1983	B.Sc. (Agriculture) Hebrew University, Rehovot, Israel
1992	Ph.D. Summa Cum Laude (Microbiology) Hebrew University, Jerusalem, Israel
1992-95	Post-Doctoral Associate, Laboratory of molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda MD, USA

Academic Positions

1995-2001	Lecturer, Dept. Molecular Microbiology and Biotechnology, Tel-Aviv University
2001-2005	Senior Lecturer, Dept. Molecular Microbiology and Biotechnology, Tel-Aviv University
2005-2008	Associate Professor, Dept. Molecular Microbiology and Biotechnology, Tel-Aviv University
2002-ongoing	Head of the B.Sc. program in Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University
2007-2011	Chairman, Department of Molecular Microbiology and Biotechnology, Tel Aviv University
2008-ongoing	Professor, Dept. Molecular Microbiology and Biotechnology, Tel-Aviv University

Academic and Professional Awards

1987-89	Ph.D. Studentship Scholarship. Levi Eshkol Foundation at the Israeli Research Council, The Ministry of Science and Technology, The Government of Israel
1992	The Prize for Excellence in Ph.D. Studies. The Sepharadic Federation and The World Zionist Agency, Jerusalem, Israel
1992	The Hebrew University Faculty of Medicine Award for Excellence in Ph.D. Studies - awarded for completion of Ph.D. Thesis Summa Cum Laude
1994	Federal Technology Transfer Award for an outstanding Scientific contribution of value to the USA. Awarded by the National Cancer Institute, National Institutes of Health., Bethesda MD
1995	The "Alon" fellowship for outstanding young researchers. Awarded by the Israeli Ministry of Education

Protein-protein interactions 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:

a) Elucidation of antibody-antigen interactions at the mechanistic and atomic levels.
b) Manipulation of antibody-folding, towards enhancement of stability and solubility.
c) Application of antibodies and peptide-aptamers as modifiers of biological processes, such as enzymatic activities.
d) Using recombinant antibodies as targeting moieties of drug-delivery platforms such as immuntoxins and targeted drug-carrying phage nanoparticles

Antibody Engineering

With over 10 billion dollars in annual sales (2007 figures), antibodies and antibody-related products currently dominate the market of protein pharmaceutics. As a result, antibody engineering (a 50 billion dollar industry, 2007 figures) is a leading biotechnological field (Figure 1). Antibody engineering is based on thorugh understanding of antibodies structure-function and the ability to manipulate those at will. The Fv or Fab parts (Figure 2) are manipulated for modifying binding affinity and specificity, while the Fc part is manipulated with the porpose of manipulating effector-functions of the antibody. In addition, antibody engineering, which was born with monoclonal antibodies in 1975, is currently applied to make (therapeutic) antibodies more human in nature (Figure 3). Antibody engineering is also about making various forms of recombinant antibody fragments, each with its unique properties and applicatios (Figure 4).

Figure 1

Figure 2

Figure 3

Figure 4

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).

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).

Figure 6

Figure 7

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).

Figure 9

Targeted immunotherapy

We applied recombinant antibody technology to clone and humanize the monoclonal antibody H23. The tumor-associated antigen MUC1 is a cell surface mucin that is expressed on the apical surface of most glandular epithelial cells, including the ducts of the breast, ovary, pancrease, lung and colon. During malignancy, epithelial tissues regularly display elevated levels of MUC1 in a non-polar fashion and in an underglycosylated form, exposing cryptic peptide and carbohydrate epitopes. As such, MUC1 is regarded a potential target for immunotherapeutical intervention. Murine monoclonal H23 antibody specifically recognizes a MUC1 epitope on the surface of human breast cancer cells. We described the cloning of the variable domains of H23 and their expression in (Escherichia coli) E. coli as maltose-binding protein-scFv (MBP-scFv) fusions. We humanized H23 and evaluated the binding properties of the murine and the humanized recombinant forms, which were similar in affinity and specificity, but lower in apparent affinity in comparison to the original monoclonal IgG. We mapped the epitope of humanized H23 by affinity-selecting a phage-displayed random peptide library on humanized H23 scFv-displaying bacteria. Our results show that humanized H23 binds an epitope corresponding to the MUC1 tandem repeat and an additional epitope not related to MUC1. These epitopes are competitive, bound with similar affinities and are recognized by the original murine H23 monoclonal antibody as well (Mazor et al., 2005) (Figure 10). In more recent work, we generated an expression system for whole IgG antibodies in transfected mammalian cells (Figure 11) and used it to prepare IgG mouse/human chimeric derivative that are being evaluated for their potential to deliver a cytotoxic payload to cancer cells.

Figure 10

Figure 11

Targeted 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.
Internalization can be studied be several biochemical and microscopy approaches. An undisputed proof of internalization can be provided by the ability of an antibody to specifically deliver a drug into the target cells and kill it. Recently we presented a novel IgG binding toxin fusion, ZZ-PE38, in which the Fc-binding ZZ domain, derived from Streptococcal protein A, is linked to a truncated Pseudomonas exotoxin A (Figure 12). The preparation of complexes between ZZ-PE38 and IgGs that bind tumor cells and the specific cytotoxicity of such immunocomplexes was also reported. Our results suggest that ZZ-PE38 could prove to be an invaluable tool for the evaluation of the suitability potential of antibodies and their cognate cell-surface antigens to be targeted by immunotherapeutics based on armed antibodies that require internalization (Mazor 2007). The data from an anti-tumor experiment in a nude mouse tumor xenograft model (Figure 13) suggested that such molecules have an impressing anti tumor potential that should be further investigated.

Figure 12

Figuer 13

Targeted drug-carrying phage nanomedicines

While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremely potent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapy could provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targeted antibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmed drug release mechanism.
Recently, we presented a novel application of filamentous bacteriophages (phages) as targeted drug carriers for the eradication of (pathogenic) bacteria. The phages are genetically modified to display a targeting moiety on their surface and are used to deliver a large payload of a cytotoxic drug to the target bacteria. The drug is linked to the phages by means of chemical conjugation through a labile linker subject to controlled release (Figure 14). In the conjugated state, the drug is in fact a prodrug devoid of cytotoxic activity and is activated following its dissociation from the phage at the target site in a temporally and spatially controlled manner.

Figure 14

Our model 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 (Yacoby 2006).
To overcome problems of limited solubility of the phages when loaded with a hydrophobic drug, such as chloramphenicol, we presented a novel drug conjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibiotics that serve as solubility-enhancing branched linkers (Figure 15). This new formulation allowed a significantly larger drug carrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carrying nanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, we present antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphenicol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward the pathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli (Figure 16) with an improvement in potency by a factor of 20,000 compared to the free drug (Yacoby 2007).

Figure 15

Figure 16

Hepatitis C virus

We are investing a lot of effort in studies of potential inhibitors of the Hepatitis C Virus (HCV) NS3 serine protease. HCV infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins (Figure 17). The viral non-structural proteins are cleaved by the HCV NS3 serine protease (Figure 18). NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance.

Figure 17

Figure 18

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).

Figure 19

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).

Figure 20

Figure 21

The antibodies 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.
We developed a novel genetic screen for inhibitors of NS3 catalysis applied it for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression
of a reporter gene, of recombinant NS3 and of stabilized scFvs in E. coli. The reporter system was constructed by inserting a peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase, with NS3 expressed from the same plasmid. The resultant beta-galactosidase enzyme was active, conferring a Lac+ phenotype that is lost upon induction of NS3 expression (Figure 22). The identification of inhibitors was demonstrated by isolating NS3 inhibiting single-chain antibodies, expressed from a compatible plasmid, was based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active) on X-gal indicative plates. Our source of inhibitory scFvs was a library prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as specific NS3 binders and as bone fide NS3 serine protease inhibitors in vitro as well (Gal-Tanamy 2005) (Figure 23).

Figure 22

Figure 23

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.

Figure 24

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.

1. Benhar, I. and Engelberg-Kulka, H. (1991). A procedure for amino acid sequencing in internal regions of proteins. Gene 103: 79-92.

2. Benhar, I., Miller, C. and Engelberg-Kulka, H. (1992). Frameshifting in the expression of the Escherichia coli trpR Gene. Mol. Microbiol. 6: 2777-2784.

3. Benhar, I. and Engelberg-Kulka, H. (1993). Frameshifting in the expression of the Escherichia coli trpR gene by the bypassing of a segment of its coding sequence. Cell 72: 121-130.

4. Benhar. I., Miller, C. and Engelberg-Kulka, H. (1993). Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation. J. Bacteriol. 175: 3204-3207.

5. Engelberg-Kulka, H., Benhar, I. and Schoulaker-Schwarz, R. (1993). Translational Introns: an additional regulatory element in gene expression. Trends in Biol. Sci. 18: 294-296.

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:

1509-1515. 

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.

13. Li. M., Dyda, F., Benhar, I., Pastan, I. and Davies, D.R. (1995). The crystal structure of Pseudomonas aeruginosa exotoxin domain III. Proc. Natl. Acad. Sci. (USA) 92: 9308-9312.

14. Benhar, I. and Pastan I. (1995). Identification of residues that stabilize the single chain Fv of MAb B3. J. Biol. Chem. 270: 3373-3380.

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.

20. Frenkel, D., Solomon, B. and Benhar, I. (1999). Modulation of Alzheimer's beta amyloid neurotoxicity by an anti-aggregating single-chain antibody. J. Neuroimmunology, 106: 23-31.

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.

33. Mazor, Y., Keydar, I., Benhar, I. (2005) Humanization and epitope mapping of the H23 anti-MUC1 monoclonal antibody reveals a dual epitope specificity. Mol. Immunol. 42: 55-69.

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.

36. Shaki-Loewenstein, S., Zfania, R., Hyland, S., Wels, W. S. and Benhar, I. (2005) A universal solution for stable intracellular antibodies. J. Immunol. Methods 303: 19-39.

37. Yacoby I, Shamis M, Bar H, Shabat D, Benhar I. (2006) Targeting antibacterial agents by using drug-carrying filamentous bacteriophages. Antimicrob Agents Chemother. 50(6):2087-97.

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.

41. Mazor Y, Barnea I, Keydar I, Benhar I. (2007) Antibody internalization studied using a novel IgG binding toxin fusion. J. Immunol. Methods 321(1-2):41-59.

42. Yacoby I, Bar H, Benhar I. (2007) Targeted drug-carrying bacteriophages as antibacterial nanomedicines. Antimicrob. Agents Chemother. 51(6):2156-63.

43. Benhar I. (2007) Design of synthetic antibody libraries. Expert Opin. Biol. Ther. 7(5):763-79.

44. Mazor, 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 Lett. 257(1):124-135.

45. Yacoby, I. and Benhar, I. (2007) Targeted anti bacterial therapy. Infectious Diseases Drug Targets. 7(3):221-229.

46. Trahtenherts, A., Gal-Tanamy, M., Zemel, R., Bachmatov, L., Loewenstein, A., Tur-Kaspa, R. and Benhar, I. (2008). Inhibition of Hepatitis C Virus RNA replicons by peptide aptamers. Antiviral Res. 77: 195-205.

47. Yacoby, I. and Benhar, I. (2008) Targeted Bacteriophages as Therapeutic Agents. Expert Opin Drug Targets. 5(3):321-329.

48. Bar, H., Yacoby, I. and Benhar, I. (2008) Killing cancer cells by targeted drug-carrying phage nanomedicines. BMC Biotech. 8:37.

49. Yacoby, I. and Benhar, I. (2008) Potential of Antibacterial Nanomedicines. Nanomedicine 3(3):329-341.

50. Artzy-Schnirman, A., Brod, E., Epel, M., Dines, M., Hammer, T., Benhar, I., Reiter, Y. and Sivan U. (2008) A two-state electronic antigen and an antibody selected to discriminate between these states. Nano Lett. 8(10):3398-3403.

51. Nahary, L., Tamarkin, A., Kayam, N., Sela, S., Fry, L., Baker, B., Powles, A., Rogers, S. 4, Benhar, I. (2008). An Investigation of Anti Streptococcal Antibody Responses in Guttate Psoriasis. Arch. Dermatol. Res. 300(8):441-449.

52. Rubinstein, D.B., Karmely, M., Pichinuk, E., Ziv, R., Benhar, I., Feng, N. and Wreschner, D.H. (2009). The MUC1 dyad oncoprotein as a functional target: Inhibiting α/β chain interaction mediates cytotoxicity of cells expressing MUC1. Int. J. Cancer. 124(1):46-54.

53. Hakim, R., Benhar, I. (2009) “Inclonals”; IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system. mAbs 1(3):1-7.

54. Trahtenherts, A., Benhar, I. (2009) Isolation and characterization of an internalizing antibody specific for the human asialoglycoprotein receptor. Hybridoma 28(4): 225-33.

55. Orgad, S., Dimant, H., Dor-On, E., Azriel-Rosenfeld, R., Benhar, I., Solomon, B. (2010) TAR1, a human anti-p53 single-chain antibody, restores tumor suppressor function to mutant p53 variants. J. Immunother. 33(2):146-54.

56. Hartmann, C., Müller, N, Blaukat, A., Koch, J., Benhar, I., Wels, W. S. (2010) Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response. Oncogene 12;29(32):4517-27.

57. Bourbeillon, J., Orchard, S., Benhar, I., Borrebaeck, C., de Daruvar, A., Dübel, S (and 20 more authors) (2010) Minimum Information about a Protein Affinity Reagent (MIAPAR). Nature Biotechnology 28(7):650-653.

58. Mendel, K., Eliaz, N., Benhar, I, Hendel, D., Halperin, N. (2010) Magnetic isolation of particles suspended in synovial fluids for diagnostics of natural joint chondropathies. Acta Biomaterialia, Acta Biomater. 6: 4430-4438.

59. Gal-Tanamy M, Zemel R, Bachmatov L, Jangra RK, Shapira A, Villanueva R, Yi M, Lemon SM, Benhar I, Tur-Kaspa R. (2010) Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies. Antiviral Res. 88(1):95-106.

60. Shapira A, Benhar I. (2010) Toxin-Based Therapeutic Approaches. Toxins 2: 2519-2583.

61. Dalken B, Jabulowsky RA, Oberoi1 P, Benhar I, Wels WS (2010) Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein. PLoS One 5 (12) e14404.

62. Hakshur K, Benhar I, Bar-Ziv Y, Halperin N, Segal D, Eliaz N (2011) The effect of hyaluronan injections into human knees on the number of bone and cartilage wear particles captured by bio-ferrography. Acta Biomaterialia. 7: 848-857.

63. Shapira A, Gal-Tanamy M, Nahary L, Litvak-Greenfeld D, Zemel R, Tur-Kaspa R, Benhar I. (2010) Engineered toxins "Zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain. PLoS One 6(1):e15916.

64. Shapira S, Shapira A, Starr A, Kazanov D, Kraus S, Benhar I, Arber N. (2010) An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice. Gastroenterology. Dec 10. [Epub ahead of print]

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.

6. Nahary, 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.

7. Nahary, L., Trahtenherts, A. and Benhar, I. (2009) Isolation of scFvs that inhibit the NS3 protease of Hepatitis C virus by a combination of phage display and a bacterial genetic screen in: Antibody phage display: methods and protocols. Rob Aitken (Ed) Humana Press. 562:115-32.

8. Benhar, I. (2009) Combinatorial libraries of arrayable single-chain antibodies. in Combinatorial methodologies for development of chemical and biological sensors. R.A. Potyrailo and V. Mirsky (Eds). Springer, Germany. 4, 223-248.

C. PATENTS

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.

3. Yacoby, Y., Ron, E.Z., Shabat, D., Shamis, M., Benhar, I. Targeted drug-carrying viruses. PCT submitted April 2006.

4. Benhar, I. Mazor Y. (2006). Recombinant immunoglobulin-Fc binding toxin fusion ZZ-PE38 and complexes thereof. US Provisional submitted May 2006.

5. Solomon, B., Orgad, S., Benhar, I., Rosenfeld, R. Human synthetic single-chain antibodies directed against the common epitope of mutant p53 and its uses. US Provisional submitted June 2006

6. Benhar, I. Hakim, R. (2008). Production of heteromeric proteins in bacteria. Provisional application submitted March 2008.