Production of Soluble ScFvs with C-Terminal-Free Thiol for Site-Specific Conjugation or Stable Dimeric ScFvs on Demand

Abstract

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.

Dynamic force spectroscopy of parallel individual Mucin1–antibody bonds

“…The scFvs for this experiment were produced in the Escherichia coli HB2151 strain. scFv clones selected from an anti-MUC1 phage display library (21) were expressed with an additional cysteine tag at the C terminus, which does not interfere with the binding domain (14).…”

“…This interaction is the main targeting mechanism for a family of experimental radioimmunotherapeutics for cancer treatment (14). These agents consist of several Ab fragments on a poly(ethylene glycol) (PEG) scaffold that preloads onto the cancer cells and then catches a subsequently administered radioactive Y 90 payload (Fig.…”

Enhancement of the Therapeutic Index: From Nonmyeloablative and Myeloablative toward Pretargeted Radioimmunotherapy for Metastatic Prostate Cancer

“…Immunohistochemistry was done as previously described (42), with the following mAb and scFv-c concentrations: BrE3 at 5 Ag/mL; B27.29 at 0.5 Ag/mL; HMFG1 were tissue culture supernatants diluted to 1:10; and scFv-c at 50 Ag/mL. Scores were assigned based on three criteria: percent of cancer cells stained, apical and/or cytoplasmic staining, and intensity of staining.…”

Antibody constructs in cancer therapy

“…The primary building blocks of Ab-based molecules are Fab fragments (55 kDa) and single-chain Fv (scFV; 25 kDa), 6,7 both of which can be used alone or as units of larger protein constructs. Fab fragments, obtained by proteolytic digestion of IgG, include a single Ab light chain linked by a disulfide bond to a heavy chain fragment consisting of the variable region and the first heavy chain constant region, and form a single Ag binding site from the noncovalent association of heavy chain and light chain variable regions.…”

“…scFv molecules can also be engineered to incorporate a carboxy terminal cysteine residue, thereby enabling formation of a (scFv) 2 fragment by virtue of disulfide bridging. 6,7 Another bivalent molecule is a ''diabody,'' which is formed from 2 scFvs linked noncovalently. Each of 2 Ag-binding sites is formed from the heavy chain variable region of 1 chain and the light chain variable region of the other, with extensive noncovalent interaction.…”