Engaging the ‘A’ Class Antibody: Variable-Heavy (VH) region influencing IgA1&2 engagement of FcαRI and superantigen proteins G, A, and L

Interest in IgA as an alternative therapeutic and diagnostic antibody has increased over the years, yet much remains to be investigated especially given their importance in activating immune cells in blood and in mucosal immunity. Recent whole antibody-based investigations have shown significant distal effects between the variable (V) and constant (C)-regions that can be mitigated by the different hinge regions of the human IgA subtypes A1 and A2. Diving deeper into the mechanisms underlying this, systematic VH manipulations retaining the CDRs were performed on a panel of 28 IgA1s and A2s across the Trastuzumab and Pertuzumab models, revealed distal effects on FcαRI binding. Further insights from structural modelling showed these effects to also be mitigated by the differing glycosylation patterns in IgA1 and 2 to explain reversal of trends of IgA1s and 2s effected by slight changes in the CDRs. IgAs bound at the Fc showed similar trends but magnitudes better binding to Her2 with that bound by ppL, showing that ppL can sterically hinder Her2 antigen binding. Contrary to canonical knowledge, we found strong evidence of IgAs binding SpG that was narrowed to be at the CH2-3 region, and that the likely binding with SpA was beyond VH3 FWR and most likely at the CH1. VH1 was found to be the most suitable framework (FWRs) for CDR-grafting for both IgA1 and 2. With relevance to interactions with the microbiome at mucosal surfaces, mechanistic insight of how these IgAs can interact bacterial superantigens proteins G, A, and L are also discovered for potential future interventions.One Sentence SummaryAn insight into the mechanism of distal V-region effects on FCAR and superantigens proteins G, A, and L by both IgA1 and A2.

Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2+ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2+ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2+ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2+ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P < 0.05) with minimal obvious toxicity in a tumor-bearing mouse model. We believe that the mPEG × HER2 can significantly improve the therapeutic efficacy, potentially reduce the relapse freqency and thereby achieve good prognosis in ovarian cancer patients.

Binding Properties and Biological Activity of a Novel Therapeutic Monoclonal Antibody Directed toward HER2 Antigen

Several biosimilars for Herceptin® (trastuzumab) and other innovator monoclonal antibodies (mAbs) against HER2 molecule have been developed and approved in the last years. Here, it is reported the binding properties of a novel anti-HER2 mAb (called 5G4) as well as the capacity of this mAb to inhibit cell proliferation and to induce Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC). mAb 5G4 was compared with Herceptin® in a panel of human normal tissues, breast tumors and cell lines using immunohistochemistry, immunocytochemistry, western blot, flow cytometry and colorimetric methods. mAb 5G4 showed weak to moderate staining in breast ductal cells (1/2), gastric glandular cells (1/3) and renal tubes (2/3). Additionally, an intense reactivity was evident with mAb 5G4 in HER2-positive breast adenocarcinomas. The tissue staining was slightly more intense with Herceptin® (trastuzumab) that was used as control. Flow cytometry analysis revealed that mAb 5G4 is able to react with HER2 overexpressing cells (SK-BR-3 and SKOV3) comparable to Herceptin®. The relative binding and the anti-proliferative activity ranges of mAb 5G4 using Herceptin® as reference were between 91-115% and between 91-106%, respectively, which are considered comparable. Moreover, preliminary results suggest that the capacity of mAb 5G4 to induce ADCC in SK-BR-3 cells is non-inferior to Herceptin®. Our data permit to consider that mAb 5G4 has similar binding properties to HER2 antigen and biological activity compared with Herceptin®. However, further determinations such as affinity and FcgRIIIa binding activity of mAb 5G4 could be useful to confirm these results.

Enhanced Immunogenicity of Engineered HER2 Antigens Potentiates Antitumor Immune Responses

For cancer vaccines, the selection of optimal tumor-associated antigens (TAAs) that can maximize the immunogenicity of the vaccine without causing unwanted adverse effects is challenging. In this study, we developed two engineered Human epidermal growth factor receptor 2 (HER2) antigens, K965 and K1117, and compared their immunogenicity to a previously reported truncated HER2 antigen, K684, within a B cell and monocyte-based vaccine (BVAC). We found that BVAC-K965 and BVAC-K1117 induced comparable antigen-specific antibody responses and antigen-specific T cell responses to BVAC-K684. Interestingly, BVAC-K1117 induced more potent antitumor activity than the other vaccines in murine CT26-HER2 tumor models. In addition, BVAC-K1117 showed enhanced antitumor effects against truncated p95HER2-expressing CT26 tumors compared to BVAC-K965 and BVAC-K684 based on the survival analysis by inducing T cell responses against intracellular domain (ICD) epitopes. The increased ICD epitope-specific T cell responses induced by BVAC-K1117 compared to BVAC-K965 and BVAC-K684 were recapitulated in human leukocyte antigen (HLA)-untyped human PBMCs and HLA-A*0201 PBMCs. Furthermore, we also observed synergistic antitumor effects between BVAC-K1117 and anti-PD-L1 antibody treatment against CT26-HER2 tumors. Collectively, our findings demonstrate that inclusion of a sufficient number of ICD epitopes of HER2 in cellular vaccines can improve the antitumor activity of the vaccine and provide a way to optimize the efficacy of anticancer cellular vaccines targeting HER2.

Аffinity properties of plant-made anti-her2 antibodies

The Her2 receptor is an important target for antitumor therapy in the treatment of breast cancer. Trastuzumab, based on anti-Her2 monoclonal antibodies, is used in clinical practice. Trastuzumab is produced by animal cells culture technology and is quite expensive. We use the technology of production of recombinant antibodies in the plants Nicotiana benthamiana with a high yield of final purified protein. Objective. The aim of following study is a comparison of monoclonal antibodies received via classic cell culture technology and produced in plant biomass. Materials and methods. Recombinant plant-made antibodies were isolated by affinity chromatography from the biomass of N. benthamiana plants agroinfiltrated by vector constructs. Comparison of affinity properties was carried out by immunocytochemical staining of cells and competitive binding using flow cytometry analysis. Results and conclusion. We show that the antibodies expressed in N. benthamiana are equal to those obtained from mammalian cells in binding to Her2 antigen localized on the surface of the SK-BR-3 cells. In the present work it was shown that the plant-made anti-Her2 antibodies do not differ in specific binding with the Her2 antigen, as well as with IV subdomain of the Her2 receptor.

Detection of HER2 through Antibody Immobilization Is Influenced by the Properties of the Magnetite Nanoparticle Coating

Considerable effort has been focused on improving the control of size, shape, and surface modifications to detect proteins. The purpose of this study was to compare the efficiencies of aminosilane-coated magnetite (As-M) nanoparticles (NPs), dextran-coated magnetite nanoparticles (Dx-M), and bare nanoparticles for conjugating single-chain variable fragment antibodies (scFvs) with the aim of detecting the human epidermal growth factor receptor 2 (HER2) protein. Dx-M and As-M NPs were characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, and Raman spectroscopy. Dx-M and As-M were conjugated with a monoclonal scFv for active targeting of the HER2 antigen. Aminosilane surface coating enhanced the scFv conjugation efficiency over twofold compared to that of the dextran-coated magnetite NPs for the detection of HER2 proteins.