoring for the 22C3 Immunohistochemistry Companion Diagnostic Test.

Programmed death-ligand 1 (PD-L1) antibody 22C3 is the approved companion diagnostic immunohistochemistry test for treatment with pembrolizumab and cemiplimab in multiple cancer types. The 22C3 and 28-8 antibodies target the extracellular domain (ECD) of PD-L1, which is known to contain N-glycosylation sites. We hypothesize that antigenicity could be affected by the degradation of the glycan part of the epitope and thus change the scoring of the assay over time. Here, we test samples over time and assess the effects of time and deglycosylation on PD-L1 signal by comparing an antibody with an ECD antigen to an antibody with an intracellular domain (ICD) antigen. Ten whole-tissue sections of non-small-cell lung cancer (NSCLC) from 2018 were selected for testing. Fresh-cut serial sections for each case were stained on DAKO Link48 for 22C3 according to the label. In parallel, a previously described laboratory-developed test using E1L3N (an ICD antibody) was performed on the Leica BondRX. Tumor proportion scores for 22C3 and E1L3N were read by a pathologist and compared to the previous clinical diagnoses. To determine the effect using a quantitative approach, a tissue microarray (TMA) cohort with 90 NSCLC cases was similarly assessed. Finally, to determine whether the possible effect of epitope glycosylation, antibodies were tested before and after enzymatic deglycosylation of specimens. We found that 6 of 7 archival positive samples showed a significant reduction in positive staining with 22C3 compared to the original diagnostic sample assessed 3 years earlier. In an older archival TMA cohort, a quantitative significant difference in signal intensity was noted when staining with 22C3 was compared to E1L3N. This loss of signal was not noted in the fresh cell line TMA consistent with a time-dependent degradation of staining. Finally, quantitative assessment of the fresh TMA showed a significant loss of signal after a deglycosylation procedure when stained with 22C3, which was not seen when stained with E1L3N. We believe that these data show that the glycan part of the 22C3 epitope is not stable over time, and that this issue should be considered when assessing archival tissue for diagnostic or research purposes.

Using imlifidase to elucidate the characteristics and importance of anti-GBM antibodies produced after start of treatment.

BACKGROUND: Autoantibodies are common in glomerulonephritis, but the clinical benefit of rapid elimination has not been determined, even in anti-glomerular basement membrane (GBM) disease. Even less is known about the importance of autoantibody characteristics, including epitope specificity and immunoglobulin G (IgG) subclass distribution. We aimed to address this by characterizing the autoantibody profile in anti-GBM patients: we utilized samples from the GOOD-IDES-01 (treating GOODpasture's disease with Imunoglobulin G Degrading Enzyme of Streptococcus pyogenous) (ClinicalTrials.gov identifier: NCT03157037) trial , where imlifidase, which cleaves all IgG in vivo within hours, was given to 15 anti-GBM patients. METHODS: In the GOOD-IDES-01 trial, plasmapheresis was (re)started if anti-GBM antibodies rebounded. Serum samples were collected prospectively for 6 months and analyzed for anti-GBM epitope specificity using recombinant constructs of the EA and EB epitopes, IgG subclass using monoclonal antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA). The results were correlated with clinical data. RESULTS: Patients with a rebound (n = 10) tended to have lower eGFR at 6 months (11 vs 34 mL/min/1.73 m2, P = .055), and patients with dialysis at 6 months had a higher EB/EA ratio at rebound (0.8 vs 0.5, P = .047). Moreover, two patients demonstrated increasing epitope restriction and several patients displayed a shift in subclass distribution at rebound. Six patients were double positive for ANCA. ANCA rebound was seen in 50% of patients; only one patient remained ANCA positive at 6 months. CONCLUSIONS: In this study, rebound of anti-GBM antibodies, especially if directed against the EB epitope, was associated with a worse outcome. This supports the notion that all means should be used to eliminate anti-GBM antibodies. In this study ANCA was removed early and long-term by imlifidase and cyclophosphamide.

Tumour neoantigen mimicry by microbial species in cancer immunotherapy.

Tumour neoantigens arising from cancer-specific mutations generate a molecular fingerprint that has a definite specificity for cancer. Although this fingerprint perfectly discriminates cancer from healthy somatic and germline cells, and is therefore therapeutically exploitable using immune checkpoint blockade, gut and extra-gut microbial species can independently produce epitopes that resemble tumour neoantigens as part of their natural gene expression programmes. Such tumour molecular mimicry is likely not only to influence the quality and strength of the body's anti-cancer immune response, but could also explain why certain patients show favourable long-term responses to immune checkpoint blockade while others do not benefit at all from this treatment. This article outlines the requirement for tumour neoantigens in successful cancer immunotherapy and draws attention to the emerging role of microbiome-mediated tumour neoantigen mimicry in determining checkpoint immunotherapy outcome, with far-reaching implications for the future of cancer immunotherapy.

Bivalent mucosal peptide vaccines administered using the LCP carrier system stimulate protective immune responses against Streptococcus pyogenes infection.

Despite the broad knowledge about the pathogenicity of Streptococcus pyogenes there is still a controversy about the correlate of protection in GAS infections. We aimed in further improving the immune responses stimulated against GAS comparing different vaccine formulations including bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and BPPCysMPEG, a derivative of the macrophage-activating lipopeptide (MALP-2), as adjuvants, respectively, to be administered with and without the universal T helper cell epitope P25 along with the optimized B cell epitope J14 of the M protein and B and T cell epitopes of SfbI. Lipopeptide based nano carrier systems (LCP) were used for efficient antigen delivery across the mucosal barrier. The stimulated immune responses were efficient in protecting mice against a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain. Moreover, combination of the LCP based peptide vaccine with c-di-AMP allowed reduction of antigen dose at the same time maintaining vaccine efficacy.

Neo-epitopes on crotonaldehyde modified DNA preferably recognize circulating autoantibodies in cancer patients.

DNA damage is one of the leading causes of various pathological conditions including carcinogenesis. Crotonaldehyde is a 4-carbon unsaturated bifunctional aldehyde which is found ubiquitously and produced both exogenously and endogenously. It reacts with deoxyguanosine and form adducts with DNA. These adducts were detected and found involved in tumor formation in rats treated with crotonaldehyde. In the present study, structural changes in DNA by crotonaldehyde were evaluated by Fourier transform infrared (FTIR) spectroscopy, differential scanning colorimetry (DSC), dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Enhanced binding was observed in cancer autoantibodies with the DNA modified by crotonaldehyde than the native counterpart. Immunological studies revealed enhanced binding of cancer autoantibodies with crotonaldehyde modified DNA, compared to the native form. Furthermore, lymphocyte DNA isolated from cancer patients demonstrated considerable recognition of anti-Cro-DNA IgG as compared to the DNA from healthy individuals. Therefore, we suggest that crotonaldehyde modified DNA presents unique epitopes, that may trigger autoantibody induction in cancer patients.

Cross-neutralization between three mumps viruses & mapping of haemagglutinin-neuraminidase (HN) epitopes.

BACKGROUND & OBJECTIVES: The reports from the countries where mumps vaccine is given as routine immunization suggest differences in mumps virus neutralizing antibody titres when tested with vaccine and wild type viruses. Such reports are unavailable from countries like India where mumps vaccine is not included in routine immunization. We, therefore, undertook this study to understand the cross-neutralization activity of Indian mumps viruses. METHODS: By using commercial mumps IgG enzyme immunoassay (EIA) and a rapid focus reduction neutralization test (FRNT), a panel of serum samples was tested. The panel consisted of 14 acute and 14 convalescent serum samples collected during a mumps outbreak and 18 archived serum samples. Two wild types (genotypes C and G) and Leningrad-Zagreb vaccine strain (genotype N) were used for the challenge experiments and FRNT titres were determined and further compared. The HN protein sequence of three mumps viruses was analyzed for the presence of key epitopes. RESULTS: All serum samples effectively neutralized mumps virus wild types and a vaccine strain. However, significantly lower FRNT titres were noted to wild types than to vaccine strain (P<0.05). The comparison between EIA and FRNT results revealed 95.6 per cent agreement. No amino acid changes were seen in the epitopes in the Indian wild type strains. All potential N-linked glycosylation sites were observed in Indian strains. INTERPRETATION & CONCLUSIONS: Good cross-neutralization activity was observed for three mumps virus strains, however, higher level of FRNT titres was detected for mumps virus vaccine strain compared to Indian wild type isolates.

Protection against respiratory syncytial virus by inactivated influenza virus carrying a fusion protein neutralizing epitope in a chimeric hemagglutinin.

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology. FROM THE CLINICAL EDITOR: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization.

Vaccine manufacturing and technology: from biotechnological platforms to syntethic epitopes, current viepoint.

The Purposes: The Purposes: the review take into account short history of vaccination practice and development of vaccine technology. Methods: In the review we include data from several monographs about manufacturing of vaccines published by authors from such companies as Merck & Co; Sanofi Pasteur; Dynavax Europe/Rhein Biotech GmbH; Latham Biopharm Group; Aridis Pharmaceuticals LLC; Genentech; Amgen; Shamir Biologics LLC; Biopharm Services US; Novartis Pharma AG, аnd several research centers: Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research; Purdue University, West Lafayette, IN, US; Department of Pharmaceutical Chemistry, Univ. Of Kansas; Max Planck Institute for dynamics of Complex Technical Systems; Fraunhofer USA Center for Molecular Biotechnology; US Dep. of Agriculture Animal and Plant Health Inspection Service, etc. Results: In historic literature there are data about inoculation practices in antique China, Persia, India, Byzantium, native Americans, some African population. In modern immunology since the end of XIX century the vaccines were produced at the in vivo platforms - in animals (rabbits, mice, cows). Since 1931 due to E. Goodpasture' elaboration most virus vaccines were and are produced at the in ovo platform. In 1949 J.F. Enders elaborated large-scale polio virus production in the primary culture of monkey kidney cells in vitro. Up to day primary culture of chiken embrio fibroblasts are used to large-scale production of vaccine viruses of measles, mumps, rabies. Since 2000-th in Western countries most part of virus vaccines were began to produced via a cultivation in continuous tumor cell lines. The last technology is the most low cost for large-scale production of vaccines. We review several new biotechnological platforms for the production of the recombinant protein or virus-like particles as subunit vaccines: plant system, algae, mushrooms, insect cells, etc. Conclusion: Beside of good purpose of vaccination - prophylactic of several infectious deseases, doctors must take into account possibility of inter-species transmission of unknown pathogens (retroviruses, prions, etc) from biotechnological platforms - animals, cell cultures - into human population, and don't ignore L.A. Zilber' theory of virus' etiology of cancer diseases.

Protective effect of a Protein Epitope Mimetic CCR10 antagonist, POL7085, in a model of allergic eosinophilic airway inflammation.

BACKGROUND: Potential involvement of the CCR10/CCL28 axis was recently reported in murine models of allergic asthma. If confirmed, blockade of the CCR10 receptor would represent an alternative to current asthma therapies. We evaluated the effect of a novel Protein Epitope Mimetic CCR10 antagonist, POL7085, in a murine model of allergic eosinophilic airway inflammation. METHODS: Mice were sensitized and challenged to ovalbumin. POL7085, a CCR10 antagonist (7.5 and 15 mg/kg), dexamethasone (1 mg/kg) or vehicle were administered intranasally once daily 1h before each allergen challenge. On day 21, airway hyperresponsiveness, bronchoalveolar lavage inflammatory cells and Th2 cytokines, and lung tissue mucus and collagen were measured. RESULTS: Allergen challenge induced airway hyperresponsiveness in vehicle-treated animals as measured by whole body barometric plethysmography, and eosinophilia in bronchoalveolar lavage. POL7085 dose-dependently and significantly decreased airway hyperresponsiveness (34 ± 16 %) and eosinophil numbers in bronchoalveolar lavage (66 ± 6 %). In addition, the highest dose of POL7085 used significantly inhibited lung IL-4 (85 ± 4 %), IL-5 (87 ± 2 %) and IL-13 (190 ± 19 %) levels, and lung collagen (43 ± 11 %). CONCLUSIONS: The Protein Epitope Mimetic CCR10 antagonist, POL7085, significantly and dose-dependently decreased allergen-induced airway hyperresponsiveness and airway inflammation after once daily local treatment. Our data give strong support for further investigations with CCR10 antagonists in asthmatic disease.

Epitope-based approaches to a universal influenza vaccine.

The development of vaccines has been one of the most important contributions of immunology to public health to date. Although several infectious diseases have all but vanished thanks to effective vaccines, the most common infectious disease, influenza, still represents a major threat to public health. This is more concerning than ever before in light of potentially virulent avian pandemic strains which have emerged in the last decade and infected human hosts, causing high morbidity and mortality. Despite considerable efforts to improve production of influenza vaccines and vaccinate large portions of the population annually, the currently available influenza vaccines are strain-specific and not effective enough. Considering the vulnerability of infants and elderly to seasonal influenza-related complications and the ever present public health threat of a deadly influenza pandemic, there is urgent need for a new kind of influenza vaccine. Ideally, such a vaccine should provide enhanced long term, multi-strain protection without compromising safety and in this way, dramatically improve global protection against seasonal and pandemic influenza viruses. This review highlights one approach to developing a universal influenza vaccine, which is based on highly conserved viral sequences, 'epitopes', that specifically activate humoral and/or cellular immune responses. This approach to vaccinology was pioneered by Prof Arnon, who initiated development of an epitope-based universal vaccine called Multimeric-001 (M-001), which has already been validated in clinical trials to induce broad immunity against A and B-Type, seasonal and pandemic strains.

α-Gal Nanoparticles in CNS Trauma: II. Immunomodulation Following Spinal Cord Injury (SCI) Improves Functional Outcomes.

BACKGROUND: Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. METHODS: α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. RESULTS: Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. CONCLUSIONS: Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.

Emerging antibody-based therapies for Huntington's disease: current status and perspectives for future development.

INTRODUCTION: Being an inherited neurodegenerative disease with an identifiable genetic defect, Huntington's disease (HD) is a suitable candidate for early intervention, possibly even in the pre-symptomatic stage. Our recent advances in elucidating the pathogenesis of HD have revealed a series of novel potential therapeutic targets, among which immunotherapies are actively pursued in preclinical experiments. AREAS COVERED: This review focuses on the potential of antibody-based treatments targeting various epitopes (of mutant huntingtin as well as phosphorylated tau) that are currently evaluated in vitro and in animal experiments. The references used in this review were retrieved from the PubMed database, searching for immunotherapies in HD, and clinical trial registries were reviewed for molecules already evaluated in clinical trials. EXPERT OPINION: Antibody-based therapies have raised considerable interest in a series of neurodegenerative diseases characterized by deposition of aggregated of aberrantly folded proteins, HD included. Intrabodies and nanobodies can interact with mutant huntingtin inside the nervous cells. However, the conflicting results obtained with some of these intrabodies highlight the need for proper choice of epitopes and for developing animal models more closely mimicking human disease. Approval of these strategies will require a considerable financial and logistic effort on behalf of healthcare systems.

High level expression, purification and characterization of recombinant CCR5 as a vaccine candidate against HIV.

Cysteine-cysteine chemokine receptor type 5 (CCR5) is an important co-receptor for human immunodeficiency virus (HIV) infection and CCR5 neutralizing agents have proven efficient in patients suffering from HIV infection. Here, we expressed and purified various CCR5 vaccines named rCCR5, PADRE-rCCR5, GST-C1 and GST-C2 composed of different epitopes of CCR5. Results showed that vaccines containing multiple epitopes (rCCR5 and PADRE-rCCR5) induced stronger immune responses than single-epitope ones (GST-C1 and GST-C2). In addition, the elicited antibodies can specifically bind CCR5(+) U937 but not CCR5(-) Wish cells. These results demonstrate that the CCR5 vaccines are useful for further research, especially for the in vitro preclinical evaluation of their potential as biological CCR5 neutralizing agents.

Activation of inflammatory cells and cytokines by peptide epitopes in vitro: a simple in-vitro screening assay for prioritizing them for in-vivo studies.

OBJECTIVE: Antigen-specific immune modulation is an attractive approach to atherosclerosis treatment. The aim of this study was to develop an in-vitro assay to screen peptide molecules for their inflammatory propensity. MATERIALS: Human dendritic cells derived from CD14(+) monocytes were activated using peptides derived from apolipoprotein B100 (ApoB), heat shock protein 60 (HSP60) and complement cascade (peptide A) in vitro, and used for priming autologous T cells. Proliferation of T cells, their differentiation to regulatory cells (Treg) and their cytokine profile were studied. The efficacy of the peptides in preventing atherosclerosis was studied in ApoB(tm2Sgy)/Ldlr(tm1Her/J) knockout mice. RESULTS AND CONCLUSION: ApoB and HSP60 peptides induced T-cell proliferation and expansion of regulatory T cells with interleukin-10 and transforming growth factor-ß secretion. In comparison, peptide A was a poor stimulator of T cells and was found to induce tumor necrosis factor-α secretion by activated T cells. ApoB and HSP60 peptides were found to reduce early atherosclerotic lesion formation in mice by 32.1 and 33.5 %, respectively, while the reduction with peptide A was 5.7 %. Thus the in-vitro assay shows an apparent correlation with in-vivo activity and can be developed as a screening assay to prioritize the candidate molecules for animal efficacy testing.

RAFTsomes containing epitope-MHC-II complexes mediated CD4+ T cell activation and antigen-specific immune responses.

PURPOSE: To develop a liposome formulation incorporating antigen-presenting cells (APCs) membrane microdomains with enriched epitope/MHC complexes to evaluate the activities of these liposomes (RAFTsomes) to activate T cells and prime immune responses. METHODS: We isolated membrane microdomain structures that contained the epitope/MHC complexes from ovalbumin (OVA) primed dendritic cells (DCs), and reconstituted them on liposomes surface by detergent dialysis. The resulted RAFTsomes were purified by density gradient centrifugation. Their T cell activation functions were evaluated by IL-2 secreting and proliferation assays in vitro. In vivo immune responses and the protective effect against OVA expressing EG.7 tumor challenge were also examined. RESULTS: Membrane microdomains containing enriched epitope/MHC complexes can be reconstituted into liposomes with defined size and composition. The integrity and activities of these complexes after reconstitution were confirmed by in vitro T cell assays. OVA epitope loaded RAFTsomes injected in vivo resulted in high anti-OVA IgG production (predominantly IgG1). The immunized mice were protected from EG.7 tumor cell inoculation challenge. CONCLUSIONS: Based on these findings, we propose that RAFTsomes can be prepared with unique properties that may be used as an antigen delivery system for immunotherapeutic applications.

Immunization with a combination of 2 peptides derived from the C5a receptor significantly reduces early atherosclerotic lesion in Ldlr(tm1Her) Apob(tm2Sgy) J mice.

OBJECTIVE: The goal of this study was to assess whether immunization of Ldlr(tm1Her) Apob(tm2Sgy) J mice with 2 peptides located at the N-terminus of the C5a receptor (C5aR), either alone or in combination, is effective in reducing atherosclerotic lesions. METHODS AND RESULTS: Five- to 6-week-old female Ldlr(tm1Her)Apob(tm2Sgy) J mice were immunized using a repetitive immunization multiple sites strategy with keyhole limpet hemocyanin-conjugated peptides derived from the C5aR, either alone (designated as C5aR-P1 [aa 1-21] and C5aR-P2 [aa 19-31]) or in combination (designated as C5aR-P1+C5aR-P2). Mice were fed a high-fat diet for 10 weeks. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Immunization of Ldlr(tm1Her)Apob(tm2Sgy) J mice with these peptides elicited high concentrations of antibodies against each peptide. Immunization with the single peptide inhibited plaque development. Combined inoculation with C5aR-P1+C5aR-P2 had an additive effect on reducing the lesion in the aorta sinus and descending aortas when compared with controls. This effect correlated with cellular infiltration and cytokine/chemokine secretion in the serum or in stimulated spleen cells as well as specific cellular immune responses when compared with controls. CONCLUSIONS: Immunization of mice with C5aR-P1 and C5aR-P2, either alone or in combination, was effective in reducing early atherosclerotic lesion development. The combined peptide is more potential than either epitope alone to reduce atherosclerotic lesion formation through the induction of a specific Treg cell response as well as blockage of monocyte differentiation into macrophages.

Unlocking the potential of agonist antibodies for treating cancer using antibody engineering.

Agonist antibodies that target immune checkpoints, such as those in the tumor necrosis factor receptor (TNFR) superfamily, are an important class of emerging therapeutics due to their ability to regulate immune cell activity, especially for treating cancer. Despite their potential, to date, they have shown limited clinical utility and further antibody optimization is urgently needed to improve their therapeutic potential. Here, we discuss key antibody engineering approaches for improving the activity of antibody agonists by optimizing their valency, specificity for different receptors (e.g., bispecific antibodies) and epitopes (e.g., biepitopic or biparatopic antibodies), and Fc affinity for Fcγ receptors (FcγRs). These powerful approaches are being used to develop the next generation of cancer immunotherapeutics with improved efficacy and safety.

Structures of Cancer Antigen Mesothelin and Its Complexes with Therapeutic Antibodies.

The tumor-associated antigen mesothelin is expressed at high levels on the cell surface of many human cancers, while its expression in normal tissues is limited. The binding of mesothelin to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. Immunotherapeutic strategies targeting mesothelin are being intensively investigated. Here, we report the crystal structures of mesothelin that reveal a compact, right-handed solenoid consisting of 24 short helices and connecting loops. These helices form a nine-layered spiral coil that resembles ARM/HEAT family proteins. Glycan attachments have been identified in the structure for all three predicted N-glycosylation sites and confirmed with samples from cell culture and patient ascites. The structures of full-length mesothelin and its complex with the Fab of MORAb-009 reveal the interaction of the antibody with the complete epitope, which has not been reported previously. The N-terminal half of mesothelin is conformationally rigid, suitable for eliciting specific antibodies, whereas its C-terminal portion is more flexible. The structure of the C-terminal shedding-resistant fragment of mesothelin complexed with a mAb 15B6 displays an extended linear epitope and helps explain the protection afforded by the antibody for the shedding sites. Significance: The structures of full-length mesothelin and its complexes with antibodies reported here are the first to be determined experimentally, providing atomic models for structural organization of this protein and its interactions with antibodies. It offers insights into the function of mesothelin and guidance for further development of therapeutic antibodies.

Anti-citrullinated protein antibodies as biomarkers in rheumatoid arthritis.

INTRODUCTION: The serological biomarker anti-citrullinated protein antibodies (ACPAs) may have several functions but is especially important for the diagnosis of rheumatoid arthritis (RA) along with clinical symptoms. AREAS COVERED: This review provides an overview of ACPAs, which are useful in RA diagnostics and may improve our understanding of disease etiology. PubMed was searched with combinations of words related to antibodies recognizing epitopes containing the post-translationally modified amino acid citrulline in combination with rheumatoid arthritis; cyclic citrullinated peptide, CCP, anti-CCP, anti-citrullinated protein antibodies, ACPA, citrullination, peptide/protein arginine deiminase, PAD, filaggrin, vimentin, keratin, collagen, perinuclear factor, EBNA1, EBNA2, and others. From this search, we made a qualitative extract of publications relevant to the discovery, characterization, and clinical use of these antibodies in relation to RA. We highlight significant findings and identify areas for improvement. EXPERT OPINION: ACPAs have high diagnostic sensitivity and specificity for RA and recognize citrullinated epitopes from several proteins. The best-performing single epitope originates from Epstein-Barr Virus nuclear antigen 2 and contains a central Cit-Gly motif, which is recognized by ACPAS when located in a flexible peptide structure. In addition, ACPAs may also have prognostic value, especially in relation to early treatment, although ACPAs' main function is to aid in the diagnosis of RA.