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Burlingame, CA, United States

Jackson D.Y.,Igenica Biotherapeutics
Organic Process Research and Development | Year: 2016

Antibody drug conjugates (ADCs) are synthesized by conjugating a cytotoxic drug or "payload" to a monoclonal antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react with lysines or cysteines on the antibody surface. A typical antibody contains over 60 lysines and up to 12 cysteines as potential conjugation sites. The desired DAR (drugs/antibody ratio) depends on a number of different factors and ranges from two to eight drugs/antibody. The discrepancy between the number of potential conjugation sites and the desired DAR, combined with use of conventional conjugation methods that are not site-specific, results in heterogeneous ADCs that vary in both DAR and conjugation sites. Heterogeneous ADCs contain significant fractions with suboptimal DARs that are known to possess undesired pharmacological properties. As a result, new methods for synthesizing homogeneous ADCs have been developed in order to increase their potential as therapeutic agents. This article will review recently reported processes for preparing ADCs with improved homogeneity. The advantages and potential limitations of each process are discussed, with emphasis on efficiency, quality, and in vivo efficacy relative to similar heterogeneous ADCs. © 2016 American Chemical Society. Source

Liao-Chan S.,Igenica Biotherapeutics | Daine-Matsuoka B.,Igenica Biotherapeutics | Heald N.,Igenica Biotherapeutics | Wong T.,Igenica Biotherapeutics | And 5 more authors.
PLoS ONE | Year: 2015

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology. © 2015 Liao-Chan et al. Source

Behrens C.R.,Igenica Biotherapeutics | Ha E.H.,Igenica Biotherapeutics | Chinn L.L.,Igenica Biotherapeutics | Bowers S.,Igenica Biotherapeutics | And 18 more authors.
Molecular Pharmaceutics | Year: 2015

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs. © 2015 American Chemical Society. Source

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