Bacterial production and functional characterization of the Fab fragment of the murine IgG1/  monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics

Abstract

Hsp70, the major stress-inducible member of the 70 kDa heat shock protein family, is frequently exposed on the plasma membrane of human tumours and, even more pronounced, on metastases but not detectable on normal tissues. The mouse monoclonal antibody cmHsp70.1, which recognizes a peptide epitope in the C-terminal substrate binding domain of both human and murine Hsp70, provides a promising reagent for the monitoring of Hsp70-positive tumours during cancer therapy. Here, we describe the variable domain sequences of the antibody produced by the hybridoma cell line and attempts to secrete the corresponding recombinant Fab fragment in Escherichia coli. Initially, the yield of soluble functional Fab fragment that could be purified from the periplasmic cell extract was extremely low, even when preparing different chimeric versions with constant domains of human or murine origin or with the light chain constant domain belonging to the kappa or lambda class. Surprisingly, this yield could be raised dramatically by more than a factor 100 in the presence of the folding helper plasmid pTUM4, which overexpresses two periplasmic disulphide oxido-reductases as well as two chaperones with proline-cis/trans-isomerase activity. Thus, more than 15 mg functional recombinant Fab fragment could be purified per litre E.coli culture from a bench top fermenter. This Fab fragment showed high and specific Hsp70 binding activity in ELISA and SPR measurements, revealing a dissociation constant of 35 nM. Notably, the Fab fragment sensitively recognizes the membrane-associated Hsp70 on tumour cell lines both in immunofluorescence microscopy and flow cytometry, thus showing potential for tumour detection in vitro and in vivo.

A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord

“…Using the pASK85 derivatives from above, recombinant Fab fragments were expressed either at the shake flask scale using E. coli strain JM83 [31] or in an 8 litre bench-top fermenter using the strain MC4100 [32] co-transformed with pTUM4 as described previously [33]. The recombinant protein was extracted from the periplasmic cell fraction and purified via IMAC (immobilized metal-ion-affinity chromatography) and SEC (sizeexclusion chromatography).…”

“…ELISA measurements were performed as described previously [33]. Briefly, a 96-well Maxisorp microtitre plate (Nunc) was coated with 10 μg/ml mL1-TEV (tobacco etch virus protease recognition sequence)-Fc (InVivo) in PBS (4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 and 115 mM NaCl).…”

Humanization of a Mouse Monoclonal Antibody Directed Against a Cell Surface-Exposed Epitope of Membrane-Associated Heat Shock Protein 70 (Hsp70)

“…They also observed low production levels in E. coli for recombinant chimeric Fab fragments including sequence-optimized derivatives, e.g., generated by correcting, as in our study, the N-terminal sequence of the cloned V H and V L domains to that obtained by Edman sequencing and by codon optimization of two rare codons (encoding V47 and V48) of the V L domain. Interestingly, in this study, yields were dramatically increased (more than 100-fold) by coexpression of two thiol-disulfide oxidoreductases (DsbA, DsbC) and two peptidyl-prolyl cis/trans-isomerases with chaperone activity (FkpA, SurA) indicating that disulfide bond formation and cis/trans-isomerization of prolyl-iminopeptide bonds are the rate-limiting steps [37]. The coexpression of these or similar chaperones might also have beneficial effects on the expression of scFv tumex, as already shown for various other scFv molecules [38,39].…”

“…Also, we did not observe a substantial improvement of expression in E. coli for variant 1. Recently, cloning of the cmHsp70.1 variable domain was also described by Friedrich et al [37]. They also observed low production levels in E. coli for recombinant chimeric Fab fragments including sequence-optimized derivatives, e.g., generated by correcting, as in our study, the N-terminal sequence of the cloned V H and V L domains to that obtained by Edman sequencing and by codon optimization of two rare codons (encoding V47 and V48) of the V L domain.…”

“…Further studies have to be performed to analyze the cellbinding activity of the chimeric and humanized antibodies in comparison to the parental antibody and to demonstrate antitumor activity. Friedrich et al [37] already demonstrated cell-binding activity of a chimeric Fab fragment derived from cmHsp70.1 containing the identical CDRs as used in our humanized version. Because mHsp70 has no proliferative activity, antitumor effects of the anti-mHsp70 antibody will be primarily exerted through ADCC and CDC and not by blocking of mHsp70 [22].…”