Carlos F. Barbas III, Christoph Rader, Mikhail Popkov, Nina Jendreyko, Subash Sinha 
The Scripps Research Institute, La Jolla, CA

Cancer—A Multidisciplinary Approach: Genes, Proteins, and Drugs

Cancers arise in a multitude of cell types as the result of diverse oncogenic mechanisms that ultimately lead to cell transformation. While it has been observed for many years that cancers that initially respond to a defined therapeutic agent often develop resistance, recent studies with a highly potent and selective inhibitor of a single molecular target, Gleevec, drive home the point that like viruses, cancers can rapidly evolve to evade therapeutic agents. In recent years my laboratory has focused on the development of diverse strategies for the development of therapeutics and the validation of molecular targets. In this presentation, I will attempt to summarize some of our recent results in this area.

Prodrug Therapy with Catalytic Antibodies
Effective chemotherapy remains a key issue for successful cancer treatment in general, and neuroblastoma in particular. We have developed a novel chemotherapeutic strategy based on catalytic antibody mediated prodrug activation. In order to study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 102 -fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell lines. Drug activity was restored following activation by antibody 38C2. Proof of principle for local antibody catalyzed prodrug activation in vivo, was established in a syngeneic model of murine neuroblastoma. Mice with established 100 mm3 subcutaneous (s.c.) tumors that received one intratumoral injection of antibody 38C2 followed by systemic intraperitoneal injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially non-immunogenic approach for targeted cancer chemotherapy.

Tumor and Vascular Targeting
Angiogenesis is required for tumor growth and metastasis, and inhibition of angiogenesis is a promising approach for anticancer therapy. In some instances, it should be possible to create therapeutic agents that target receptors found on both tumors as well as there supporting vasculature. Such agents would have the potential to mediate a double-strike against the cancer. As a model for the development of such therapeutic reagents we have initially studied Kaposi’s sarcoma. Kaposi’s sarcoma (KS) is a cancer that was first described in 1872 by dermatologist Moritz Kaposi. The disease was once relatively rare, presenting primarily in older males of Mediterranean and Eastern European decent at a frequency in the general population of less than 1 case per 100,000. With the emergence of the HIV-1 pandemic this has, however, changed dramatically. Several different clinical and epidemiological forms of KS have now been recognized, the most aggressive of which is AIDS-associated KS. KS is the most common AIDS-associated malignancy. While the variety of different epidemiological forms of KS progress over different clinical courses, all forms of KS present lesions whose histopathology is characterized by intense angiogenesis, edema, proliferation, and infiltration by inflammatory cells. The predominant cell type in the tumor is a spindle-shaped cell typically of endothelial origin, which is considered to be the tumor element of KS. KS tumors are highly vascularized as a result of the intense angiogenesis characteristic of this disease and the blood vessels are often abnormal and leaky. All forms of KS are also associated with infection by the human herpes virus 8 (HHV8), also known as Kaposi sarcoma herpes virus (KSHV). We have studied integrin avb3 as a molecular target for antibody therapy of Kaposi’s sarcoma. Using a new phage display strategy based on designed combinatorial V gene libraries, we previously reported the humanization of mouse monoclonal antibody LM609 directed to human integrin avb3. We now report the in vitro affinity maturation of humanized LM609 by phage display and the conversion of the evolved Fab into IgG1 using a new mammalian expression vector. The resulting whole antibody, designated JC-7U IgG1, was found to inhibit the growth of human Kaposi’s sarcoma in a nude mouse model at a therapeutically relevant dose. Our findings suggest that in addition to vascular targeting, integrin avb3 expressed on the surface of tumor cells can serve as a therapeutic target.

Expanding on these studies, we are beginning to assess the potential of targeting both integrins avb3 and avb5 with a single novel antibody. The advantages of dual targeting include the ability to block both the bFGF as well as the VEGF angiogenic pathways. This approach also provides the potential to target both tumor and its supporting vasculature. One or both of these integrins are expressed on a wide variety of cancers such as Kaposi’s, melanoma, breast, and prostate. We believe that dual targeting also reduces the potential for the cancer to escape the therapeutic regimen as well as increases the loading of antibody onto the target cells since two receptor types are available for binding. I will present recent studies involving this unique approach.

In addition to the development of therapeutics based on the administration of proteins, we are also exploring gene-based antibody strategies. Recently, we developed a CCR5-specific single-chain antibody that was expressed intracellularly and retained in the endoplasmic reticulum. This CCR5-intrabody efficiently blocked surface expression of human and rhesus CCR5 and thus prevented cellular interactions with CCR5-dependent HIV-1 and SIV env. Intrabody expressing cells were shown to be highly refractory to challenge with R5 HIV-1 viruses or infected cells. These results suggest that gene therapy approaches that deliver this intracellular antibody could be of benefit to infected individuals. We have now extended this approach to cancer therapy and present the potential of this approach in target validation. One of the receptors that we have targeted is Tie2. Tie2 is a receptor tyrosine kinase known to play a role in tumor angiogenesis. This receptor is expressed almost exclusively by vascular endothelium. To explore the therapeutic potential of blocking the Tie2 pathway, an adenoviral vector was constructed to deliver a recombinant intrabody (pAd-mTie2) capable of blocking mouse and human Tie2 expression. Administration of pAd-Tie2 to mice with two different well-established tumor lines, a human Kaposi’s sarcoma (SLK) or a human colon carcinoma (SW1222), has been studied. I will present the potential utility of targeting an endothelium-specific receptor as a gene-based therapeutic approach to cancer.

Recent Publications relevant to this presentation:

1. Prodrug Activation with Catalytic Antibodies
2. Shabat, D.; Lode, H.N.; Pertl, U.; Reisfeld, R.A.; Rader, C.; Lerner, R.A.; Barbas, III, C.F. (2001) In vivo activity in a catalytic antibody- prodrug system: antibody catalyzed etoposide prodrug activation for selective chemotherapy. Proc. Natl. Acad. Sci., USA 98 (13), 7528- 7533.
3. Shabat, D.; Rader, C.; List, B.; Lerner, R.A.; Barbas III, C.F. (1999) Multiple event activation of a generic prodrug trigger by antibody catalysis. Proc. Natl. Acad. Sci, USA, 96, 6925-6930.
4. Warrall, D.S.; McDunn, J.E.; List, B.; Reichart, D.; Hevener, A.; Gustafson, T.; Barbas III, C.F.; Lerner, R.A.; Olefsky, J.M. (2001) Synthesis of an organoinsulin molecule that can be activated by antibody catalysis. Proc. Nat. Acad. Sci., USA, 98 (24), 13514-13518.
5. Tanaka, F.; Lerner, R.A.; Barbas III, C.F. (2000) Reconstructing Aldolase Antibodies to Alter Their Substrate Specificity and Turnover. J. Am. Chem. Soc., 122, 4835-4836.
6. Barbas III, C.F.; Rader, C.; Segal, D.J.; List, B.; Turner, J.M. (2000) From Catalytic Asymmetric Synthesis to the Transcriptional Regulation of Genes: In Vivo and In Vitro Evolution of Proteins, in Advances in Protein Chemistry, 55: 317-66.
7. Generation of Therapeutic Antibodies and Intrabodies
8. Steinberger, P.; Andris-Widhopf, J.; Bühler, B.; Torbett, B.E.; Barbas III, C.F. (2000) Functional Deletion of the CCR5 Receptor by Intracellular Immunization Produces Cells that are Refractory to CCR5-dependent HIV-1 Infection and Cell Fusion. Proc. Natl. Acad. Sci. USA, 97, 805-810.
9. Steinberger, P.; Sutton, J.K.; Rader, C.; Elia, M.; and Barbas III, C. F. (2000) Generation and Characterization of a Recombinant Human CCR5-specific Antibody: A Phage Display Approach for Rabbit Antibody Humanization, J. Biol. Chem., 275, 36073-36078.
10. Rader, C.; Cheresh, D.; Barbas III, C.F. (1998) Phage display approach for rapid antibody humanization: Designed combina- torial V gene libraries. Proc. Natl. Acad. Sci. USA, 95, 8910-8915.
11. Rader, C.; Ritter, G.; Nathan, S.; Elia, M.; Gout, I.; Jungbluth, A.A.; Cohen, L.S.; Welt, S.; Old, L.J.; Barbas III, C.F. (2000) The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J. Biol. Chem., 275, 13669-13676.
12. Barbas, C. F., III; Burton, D. R.; Scott, J.K., Silverman, G.J. Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 736 pages.
13. Rader, C; Popkov, M.; Neves, J.A.; Barbas III, C.F. Targeting Kaposi’s Sarcoma with an In Vitro Evolved Antibody Directed to Integrin avb3. Submitted
14. Development of Zinc Finger Technology for the Regulation of Endogenous Genes
15. Beerli, R.R.; Barbas III, C.F. (2002) Engineering Polydactyl Zinc Finger Transcription Factors. Nature Biotechnology, 20 (2), 135-41.
16. David J. Segal and Carlos F. Barbas III (2001) Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Current Opinion in Biotechnology, 12, 632-637
17. Beerli, R. R.; Dreier, B.; Barbas III, C.F. (2000) Selective Positive and Negative Regulation of Endogenous Genes by Designed Transcription Factors. Proc. Natl. Acad. Sci. USA, 97, 1495-1500.