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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.

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.

International audience; VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2-Fc-VHH2. The structure of VHH2-Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2-Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2-Fc). This new type of molecules, VHH2-Fc-VHH2, could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Highly selective nanoprobes have been developed based on SERS-active Au@Ag nanoparticles protected by a PEG coating and functionalized with monoclonal antibodies against human epidermal growth factor receptor 2 (HER2). The PEG coating allows to drastically reduce unspecific interactions during incubation on tissues, while the monoclonal antibodies allow a highly specific targeting of HER2. Using the designed SERS nanoprobes combined with a spectral imaging and data weighting approach, we demonstrate the proportionality between the SERS signal and the amount of HER2 antigen on the cell membranes as measured by digital image analysis of IHC staining in microscopic breast tumors (linear fit R2 = 0.87). We also show that the level of expression of HER2 measured by SERS is significantly different between several microscopic tumor parts of the same tissue slide. Therefore, SERS is proving to be a suitable technique for the localized quantitative measurement of specific markers in breast cancerous tissues. Owing to its high multiplexing capabilities, SERS could be a future tool of choice for characterizing the molecular heterogeneity of tumors at the microscopic scale.

HER2 antigen is a marker used for breast cancer diagnosis and prevention. Its determination has great importance since breast cancer is one of the most insidious types of cancer in women. HER2 antigen assessment in human serum is traditionally achieved by enzyme-linked immunosorbent assay (ELISA method), but it has some disadvantages, such as suppressing the thermodynamic–kinetic studies regarding the antibody–antigen interaction, and the use of labeled molecules that can promote false positive responses. Biosensors based on surface plasmon resonance (SPR) are sensitive optical techniques widely applied on bioassays. The plasmonic devices do not operate with labeled molecules, overcoming conventional immunoassay limitations, and enabling a direct detection of target analytes. In this way, a new SPR biosensor to assess HER2 antigen has been proposed, using nanohole arrays on a gold thin film by signal transduction of transmitted light measurements from array image acquisitions. These metallic nanostructures may couple the light directly on surface plasmons using a simple collinear arrangement. The proposed device reached an average sensitivity for refractive index (RI) variation on a metal surface of 4146 intensity units/RIU (RIU = RI units). The device feasibility on biomolecular assessment was evaluated. For this, 3 ng ml−1 known HER2 antigen concentration was efficiently flowed (using a microfluidic system) and detected from aqueous solutions. This outcome shows that the device may be a powerful apparatus for bioassays, particularly toward breast cancer diagnosis and prognosis.

The objectives of this phase I/II study (NCT00140738) were to evaluate the safety and clinical activity of a cancer immunotherapeutic agent (recombinant HER2 protein (dHER2) and the immunostimulant AS15) in patients with HER2-overexpressing metastatic breast cancer (MBC). Forty HER2-positive MBC patients received up to 18 doses (12q2w, 6q3w) of dHER2 immunotherapeutic, as first- or second-line therapy following response to trastuzumab-based treatment as maintenance. Toxicity was graded by the Common Terminology Criteria for Adverse Events (CTCAE) and clinical activity was evaluated by target lesion assessment according to the Response Evaluation Criteria in Solid Tumors (RECIST). Immunogenicity was assessed. The dHER2 immunotherapeutic was well tolerated: grade 1/2 adverse events (AEs) were most common. No cardiac events were observed and one patient experienced an asymptomatic decrease of left ventricular ejection fraction below the normal range (47 %). Both humoral and cellular immunogenicity to the dHER2 antigen was observed. No patient discontinued the immunizations because of AEs but 35/40 withdrew prematurely, 34 because of disease progression (24/34 before or at the tumor assessment after dose 6). One patient achieved a complete response lasting 11 months and one patient had a partial response lasting 3.5 months. Ten patients experienced stable disease ≥26 weeks with 4/10 still in stable disease at the last tumor assessment after 47 weeks. Immunization of MBC patients with the dHER2 immunotherapeutic was associated with minimal toxicity and no cardiac events. Clinical activity was observed with two objective responses and prolonged stable disease for 10/40 patients.

The studies reported herein demonstrate the efficacy of α-particle–targeted radiation therapy of peritoneal disease with Herceptin as the targeting vehicle. Using the CHX-A-DTPA linker, Herceptin was radiolabeled with indium-111 and bismuth-213 with high efficiency without compromising immunoreactivity. A pilot radioimmunotherapy study treating mice bearing 5-day LS-174T (i.p.) xenografts, a low but uniform HER2 expressing, human colon carcinoma, with a single dose of 213Bi-CHX-A”-Herceptin, proved disappointing. This defined the effect of tumor burden/size on tumor response to radioimmunotherapy with α-radiation. A more successful experiment with a lower tumor burden (3 days) in mice followed. A specific dose-response (P = 0.009) was observed, and although a maximum-tolerated dose was not determined, a dose of 500 to 750 μCi was selected as the operating dose for future experiments based on changes in animal weight. Median survival was increased from 20.5 days for the mock-treated mice to 43 and 59 days with 500 and 750 μCi, respectively. The therapeutic effectiveness of 213Bi-CHX-A”-Herceptin was also evaluated in a second animal model for peritoneal disease with a human pancreatic carcinoma (Shaw). The results of this study were not as dramatic as with the former model, and higher doses were required to obtain an increase in survival of the mice (P = 0.001).