Methods and compositions for inhibition of metastasis

This application claims the benefit of U.S. Application No. 60/544,807, filed Feb. 13, 2004, and U.S. Application No. 60/626,726, filed Nov. 10, 2004, the entire disclosures of which are incorporated herein by reference.

This invention was made with Government support by Grant Nos. NCI-CA95458 and NIAID-AI147027, awarded by the National Institutes of Health and Grant No. DAMD 17-99-1-9368, awarded by the U.S. Army Breast Cancer Research Program. The Government has certain rights in this invention.

This invention generally relates to methods of producing an antibody phage population having affinity for a tumor cell target expressing a metastatic phenotype. The invention further relates to antibody compositions that specifically bind to a cell surface receptor on the metastatic cell.

Breast cancer metastasis to lungs, liver, bone and brain is the primary cause of death in breast cancer patients. It involves cancer cell dissemination via the blood stream and depends on adhesive and invasive tumor cell functions and their ability to survive and proliferate at the target site. Bogenrieder et al., Oncogene 22: 6524-6536, 2003. These events are supported by integrins, a family of transmembrane adhesion receptors composed of α and β subunits. Felding-Habermann, Clin. Exp. Metastasis 20: 203-213, 2003; Hood et al., Nat. Rev. Cancer 2: 91-100, 2002. Integrins exist in distinct states of activation, which determine the affinity for ligand and regulate whether soluble ligands are bound, which matrix proteins are recognized, and the degree to which cells can adhere, migrate and arrest under dynamic flow conditions as found in the circulation. Tadokoro et al., Science 302: 103-106, 2003; Shattil et al., J. Clin. Invest. 100:1-5, 1997; Felding-Habermann, et al., Proc. Natl. Acad. Sci. U. S. A 98: 1853-1858, 2001.

An integrin found on breast cancer cells, but not on normal breast epithelium is αvβ3. Expressio[n of this receptor correlates with invasion and cancer progression. Liapis et al., Diagn. Mol. Pathol. 5: 127-135, 1996; Pignatelli et al., Hum. Pathol. 23: 1159-1166, 1992. Human breast cancer cells can express αvβ3 in an activated or a non-activated functional state. Activated αvβ3 supports breast cancer cell attachment during blood flow, and strongly promotes invasive tumor cell migration. In particular, only the activated state supports target organ colonization by circulating breast cancer cells in a mouse model, and metastatic cells isolated from breast cancer patient blood express αvβ3 in a constitutively activated form. Felding-Habermann et al.; Proc. Natl. Acad. Sci. U.S.A 98: 1853-1858, 2001; Rolli et al., Proc. Natl. Acad. Sci. U.S. A 100: 9482-9487, 2003. Integrin transition between distinct states of activation is associated with conformational changes within the heterodimer. Calzada et al., J. Biol. Chem. 277: 39899-39908, 2002; Beglova et al., Nat. Struct. Biol. 9: 282-287, 2002; Xiong et al., Science 294: 339-345, 2001; Xiong et al., Science 296: 151-155, 2002; Pampori et al., J. Biol. Chem. 274: 21609-21616, 1999.

Like other integrins, αvβ3 can exist in distinct states of activation and functional affinity. The activated or high affinity state of αvβ3 has a unique molecular conformation that is distinct from the non-activated state. Xiong et al., Science, 294: 339-345, 2001; Xiong et al., Science, 296: 151-155, 2002; Xiong et al., Blood, 102: 1155-1159, 2003.

In contrast to the non-activated receptor, activated αvβ3 is functionally characterized primarily through its ability to bind soluble ligand proteins, to support tumor cell interaction with platelets during blood flow and thereby mediated tumor cell arrest under conditions as found in the vasculature, and to promote invasive tumor cell and endothelial cell migration very strongly. The latter is probably very important for angiogenesis. The ability of the activated αvβ3 integrin to bind soluble ligands is a key property that enables the ligand-mimetic scFv antibodies to bind specifically and exclusively to the activated conformation of αvβ3.

No therapy is known today that prevents cancer, for example, breast cancer, from becoming systemic, and there is little understanding of even how to design and test such drugs; yet metastases ultimately are responsible for much of the suffering and mortality from breast cancer. A need exists to identify and target molecular and functional markers that identify metastatic breast cancer cells and to generate reagents for their specific inhibition.

The invention is generally related to methods of producing an antibody phage population having affinity for a tumor cell target which is a tumor cell expressing a metastatic phenotype. The tumor cell expressing the metastatic phenotype can be a cell line expressing an activated cell surface receptor, for example, an activated integrin receptor or an αvβ3 integrin receptor. The invention further relates to an antibody composition that specifically binds to a cell surface receptor on a metastatic cell. The antibody composition specifically binds to an activated cell surface receptor on a metastatic cell, for example, an activated integrin receptor or an αvβ3 integrin receptor. The invention further relates to methods for alleviating a disease state in a mammal by treatment with a cancer therapeutic comprising the step of administering to the mammal a therapeutic amount of the pharmaceutical composition of the antibody composition. The invention further relates to methods of detecting an activated cell surface receptor on a metastatic tumor cell surface in a mammalian tissue sample and to methods of identifying cells liable to undergo metastasis associated with a disease state comprising contacting a patient suspected of being at risk for metastasis with the antibody composition, the antibody having associated therewith an imaging moiety.

Cells or tumor cells with a non-activated αvβ3 integrin receptor refers to a conformation of αvβ3 that is unable to bind soluble ligands and does not support tumor cell arrest under dynamic flow conditions as during blood flow under conditions found in the vasculature. This non-activated conformation of αvβ3 can be associated with non-metastatic tumor cells and it is not recognized by an antibody, for example, scFv antibodies Bc-12 or Bc-15.

In one embodiment, an antibody which specifically binds to an activated αvβ3 integrin receptor which is differentially produced on a cell in a metastatic state compared to a similar, non-metastatic cell. In a detailed embodiment, the antibody comprises scFv antibody Bc-12. In a further detailed embodiment, the antibody comprises SEQ ID NO: 2. In a detailed embodiment, the antibody comprises scFv antibody Bc-15. In a further detailed embodiment, the antibody comprises SEQ ID NO: 4. In a further embodiment, the antibody comprises an R-G-D sequence in a complementary determining region (CDR). In a detailed aspect, the CDR can be CDR-H3. In a further detailed aspect, the metastatic cell targets to a tissue selected from breast, brain, lung, liver, or bone. In a further detailed aspect, a pharmaceutical composition comprises the antibody.

The present invention further provides a cDNA encoding scFv Bc-12 in a phage display vector for expression and production of scFv antibody Bc-12. In a detailed embodiment, the cDNA encoding scFv Bc-12 comprises SEQ ID NO: 1. The cDNA encoding scFv Bc-12 has an ATCC accession number PTA-6303, date of deposit: Nov. 12, 2004. The present invention further provides a cDNA encoding scFv Bc-15 in a phage display vector for expression and production of scFv antibody Bc-15. In a detailed embodiment, the cDNA encoding scFv Bc-15 comprises SEQ ID NO: 2. The cDNA encoding scFv Bc-15 has an ATCC accession number PTA-6304, date of deposit: Nov. 12, 2004.

In another embodiment, an antibody specifically binds to an activated αvβ3 integrin receptor and does not bind to a non-activated αvβ3 integrin receptor. In a further aspect, the activated αvβ3 integrin receptor is differentially produced on a cell in a metastatic state compared to a similar, non-metastatic cell.

In another embodiment, an antibody comprises a ligand mimetic which specifically binds to an activated αvβ3 integrin receptor which is differentially produced on a cell in a metastatic state compared to a similar, non-metastatic cell.