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Analytical Biochemistry, November 29, 2009, [Epub ahead of print]; Volume 399, Issue 1, Pages 144-146, April 1, 2010.

Antagen Pharmaceuticals, Inc. et al.
2A Peptide sequences are now being widely used to construct multicistronic expression vectors. It is suggested that when only the first 2A-linked protein bears a signal sequence, the signal-less protein(s) downstream of 2A can also be translocated into the mammalian endoplasmic reticulum system through a “slipstreaming” mechanism. By using flow cytometry and cell surface CD90 as a localization indicator, we show here that slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires signal sequence for secretary or membrane-anchored expression.

Journal of Biosciences, Volume 38, Issue 2, Pages 245-9, 2013.

Antagen Pharmaceuticals, Inc. et al.

Antagen Pharmaceuticals, Inc. and Harvard Medical School, et al.

Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression.

Antibody Therapeutics, Volume 3, Issue 3, July 2020, Pages 157–162

Antagen Pharmaceuticals, Inc. and Boston University School of Medicine

Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Statement of Significance

Unlike sIgA, translocation of intravenously administrated therapeutic IgG onto mucosal surface is limited and, if any, is mediated by FcRn. Among all the IgG-IgA hybrids, the IgG(tp)J form contains sufficient structural requirements for full binding to pIgR, mimicking sIgA function in mucosa transcytosis against infectious agents and their products.

Antibody Therapeutics, tbab016, Published on August 19, 2021

Antagen Pharmaceuticals, Inc. and Boston University School of Medicine

Expressing afucosylated human IgG1 antibodies with Chinese hamster ovary (CHO) cells deficient of α-(1,6)-fucosyltransferase (FUT8) is being more and more accepted as a routine method to enhance antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies, especially for anti-cancer regimens. However, in pre-clinical studies relying on disease models other than mice and primates, e.g., those underrepresented species for infectious diseases, it is less clear whether such afucosylated antibodies can demonstrate enhanced therapeutic index. This is because the orthologues of human FcγRIIIA or mouse FcγRIV from those species have not been well characterized. We set up a luciferase-based ADCC assay with Jurkat reporter cells expressing FcγRIIIA/FcγRIV from human, mouse, rat, hamster, guinea pig, ferret, rabbit, cat, dog, pig and monkey, and also produced human, mouse, hamster, rabbit and pig IgG from wild type and Fut8−/− CHO cells or hybridomas. We confirmed that enhanced stimulation through FcγRIIIA/FcγRIV by afucosylated IgG, as compared with wild type IgG, is a cross-species phenomenon. Thus, efficacy and toxicology studies of the next generation afucosylated therapeutic IgG and Fc fusion proteins in these underrepresented animal models should be expected to generate translatable data for treating human diseases, leading to the expanded applications of this new class of glycoengineered biologics.

Statement of Significance

Orthologues of hFcγRIIIA from mouse, rat, hamster, guinea pig, ferret, rabbit, cat, dog, pig and monkey exhibit higher sensitivities towards afucosylated IgG for enhanced ADCC, facilitating efficacy, toxicity, and MOA studies of the next generation afucosylated therapeutic IgG and Fc fusion proteins directly in underrepresented animal models of human diseases.

Our Finding Confirmed

Our finding is confirmed by a recent study, showing afucosylated human IgG1 exerts increased binding towards murine FcγRIV, the mouse orthologue of human FcγRIIIA, with enhanced tumor killing in mouse models. Furthermore, what we showed in our study that afucosylated mouse IgG2 demonstrating higher ADCC effector functions across multiple animal species is a direct advocate for more efficient antibody discovery via tapping into the vast resources of mouse hybridoma lines in combination with our Fut8-/- expression system.

Frontier in Immunology, October 28, 2022

Antagen Pharmaceuticals, Inc. and Harvard Medical School

Despite the initially reported high efficacy of vaccines directed against ancestral SARS-CoV-2, repeated infections in both unvaccinated and vaccinated populations remain a major global health challenge. Because of mutation-mediated immune escape by variants-of-concern (VOC), approved neutralizing antibodies (neutAbs) effective against the original strains have been rendered non-protective. Identification and characterization of mutation-independent pan-neutralizing antibody responses are therefore essential for controlling the pandemic. Here, we characterize and discuss the origins of SARS-CoV-2 neutAbs, arising from either natural infection or following vaccination. In our study, neutAbs in COVID-19 patients were detected using the combination of two lateral flow immunoassay (LFIA) tests, corroborated by plaque reduction neutralization testing (PRNT). A point-of-care neutAb LFIA, NeutraXpress™, was validated using serum samples from historical pre-COVID-19 negative controls, patients infected with other respiratory pathogens, and PCR-confirmed COVID-19 patients. Surprisingly, potent neutAb activity was mainly noted in patients generating both IgM and IgG against the Spike receptor-binding domain (RBD), in contrast to samples possessing anti-RBD IgG alone. We propose that low-affinity, high-avidity, germline-encoded natural IgM and subsequent generation of class-switched IgG may have an underappreciated role in cross-protection, potentially offsetting immune escape by SARS-CoV-2 variants. We suggest Reverse Vaccinology 3.0 to further exploit this innate-like defense mechanism. Our proposition has potential implications for immunogen design, and provides strategies to elicit pan-neutAbs from natural B1-like cells. Refinements in future immunization protocols might further boost long-term cross-protection, even at the mucosal level, against clinical manifestations of COVID-19.

Significance of the Study:

Antagen scientists discovered that potent neutralizing antibodies against infectious agents, including SARS-CoV-2, influenza and malaria often utilize germline-encoded antibody sequences, which do not have many somatic hypermutations and can be isolated in the general public even before encountering the pathogen. Moreover, germline-encoded sequences are the precursor pan-neutralizing antibodies that can recognize future variants. Just as the Chinese proverb, “Coping with the shifting events with underpinning principles”, our immune system has its own wisdom dealing with pathogens. And that is what the current pharmaceutical industry should really learn from.

Antibody Therapeutics, March 2, 2023

Antagen Pharmaceuticals, Inc., Florida International University, Shandong First Medical University and Shandong Academy of Medical Sciences

Aberrant post-translational glycosylation is a well-established hallmark of cancer. Altered core fucosylation mediated by α-(1,6)-fucosyltransferase (Fut8) is one of the key changes in tumor glycan patterns that contributes to neoplastic transformation, tumor metastasis, and immune evasion. Increased Fut8 expression and activity are associated with many types of human cancers, including lung, breast, melanoma, liver, colorectal, ovarian, prostate, thyroid, and pancreatic cancer. In animal models, inhibition of Fut8 activity by gene knockout, RNA interference, and small analogue inhibitors led to reduced tumor growth/metastasis, downregulation of immune checkpoint molecules PD-1, PD-L1/2, and B7-H3, and reversal of the suppressive state of tumor microenvironment. Although the biologics field has long benefited tremendously from using FUT8^−/− Chinese hamster ovary cells to manufacture IgGs with greatly enhanced effector function of antibody-dependent cellular cytotoxicity for therapy, it is only in recent years that the roles of Fut8 itself in cancer biology have been studied. Here, we summarize the pro-oncogenic mechanisms involved in cancer development that are regulated by Fut8-mediated core fucosylation, and call for more research in this area where modifying the activity of this sole enzyme responsible for core fucosylation could potentially bring rewarding surprises in fighting cancer, infections, and other immune-related diseases.