In vitro Evolution of Antibody Affinity via Insertional Mutagenesis Scanning of an Entire Antibody Variable Region

We report the first systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD was used to generate large libraries with random in-frame InDels across the entire scFv gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (ISM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach and suggests that the results of this InDel approach and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.SignificanceInsertion/deletion (InDel) mutations play key roles in genome and protein evolution. Despite their prominence in evolutionary history, the potential of InDels for changing function in protein engineering by directed evolution remains unexplored. Instead point mutagenesis is widely used. Here we create antibody libraries containing InDels and demonstrate that affinity maturation can be achieved in this way, establishing an alternative to the point mutation strategies employed in all previous in vitro selections. These InDels mirror the observation of considerable length variation in loops of natural antibodies originating from the same germline genes and be combined with point mutations, making both natural sources of functional innovation available for artificial evolution in the test tube.

Construction Hybrid immunoglobulin All Four Dengue serotype Using Mesenchymal Stem

The dengue virus is a member of vector-borne diseases that causes zoonotic disease and spreads rapidly in the world. No single treatment or vaccine yet is available that is recommended and there is no correlation with protectiveness against this disease. The heavy chain (VH) and light chain (VL) variables are molecules of immunoglobulin G (IgG) is the smallest part of the antibody. Although the part-time domain variable is short, it can be used as a long-term and rapid immune booster in the immune system. In this study we tried to clone an encoding gene that was able to influence the adaptive immune response to dengue 1-4 by using MSC as a gene carrier. The target scFv-IgG gene has been successfully integrated into the plasmid. Plasmids that we have linearly transfected into the MSC. From the cDNA synthesis results continued with PCR synthesis with primer FGHV and RGHA obtained bands in accordance with the target of 404 bp. The scFv gene encoding IgG can be integrated with MSC Keywords: immunetherapy; dengue; hybrid; scFv-IgG; mesenchymal

Construction and targeting of a 5F11-scFv-methioninase fusion protein for antibody-directed enzyme pro-drug therapy (ADEPT) against neuroblastoma

3043 Background: Recombinant methioninase (METase) depletes methionine and inhibits tumor growth in preclinical models. 5F11 single-chain variable fragment (scFv) can target fusion proteins (e.g. streptavidin (SA), J. Nucl. Med., 45:867, 2004) preferentially to GD2- positive human neuroblastoma (NB). METase targeted to tumors may release methylselenol from the prodrug selenomethionine for tumor therapy. Methods: 5F11-scFv gene fused upstream of METase in pKK-223–3 to form 5F11-scFv-METase. Fusion protein produced in E. coli and purified by chromatography and endotoxin removal retained enzyme activity. Binding of 5F11-scFv-METase to tumor cells was assayed by indirect immunofluorescence and binding kinetics to GD2 analyzed by surface plasmon resonance. Cytotoxicity against human NB cell lines LAN-1, NMB-7 and SK-N-ER was tested using proliferation inhibition and apoptosis assays. Targeting to NB xenografts was studied by biodistribution using 125I-anti-idiotype 1G8 as the secondary tracking antibody. Results: 5F11-scFv-METase was purified to 84% and 94.1% homogeneity by SDS-PAGE and HPLC, respectively. Enzyme activity was 3.6 units/mg protein. Its avidity (KD=1.72×10-7M) as a dimer compared favorably with the monomeric 5F11-scFv (KD=1.07×10- 7M) and tetrameric 5F11-scFv-SA (KD=2.62×10-9M). Immunofluorescent staining of 5F11-scFv-METase was comparable to 5F11-scFv-SA for LAN-1 and NMB-7. In the presence of 20 μM selenomethionine, IC50 of 5F11-scFv-METase was 0.03, 0.02 and 0.02 units/ml for LAN-1, NMB-7 and SK-N-ER, respectively. When treated with 0.3 units/ml of 5F11-scFv-METase and 20 μM selenomethionine, apoptosis was induced in SK-N-ER by 8 hours, peaking at 24 hours. In biodistribution studies, tumor uptake in LAN-1 xenograft averaged 4.07±0.6% injected dose per gram at 48 hours. Conclusions: 5F11-scFv-METase fusion protein retains enzyme activity and immunoreactivity. It targets to tumors in vivo and activates the pro-drug selenomethionine to effect tumor cytotoxicity. Its potential for tumor-selective methionine depletion as well as in ADEPT applications deserves further studies. No significant financial relationships to disclose.

Antiidiotypic DNA vaccination induces serum bactericidal activity and protection against group B meningococci

No vaccine is available for preventing infections by serogroup B Neisseria meningitidis (MenB), which accounts for a major portion of meningococcal cases in developed countries, because of the poor immunogenicity of the capsular polysaccharide (CP) even after protein conjugation. We have previously induced anticapsular antibodies by immunization with a single chain variable fragment (scFv), which mimics a protective CP epitope. This surrogate antigen, however, was ineffective at inducing serum bactericidal activity, an accepted marker of protection in humans. Serum bactericidal activity was consistently achieved by immunizing mice with the scFv-encoding gene. Immunization with vectors without a secretory signal sequence before the scFv resulted in markedly higher bactericidal activity relative to those with such a sequence. The induced antibodies were capsule specific, as shown by complete inhibition of bactericidal activity by purified MenB CP and by resistance to killing of MenA or MenC. Moreover, these antibodies were predominantly of the IgG2a isotype, reflecting a T helper type 1 response. Administration of sera from scFv gene–vaccinated animals protected infant rats against MenB bacteremia. These data illustrate the potential of vaccination with genes encoding capsular mimics in providing protection against MenB and other encapsulated bacteria.

Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

Surface Display of Recombinant Proteins on Bacillus thuringiensis Spores

ABSTRACT The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically.