Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody's binding to its target. Moreover, an emerging field for therapeutics is antibody-drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.
Conjugation of various
reagents to antibodies has long been an
elegant way to combine the superior binding features of the antibody
with other desired but non-natural functions. Applications range from
labels for detection in different analytical assays to the creation
of new drugs by conjugation to molecules which improves the pharmaceutical
effect. In many of these applications, it has been proven advantageous
to control both the site and the stoichiometry of the conjugation
to achieve a homogeneous product with predictable, and often also
improved, characteristics. For this purpose, many research groups
have, during the latest decade, reported novel methods and techniques,
based on small molecules, peptides, and proteins with inherent affinity
for the antibody, for site-specific conjugation of antibodies. This
review provides a comprehensive overview of these methods and their
applications and also describes a historical perspective of the field.
Albumin-binding fusion partners are
frequently used as a means
for the in vivo half-life extension of small therapeutic
molecules that would normally be cleared very rapidly from circulation.
However, in applications where small size is key, fusion to an additional
molecule can be disadvantageous. Albumin-derived affinity proteins
(ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding
domains of streptococcal protein G, with engineered binding specificities
against numerous targets. Here, we engineered this scaffold further
and showed that this domain, as small as 6 kDa, can harbor two distinct
binding surfaces and utilize them to interact with two targets simultaneously.
These novel ADAPTs were developed to possess affinity toward both
serum albumin as well as another clinically relevant target, thus
circumventing the need for an albumin-binding fusion partner. To accomplish
this, we designed a phage display library and used it to successfully
select for single-domain bispecific binders toward a panel of targets:
TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP),
renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin.
Apart from successfully identifying bispecific binders for all targets,
we also demonstrated the formation of the ternary complex consisting
of the ADAPT together with albumin and each of the five targets, TNFα,
PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of
albumin and other targets presents an opportunity to combine the advantages
of small molecules with those of larger ones allowing for lower cost
of goods and noninvasive administration routes while still maintaining
a sufficient in vivo half-life.
Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with 99mTc, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.
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