Academic and Professional Awards
are the foundation of many biological processes. As
we enter the post-Genomic era, the elucidation of
such interactions becomes central to biological research.
My research is focused on studying protein-protein
interactions by developing and applying novel protein
biotechnology tools. In particular, I am interested
in the mechanisms of antibody-antigen recognition
and have concentrated on the following research goals:
Display technologies are a central part of antibody engineering as antibody discovery-isolation-optimization tools (Benhar 2001, Benhar 2007). Display technology is based on generating a large repertoire of potential binders (an antibody library) which contains million to billions of individual clones. Selective pressure, usually in the form of affinity selection is than applied to pan out the clones with desired specificities (Figure 5).
With regard to antibody technology, we had recently introduced the concept of “antibody arrays for functional genomics”. We developed a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter (Figure 6). These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders (Figure 7). We screened our library against a number of cancer-related peptides, proteins, and peptide–protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range (Azriel-Rosenfeld et al., 2004). Our new antibody phage library, called: the “Ronit 1 library” became a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics. That potential was recently demonstrated when antibodies we isolated from the library were used to characterize a new HLA-A*0201-restrected CTL epitope of PAP, a putative tumor associated antigen relevant to prostate cancer. This epitope, designated as PAP-3 (ILLWQPIPV), induced CTLs capable to lyse in-vitro HLA-A*0201 and PAP-positive tumor cells. We isolated from the “Ronit 1 library” recombinant single-chain Fv antibodies against HLA-A*0201/PAP-3 complexes and confirmed by confocal microscopy the presentation of PAP-3 by tumor cells in the context of HLA-A*0201 molecules (Machlenkin et al., 2007) (Figure 8).
Further, we recently demonstrated the potential of our antibodies to be incorporated into antibody chips. In that study we described a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM, formally CBD) for its three-dimensional substrate, i.e., cellulose. Various microarray formats were described, e.g., conventional and single chain antibody and peptide microarray for serodiagnosis of HIV patients (Ofir et al., 2005) (Figure 9).
therapy encompasses a wide variety of different strategies,
which can be divided into direct or indirect approaches.
Direct approaches target tumor-associated antigens
by monoclonal antibodies (mAbs) binding to the relevant
antigens or by small molecule drugs that interfere
with these proteins. Indirect approaches rely on tumor-associated
antigens expressed on the cell surface with antibody–drug
conjugates or antibody-based fusion proteins containing
different kinds of effector molecules. To deliver
a lethal cargo into tumor cells, the targeting antibodies
should efficiently internalize into the cells. Similarly,
to qualify as targets for such drugs newly-discovered
cell-surface molecules should facilitate the internalization
of antibodies that bind to them.
drug-carrying phage nanomedicines
target was Staphylococcus aureus, and the model drug
was the antibiotic chloramphenicol. We demonstrated
the potential of using filamentous phages as universal
drug carriers for targetable cells involved in disease.
Our approach replaces the selectivity of the drug
itself with target selectivity borne by the targeting
moiety, which may allow the reintroduction of nonspecific
drugs that have thus far been excluded from antibacterial
use (because of toxicity or low selectivity). Reintroduction
of such drugs into the arsenal of useful tools may
help to combat emerging bacterial antibiotic resistance
A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. We have developed a novel assay for measurement of HCV NS3 serine protease activity for screening of potential NS3 serine protease inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry (Figure 19). Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors (Berdichevsky 2003).
Using antibody phage display we turned to isolate single-chain antibodies (scFvs) that, as intracellular antibodies will inhibit NS3 within cells. A few year ago we reported that in addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation (Zemel 2001). NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be of importance. With the aim of inhibiting NS3-mediated cell transformation we isolated and characterized eight anti-NS3 scFvs from a human synthetic scFv library. We investigated the phenotypic changes that NS3-expressing cells undergo upon intracellular expression of these antibodies in different subcellular compartments (intracellular immunization), assayed by their proliferation rate and their ability to grow anchorage independently. The intracellular location of NS3 and the scFvs were analyzed by immunofluorescent staining using confocal microscopy (Figure 20). We found that nuclear-targeted anti-NS3 intrabodies shuttled NS3 from the cytosol to the nucleus with concomitant inhibition of cell proliferation and loss of the transformed phenotype (Figure 21). We concluded that intracellular immunization-based gene therapy strategies may emerge as a promising antiviral approach to interfere with the life cycle and tumorigenicity of HCV (Zemel 2004).
we isolated in that study were inhibitors of NS3 serine
protease activity. To isolated scFvs that are true
inhibitors, we sorted to a different screening strategy.
Our scFvs were further explored as potential intervention towards the eradication of HCV infection using advanced HCV RNA replicons. The potential of the NS3-neutralizing scFvs to suppress HCV RNA replication was evaluated using SEAP secreting replicon-bearing Huh7 cells. SEAP secretion was suppressed in replicon cells transiently transfected with NS3-neutralizing but not in a control cell line nor by control scFvs (Figure 24). This indicates that the effect is due to specific suppression of the HCV RNA replication by the NS3-neutralizing scFvs. These inhibitors suppress the replication of drug resistant mutants replicons as well, emphasizing their advantage over small molecule inhibitors. These Single-chain antibodies may emerge as useful clinical reagents as more specific and boadly administrated for the treatment of infectious diseases and cancer.
In addition to antibodies we turned to search for peptide aptamers as NS3 inhibitors. Peptide aptamers are short (in our case 8 amino-acid long) peptides that are stabilized by their presentation on a stable protein scaffold. We had initiated a study where a novel high-throughput in vivo genetic screen for NS3 catalysis and its inhibition was applied for inhibitors isolation. Here the screen was based on the concerted co-expression of a the reporter gene, recombinant NS3 and stabilized candidate molecules (MBP-scFvs and peptide aptamers) in E. coli. The peptide-aptamers were isolated from libraries where random sequences were inserted at the C-terminus of the E. coli MBP as linear peptides. Here too, the initial identification of inhibitory peptide aptamers was based on the their expression in bacteria that express the enzyme-substrate combination as well, by the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active) on X-gal indicative plates. The peptide-aptamer inhibitors were validated as NS3 binders and as in vitro inhibitors of catalysis as well. We are currently evaluating the aptamers using the RNA replicons described above.
A. ARTICLES IN REFEREED JOURNALS
6. Benhar, I., Wang, Q-C., FitzGerald, D. and Pastan, I. (1994). Pseudomonas Exotoxin A Mutants: replacement of surface-exposed residues in domain III with cysteine residues that can be modified with Polyethylene glycol in a site specific manner. J. Biol. Chem. 269: 13398-13404.
7. Benhar, I., Brinkmann, U., Webber, K.O. and Pastan, I. (1994). Mutations in the CDR loops of a recombinant immunotoxin that reduce its sensitivity to chemical derivatization. Bioconjug. Chem. 5: 321-326.
8. Roscoe, D.M., Jung, S-h., Benhar, I., Pai, L., Lee, B.K. and Pastan, I. (1994). Primate antibody response to immunotoxin: serological and computer-aided analysis of epitopes on a truncated form of Pseudomonas exotoxin. Infect. Immun. 62: 5055-5065.
9. Benhar, I., Padlan, E.D., Jung, S-H., Lee, B. and Pastan, I. (1994). Rapid humanization of the Fv of monoclonal antibody B3 by using framework exchange of the recombinant immunotoxin B3(Fv)-PE38. Proc. Natl. Acad. Sci. (USA) 91: 12051-12055.
10. Benhar, I. and Pastan, I. (1994). Cloning, expression and characterization of the Fv fragment of the anticarbohydrate monoclonal antibodies B1 and B5 as single-chain immunotoxins. Protein Eng. 7:
11. Benhar, I. and Pastan, I. (1995). Characterization of B1(Fv)PE38 and B1(dsFv)PE38: single-chain and disulfide stabilized Fv immunotoxins with increased activity that cause complete remissions of established human carcinoma xenografts in nude mice. Clin. Cancer Res. 1: 1023-1029.
12. Benhar, I., Reiter, Y., Pai, L.H. and Pastan, I. (1995). Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenogragfts in nude mice. Int. J. Cancer 62: 351-355.
15. Li. M., Dyda, F., Benhar, I., Pastan, I. and Davies, D.R. (1996). Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. Proc. Natl. Acad. Sci. (USA) 93: 6902-6906.
16. Scherf, U., Benhar, I., Webber, K.O.W., Pastan, I. and Brinkmann, U. (1996) Cytotoxic and Antitumor activity of a Recombinant Tumor Necrosis Factor-B1(Fv) Fusion Protein on LeY-antigen expressing Human Cancer Cells. Int. J. Cancer 2: 1523-1531.
17. Almog, O., Benhar, I., Vasmatzis, G., Tordova, M., Lee, B., Pastan, I. and Gilliland, G.L. (1998) Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: conformational influence of an engineered disulfide bond. Proteins 31: 128-138.
18. Berdichevsky, Y., Ben-Zeev, E., Lamed, R. and Benhar, I. (1999). Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering. J. Immunol. Methods 228: 151-162.
19. Berdichevsky, Y., Lamed, R., Frenkel, D., Gophna, U., Bayer, E., Yaron, S., Shoham, Y. and Benhar, I. (1999) Matrix-assisted refolding of single-chain Fv-cellulose binding domain fusion proteins. Protein Express. Purif. 17: 249-259.
21. Benhar, I., Nahary, L., Shaky, S., Azriel, R., Berdichevsky, Y., Tamarkin, A. and Wels, W. (2000) Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria. J. Mol. Biol. 301: 893-904.
22. Benhar,I. (2001) Biotechnological Applications of Phage and Cell Display. Biotechnology Advances. 19: 1-33.
23. Zemel, R.,Gerechet, S., Greif, H., Bachmatove, L., Birk, Y., Golan-Goldehirsh, A., Berdichevsky, Y., Benhar, I., and Tur-Kaspa, R. (2201). Cell transformation induced by Hepatitis C virus NS3 serine-protease. J. Viral. Hepatitis, 8:96-102.
24. Bach, H., Mazor, Y., Shaky, S., Shoham-Lev, A., Berdichevsky, Y., Gutnik, D.L. and Benhar, I. (2001). E. coli maltose-binding protein as a molecular chaperone for intracellular antibodies. J. Mol. Biol. 312:79-93.
25. Benhar, I., Eshkenazi, I., Neufeld, J. Opatowsky, J., Shaky, S. and Rishpon, J. (2001). Phage displaying a recombinant single-chain antibody in electrochemical detection of the pathogenic bacterium Listeria monocytogenes. Talanta. 55: 899-907.
26. Mazor, Y., Gilad, S., Benhar, I. And Gazit, E. (2002). Identification and chracacterization of a novel molecular recognition and self-assembly domain within the islet amyloid polypeptide. J. Mol. Biol. 322: 1013-1024.
27. Berdichevsky, Y., Zemel, R., Bachmatov, L., Abromovich, A., Koren, R., Golan-Goldhirsh, A., Tur-Kaspa, R. and Benhar, I. (2003). A novel high throughput screening assay for HCV NS3 serine protease inhibitors. J. Virol. Methods 107: 245-255.
28. Denkberg, G., Lev, A., Eisenbach, L., Benhar, I. and Reiter, Y. (2003) Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen. J. Immunol. 171: 2197-2207.
29. Azriel-Rosenfeld, R., Valensi, M. and Benhar, I. (2004) A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. J. Mol. Biol. 335(1):177-192.
30. Haus-Cohen, M., Assaraf, Y., Binyamin, L., Benhar, I. and Reiter, Y. (2004) Disruption of p-glycoprotein anticancer drug efflux activityby a small recombinant single-chain fv antibody fragment targeted to an extracellular epitope. Int. J. Cancer 109: 750-758.
31. Zemel, R., Berdichevsky, Y., Bachmatov, L., Benhar, I. and Tur-Kaspa, R. (2004) Inhibition of Hepatitis C Virus NS3-mediated cell transformation by recombinant intracellular antibodies. J. Hepatol. 40: 1000-1007.
32. Zilberman-Peled, B., Benhar, I., Coon, S. L., Ron, B., Gothilf, Y. (2004) Duality of serotonin-N-acetyltransferase in the gilthead seabream (Sparus aurata): molecular cloning and characterization of recombinant enzymes. Gen. Comp. Endocrinol. 138: 139-147.
34. Gal-Tanamy, M., Zemel, R., Berdichevsky, Y., Bachmatov, L., Tur-Kaspa, R. and Benhar, I. (2004). HCV NS3 Serine Protease-Neutralizing Single-Chain Antibodies Isolated By a Novel Genetic Screen. J. Mol. Biol. 347: 991–1003.
35. Ofir, K., Berdichevsky, Y. Benhar, I., Azriel-Rosenfeld, R., Lamed, R., Barak, Y., Bayer, E. A. and Morag, E. (2005) Versatile protein microarray based on carbohydrate-binding molecules. Proteomics 5:1806-1814.
38. Artzy Schnirman, A., Zahavi, E., Yeger, H.,, Rosenfeld, R., Benhar, I., Reiter1, Y. and Sivan, U. (2006) Antibody Molecules Discriminate Between Crystalline Facets of Gallium Arsenide semiconductor. Nano Lett. 6: 1870-1874.
39. Rubinstein, D.B., Karmely, M., Ziv, R., Benhar, I., Leitner, O., Baron, S., Katz, B.Z., Wreschner, D.H. (2006) MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells. Cancer Res. 66:11247-11253.
40. Machlenkin, A., Azriel-Rosenfeld, R., Volovitz, I., Vadai, E., Lev, A., Paz, A., Goldberger, O., Reiter, Y., Tzehoval, E., Benhar, I. and Eisenbach, L. (2007) Active immunization with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice. Cancer Immunol. Immunother. 56:217-226.
Y., Noy, R., Wels, W.S. and Benhar, I. (2007) chFRP5-ZZ-PE38,
a large IgG-toxin immunoconjugate outperforms the
corresponding smaller FRP5(Fv)-ETA immunotoxin in
eradicating ErbB2-expressing tumor xenografts. Cancer
B. BOOK CHAPTERS
1. Benhar I., Miller, C., and Engelberg-Kulka, H. (1990). Frameshifting in the expression of the trpR gene of Escherichia coli. In: McCarthy, J.E.G., and Tuite, M.F. (eds): Post Transcriptional Control of Gene Expression. Springer Verlag, Berlin, pp. 591-602.
2. Benhar I. and Pastan, I. (1997). Tumor Targeting by Antibody-Drug Conjugates. In: Harris, W.J. and Adair, J.R. (eds): Antibody Therpeutics. CRC Press, Boca Raton, pp. 73-85.
3. Benhar, I., Tamarkin, A., Marash, L., Berdichevsky, Y., Yaron, S., Shoham, Y., Lamed, R. and Bayer, E. A. (2001). Phage display of cellulose binding domains for biotechnological application. In Glycosyl Hydrolases for Biomass Conversion. ACS Symposium Series 769 (M. E. Himmel, J. O. Baker and J. N. Saddler, ed.), pp. 168-189. American Chemical Society, Washington, DC.
4. Benhhar, I. and Reiter Y. (2001). Phage display of single-chain antibodies (scFvs). Current Protocols in Immunology. Chapter 10.19B. John Colligan (Ed). John Wiley & Sons, Inc, USA.
5. Benhar, I. and Berdichevsky Y. (2002). Large Scale Production of Recombinant Antibodies By Utilizing Cellulose Binding Domains. In: Welschof, M. and Krauss, J. (eds): Methods in Molecular Biology Vol. 207; Recombinant Antibody Technology for Cancer Therapy. Humana Press Series. pp. 443-454.
L. and Benhar, I. (2009) Design of Human Synthetic
Combinatorial Library of Single-chain Antibodies.
In: Therapeutic Antibodies, Methods In Molecular Biology.
Humana Press 525:61-80.
1. Pastan, I., Benhar, I., Padlan, E. A., Jung, S-H. and Lee, B. (1999) Humanized B3 antibody fragments, fusion proteins, and uses thereof. US Patent Number 5889157. Issue date 30/3/1999.
2. Pastan, I. And Benhar, I. (1999) Chimeric and mutationally stabilized tumor-specific B1, B3 and B5 antibody fragments; immunotoxic fusion proteins; and uses thereof. US Patent Number 5981726. Issue Date 9/11/1999.