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Integrin _vß₃ targeted therapy for Kaposi’s sarcoma with an in vitro evolved antibody¹

Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

²Correspondence: Department of Molecular Biology, BCC-526, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: crader@scripps.edu and carlos{at}scripps.edu

SPECIFIC AIMS

The goal of this study was to evaluate integrin _vß₃ as a molecular target for antibody therapy for Kaposi’s sarcoma (KS). We used in vitro evolution based on phage display to generate a humanized and affinity-matured antibody directed to integrin _vß₃ to evaluate tumor targeting and tumor growth inhibition in a mouse model of KS.

PRINCIPAL FINDINGS

1. In vitro evolution of antibody JC-7U directed to integrin _vß₃
Using a phage display strategy that preserves only the original CDR3 sequences of light and heavy chain while subjecting the remaining sequence to selection from naive human V gene libraries, we previously reported the full humanization of mouse monoclonal antibody (mAb) LM609 directed to human integrin _vß₃. Here we further improved the affinity of humanized LM609 using a phage display strategy for sequential and parallel optimization of three complementarity determining regions (LCDR1, HCDR1, and HCDR3) of the antibody molecule. The evolved Fab, designated JC-7U, had an affinity of 150 pM and was converted into IgG1 using a new mammalian expression vector (Fig. 1 ).

View larger version (30K): [in this window] [in a new window]   Figure 1. Tumor growth inhibition in a nude mouse model of KS in the presence of JC-7U IgG1. JC-7U IgG1 (black) or control antibody b12 IgG (white circles) was given in five doses of 5 mg/kg every third day starting on day 1 after tumor induction. Statistically significant differences in tumor volume were observed 14 days after the last treatment until the end of the experiment. Shown are average tumor volumes ± SD (n=5 for both groups).

2. Tumor targeting of antibody JC-7U in a mouse model of Kaposi’s sarcoma
A previously described nude mouse model of KS based on human KS cell line SLK was used for our studies. As an isotype control for JC-7U IgG1, we used human b12 IgG1, which binds to gp120 of HIV-1. Flow cytometry revealed strong binding of JC-7U IgG1 to SLK cells, whereas no binding was detected for b12 IgG1. For in vivo studies, tumor induction was performed by subcutaneous injection of 5 x 10⁶ SLK cells in the right flank of nude mice. We first analyzed tumor targeting by injecting 100 µg of JC-7U IgG1 or b12 IgG1 into the tail vein of mice with an established SLK xenograft. After 24 h, tumor, lung, liver, and spleen were removed and processed for the detection of bound human IgG1 by immunohistochemistry. As shown in Fig. 2 , JC-7U IgG1 was found to have intensely penetrated the tumor tissue whereas control antibody b12 IgG1 revealed only slight nonspecific uptake. By contrast, neither JC-7U IgG1 nor b12 IgG1 were found in normal tissue.

View larger version (166K): [in this window] [in a new window]   Figure 2. Schematic diagram illustrating the in vitro evolution of antibody JC-7U. Because of its high affinity and its high degree of humanization, JC-7U IgG1 is an excellent drug candidate for therapeutic applications that involve integrin vß3 as the molecular target. Of particular interest is therapy for KS, breast cancer, melanoma, and other cancers in which integrin vß3 is expressed on both angiogenic endothelial cells and tumor cells, a dual antiangiogenic and antitumor strike with a single drug. Mouse sequences are shown in yellow, human sequences in blue, optimized sequences in red.

3. Antibody JC-7U inhibits tumor growth in a mouse model of Kaposi’s sarcoma
With the selective tumor targeting of JC-7U IgG1 established, we next analyzed whether JC-7U IgG1 can inhibit tumor growth. Three different groups of five animals each were treated on days 1, 4, 7, 10, and 13 after tumor induction. Each treatment involved a 200 µl tail vein injection of 1) 0.5 mg/mL JC-7U IgG1 in PBS, 2) 0.5 mg/mL b12 IgG1 in PBS, or 3) PBS alone. Thus, animals in the antibody groups received five doses of 100 µg or 5 mg/kg. For comparison, clinical studies with Vitaxin, another humanized derivative of LM609, have used doses up to 4 mg/kg with little or no toxicity, which suggests that our dosing regimen is in a clinically relevant range. Study of animals treated with JC-7U IgG1 revealed no obvious signs of toxicity for this protein, as indicated by no weight loss or behavioral change during the course of therapy. Tumor volumes of treated animals were measured every third day starting on day 9 and ending on day 54 (Fig. 3 ). Tumor growth in animals treated with PBS or b12 IgG1 revealed no difference compared with tumor growth in untreated animals, resulting in an average tumor volume of 650 and 685 mm³, respectively, tumor growth in animals treated with JC-7U IgG1 was notably slower (Fig. 3) . The difference in tumor growth was statistically significant (P<0.05) from day 27 until the end of the experiment 27 days later. On day 54, average tumor volume in the animals treated with JC-7U IgG1 was 292 mm³, which is 45% of the average tumor volume observed in the control groups. As an additional measurement, all tumors were surgically removed on day 54 and weighed. The average tumor weight in the three treatment groups confirmed the results obtained for the measurement of the tumor volume. We did not observe growth inhibition in vitro at or beyond the administered in vivo concentration of JC-7U IgG1, suggesting that antibody-dependent cellular cytotoxicity (ADCC) is the main mechanism of tumor growth inhibition of human JC-7U IgG1 in our nude mouse model of KS. Like its parental antibody, mouse mAb LM609, JC-7U binds to human but not mouse integrin _vß₃. In the SLK xenograft, JC-7U recognizes human integrin _vß₃ expressed by tumor cells but not mouse integrin _vß₃ expressed by angiogenic endothelial cells. Thus, the reduction of KS tumor growth in the presence of JC-7U is not an antiangiogenic effect but can be attributed to a specific, direct response against the tumor, which allows us to dissect out the antitumor effect of this antibody therapy.

View larger version (27K): [in this window] [in a new window]   Figure 3. Tumor targeting of JC-7U IgG1. JC-7U IgG1 (A, E) and control antibody b12 IgG1 (B, F) were injected into the tail vein of nude mice with an established human KS tumor. After 24 h, tumor (A, B) and lung tissue (E, F) were removed and processed for the detection of bound human IgG using goat anti-human IgG secondary antibodies. C, D) Mouse endothelial cells that infiltrate the human KS tumor were detected with a rat anti-mouse CD31 mAb, followed by rabbit anti-rat IgG secondary antibodies. Note that the sites of CD31 expression and JC-7U IgG1 tumor penetration overlap. Scale bar = 50 µm for panels A–F.

4. Antibody JC-7U partially blocks the binding of HIV-1 Tat protein to integrin _vß₃
Based on published evidence that the HIV-1 Tat protein can act as a growth factor for KS by binding to RGD receptors, including integrin _vß₃, that are expressed on the surface of KS cells, we investigated whether JC-7U can interfere with this interaction. We established a binding assay in which a mixture of human integrin _vß₃ and synthetic HIV-1 Tat-biotin was incubated in solution in the presence or absence of JC-7U Fab. Complexes of integrin _vß₃ and Tat-biotin were then captured with immobilized streptavidin and were detected by using the anti-ß₃ mouse mAb AP-3, which does not interfere with JC-7U binding, followed by goat anti-mouse secondary antibodies. Background signals were determined by running the same assay in the absence of Tat-biotin. In the absence of JC-7U, a strong signal above background was observed, which revealed an interaction of integrin _vß₃ and Tat-biotin. In the presence of a threefold molar excess of JC-7U Fab over Tat-biotin or at equimolar concentrations, the signal was reduced to 45%. The same reduction was observed in the presence of JC-7U IgG1. Thus, JC-7U partially blocks the binding of HIV-1 Tat protein to human integrin _vß₃.

CONCLUSIONS AND SIGNIFICANCE

In view of the fact that KS tumors are of endothelial origin and characterized by intense angiogenesis, it is logical that many of the recently developed antiangiogenic drugs are being studied for the treatment of KS. With the emerging picture of KS pathogenesis and angiogenesis, molecular targets are being revealed that may provide effective targeting of drugs to both the KS vasculature and the KS tumor itself. We hypothesized that integrin _vß₃ might fulfill these criteria. Integrin _vß₃ is up-regulated on endothelial cells in response to angiogenic growth factors and has been established as a target for antiangiogenic therapy. Soluble antagonists of integrin _vß₃ such as mAb LM609, RGD peptides, or peptidomimetics, initiate endothelial cell apoptosis and thereby inhibit angiogenesis.

In addition to its expression on the surface of angiogenic endothelial cells, integrin _vß₃ is expressed on the surface of tumor cells in a variety of cancers. In melanoma and breast cancer, for example, tumor progression and metastasis correlate with integrin _vß₃ expression. Thus, independent of its role in tumor angiogenesis, integrin _vß₃ is likely to be functionally implicated in the pathogenesis of a variety of cancers. This is particularly apparent for KS, where a central position of integrin _vß₃ within angiogenesis and KS pathogenesis has emerged, suggesting that integrin _vß₃ may become a prime molecular target for KS therapy. The hypervascular morphology of KS makes it highly accessible to therapeutic macromolecules, such as antibodies, whose general applicability for cancer therapy is limited by poor tumor penetration.

With JC-7U, we developed a highly advanced drug candidate for therapeutic applications that involve integrin _vß₃ as the molecular target. JC-7U IgG1 was found to selectively target tumors in a mouse model of KS and inhibit tumor growth at a therapeutically relevant dose. We anticipate that the tumor growth-inhibiting activity of JC-7U will be even more effective in KS patients as compared with mouse models of KS. First, in the presence of human angiogenic endothelial cells, JC-7U is expected to exert an antiangiogenic effect based on initiation of endothelial cell apoptosis and ADCC in addition to an antitumor effect. Second, in AIDS-associated KS, JC-7U will partially block the binding of HIV-1 Tat protein to human integrin _vß₃ and, thus, potentially diminish its tumor growth-promoting activity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0281fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0281fje

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