Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

Stimmel, Julie B.; Merrill, Barbara M.; Kuyper, Lee F.; Moxham, Christopher M.; Hutchins, Jeff; Fling, Mary E.; Kull, Frederick C.

doi:10.1074/jbc.m001672200

Cited by 69 publications

(51 citation statements)

References 33 publications

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“…A simple approach pioneered by Lyons et al and later built upon by Kull and co-workers introduces a single solvent-accessible cysteine into an IgG4 scaffold, providing a site-specific handle for conjugation (18,19). After expression and reduction to remove cysteine or glutathione adducts, the mutant antibodies were functionalized using a bromoacetyllinked payload.…”

Section: Site-specific Conjugation Through Genetically Engineered Sitesmentioning

confidence: 99%

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

McCombs

Owen

2015

AAPS J

268

199

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Abstract. Antibody drug conjugates (ADCs) have emerged as an important pharmaceutical class of drugs designed to harness the specificity of antibodies with the potency of small molecule therapeutics. The three main components of ADCs are the antibody, the linker, and the payload; the majority of early work focused intensely on improving the functionality of these pieces. Recently, considerable attention has been focused on developing methods to control the site and number of linker/drug conjugated to the antibody, with the aim of producing more homogenous ADCs. In this article, we review popular conjugation methods and highlight recent approaches including "click" conjugation and enzymatic ligation. We discuss current linker technology, contrasting the characteristics of cleavable and noncleavable linkers, and summarize the essential properties of ADC payload, centering on chemotherapeutics. In addition, we report on the progress in characterizing to determine physicochemical properties and on advances in purifying to obtain homogenous products. Establishing a set of selection and analytical criteria will facilitate the translation of novel ADCs and ensure the production of effective biosimilars.

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Section: Site-specific Conjugation Through Genetically Engineered Sitesmentioning

confidence: 99%

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

McCombs

Owen

2015

AAPS J

268

199

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“…Stimmel et al used this approach and replaced a serine in the CH3 domain with a cysteine. Controlled reduction conditions enabled them to generate RICs by site-specifically attaching a chelating system exclusively to the two additional cysteines while all other cysteines in the antibody backbone remain unaffected [55]. A similar approach was exploited by Junutula and co-workers to develop homogeneous ADCs.…”

Section: Engineered Cysteinesmentioning

confidence: 97%

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

2015

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Monoclonal antibodies (mAbs) and their derivatives are currently the fastest growing class of therapeutics. Even if naked antibodies have proven their value as successful biopharmaceuticals, they suffer from some limitations. To overcome suboptimal therapeutic efficacy, immunoglobulins are conjugated with toxic payloads to form antibody drug conjugates (ADCs) and with chelating systems bearing therapeutic radioisotopes to form radioimmunoconjugates (RICs). Besides their therapeutic applications, antibody conjugates are also extensively used for many in vitro assays. A broad variety of methods to functionalize antibodies with various payloads are currently available. The decision as to which conjugation method to use strongly depends on the final purpose of the antibody conjugate. Classical conjugation via amino acid residues is still the most common method to produce antibody conjugates and is suitable for most in vitro applications. In recent years, however, it has become evident that antibody conjugates, which are generated via site-specific conjugation techniques, possess distinct advantages with regard to in vivo properties. Here, we give a comprehensive overview on existing and emerging strategies for the production of covalent and non-covalent antibody conjugates.

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“…The use of Fab' fragments yields a lower blood-pool activity and improves image contrast compared with intact MoAb and F(ab') 2 fragments. 67 Cu-labeled Fab' fragments may be more sensitive agents for detecting tumors, and cause the formation of fewer human antimouse antibodies (HAMA) [9,14,15] . Current techniques for preparing Fab' fragments necessitate purification steps that may cause irreversible damage to the antigen-binding sites due to denaturation of the antibody [16,17] .…”

Section: Discussionmentioning

confidence: 99%

“…This method of sitespecific conjugation has been widely used for conjugating antibodies with enzymes such as peroxidase and glucosidase with minimal polymerization and without impairing the activity of Fab' [5,6] . The labeling of antibodies with thiol-specific bifunctional chelating agents for metal ions has a number of potential new applications, including in vivo diagnosis and therapy and in vitro immunoassays [7][8][9] . In the past linking agents such as 2-iminothiolane(2-IT), which generates a linkage containing a disulfide bond and an amidinium bond, were always used as "bridges" to connect bifunctional chelating agents, such as 6-[p-(bromoacetamido) benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT) and antibodies containing no free thiol groups [10,11] .…”

Section: Introductionmentioning

confidence: 99%

Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab1 fragment

Wang

et al. 2006

Acta Pharmacologica Sinica

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Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG 1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method. First, stable F(ab') 2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab') 2 . Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab') 2 , Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

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Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

Cited by 69 publications

References 33 publications

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab1 fragment

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