Methods of selecting internalizing antibodies

This application is a continuation of U.S. Ser. No. 10/855,755 filed May 26, 2004, which application is a divisional of and claims benefit of U.S. Ser. No. 09/249,529 filed Feb. 12, 1999, which claims benefit under 35 U.S.C. §119(e) of provisional application U.S. Ser. No. 60/082,953, filed on Apr. 24, 1998, which application are herein incorporated by reference in their entirety for all purposes.

This work was supported, in part, by Department of Defense Grants DAMD17-96-1-6244 and DAMD17-94-4433. The government of the United States of America has certain rights in this invention.

This invention pertains to the fields of immunodiagnostics and immunotherapeutics. The invention provides methods of identifying internalizing antibodies and internalizing receptor ligands, as well as the internalizing receptors bound.

Growth factor receptors, and other signal transduction receptors, are frequently overexpressed in human carcinomas and other diseases and thus have been utilized for the development of targeted therapeutics. The HER2/neu gene, for example, is amplified in several types of human adenocarcinomas, especially in tumors of the breast and the ovary (Slamon et al. (1989) Science 244: 707-712) leading to the overexpression of the corresponding growth factor receptor ErbB2. Targeting of ErbB2 overexpressing cells has been accomplished primarily using anti-ErbB2 antibodies in different formats, including conjugation to liposomes containing chemotherapeutics (Kirpotin et al. (1997). Biochem. 36: 66-75), fusion to DNA carrier proteins delivering a toxic gene (Forminaya and Wels (1996) J. Biol. Chem. 271: 10560-10568), and direct fusion to a toxin (Altenschmidt et al. (1997) Int. J. Cancer 73: 117-124).

For many of these targeted approaches, it is necessary to deliver the effector molecule across the cell membrane and into the cytosol. In some cases, this can be facilitated by taking advantage of receptor mediated endocytosis (Ullrich and Schlessinger (1990) Cell 61: 203-212). Receptor-mediated endocytosis is often caused when ligand binding causes receptor activation via homo- or heterodimerization, either directly for bivalent ligand or by causing a conformational change in the receptor for monovalent ligand. Antibodies can mimic this process, stimulate endocytosis, become internalized and deliver their payload into the cytosol. In addition, the efficiency with which antibodies mediate internalization differs significantly depending on the type of the antibody (e.g. whole antibody, fragment, single chain, monomeric, dimeric, etc.) and on the epitope recognized (Yarden (1990) Proc. Natl. Acad. Sci. USA 87: 2569-2573; Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357.). Thus for some applications, such as liposomal targeting, only antibodies that bind specific epitopes are rapidly internalized and yield a functional targeting vehicle.

Internalizing antibodies have also been shown to cause cell growth inhibition or enhanced cell growth, depending on the epitope recognized. Thus selection for internalization should lead to the isolation of growth inhibitory or stimulatory (agonist) antibodies. Such inhibitory antibodies might be used as cancer treatments or for the treatment of other conditions characterized by cell hyperproliferation, and for the treatment of inflammation (anti-inflammatories). Agonist antibodies could be used for stimulating growth of relevant cells (for example stem cells). Targeting of cells besides cancer cells for gene delivery will also have many application

Currently, antibodies that mediate internalization are identified by screening hybridomas. Screening of hybridoma-produced antibodies, however, is laborious, time-consuming, and expensive.

This invention is based, in part, on the discovery that it is possible to directly select internalizing antibodies from large non-immune phage libraries by recovering infectious phage particles from within cells after receptor mediated endocytosis.

Thus, in one embodiment, this invention provides methods of selecting polypeptide or antibody binding moieties that are internalized into target cells. The methods preferably involve i) contacting one or more of target cells with one or more members of a phage display library; iv) culturing the target cells under conditions where members of the display library can be internalized if bound to an internalizing marker; and v) identifying internalized members of the phage display library if members of the phage display library are internalized into one or more of the target cells. The methods also optionally, and preferably additionally involve contacting members of the phage display library with a cells of a subtractive cell line; and then washing the target cells to remove the cells of a subtractive cell line and to remove members of the phage display library that are non-specifically bound or weakly bound to the target cells. In a preferred embodiment, the phage display library is an antibody phage display library, more preferably an antibody phage display library displaying single chain antibodies (e.g. scFv, scFab, etc.).

In a preferred embodiment, the “identifying” step comprises recovering internalized phage and repeating steps the process again to further select for internalizing binding moieties. In one embodiment, the “recovering” step involves lysing the target cells to release internalized phage; and infecting a bacterial host with the internalized phage to produce phage for a subsequent round of selection. The recovering step can involve recovering infective phage, and/or recovering a nucleic acid encoding a phage-displayed antibody and/or selection of phage expressing a selectable marker (e.g. an antibiotic resistance gene or cDNA). The identifying step can involve detecting expression of a reporter gene, detecting the presence absence or quantity of a particular nucleic acid, or selection of phage via a selectable marker. In preferred methods the cells of a subtractive cell line are present in at least 2-fold excess over the target cells. In preferred methods, the target cells form an adherent layer. In preferred methods the target cell line is grown adherent to a tissue culture plate and co-incubated with the subtracting cell line in suspension in a single cell culture flask. In particular preferred methods, the contacting with a subtractive cell line is performed at a temperature (e.g. at about 4° C.) lower than the internalization culture conditions (e.g. at about 37°)

In particularly preferred embodiments, the phage express a selectable maker and/or a reporter gene. Preferred selectable markers include, but are not limited to genes (or cDNAs) encoding fluorescent protein(s), an antibiotic resistance gene or cDNA, and a chromagenic gene or cDNA (e.g., horse radish peroxidase, β-lactamase, luciferase, and β-galactosidase. In certain embodiments the target cells can include solid tumor cells, members of a cDNA expression library, cells that overexpress a cytokine receptor, cells that overexpress a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA encoding a specific surface target receptor, and neoplastic cells derived from outside a solid tumor. In one particularly preferred embodiment, the said cells of a subtractive cell line are selected from the same tissue type as the target cells. Suitable s subtractive cell line cells include, but are not limited to fibroblasts, monocytes, stem cells, and lymphocytes.

The methods of this invention can also be used to identify internalizing receptors and/or internalizing receptor epitopes (regions of the receptor that when bound induce internalization of the binding moiety). The methods generally involve any of the methods for identifying internalizing antibodies or polypeptides as described herein with the additional steps whereby the internalizing antibodies or polypeptides identified are used to probe the original target cells, or different cells. When the internalizing antibodies or polypeptides so bind, they permit isolation of the cell bearing the internalizing receptor and isolation of the receptor and/or receptor epitope itself. Thus in one embodiment the methods involve i) contacting one or more of the target cells with one or more members of a phage display library; ii) optionally, but preferably, contacting members of the phage display library with a cells of a subtractive cell line; iii) optionally, but preferably washing the target cells to remove said cells of a subtractive cell line and to remove members of the phage display library that are non-specifically bound or weakly bound to said target cells; iv) culturing the cells under conditions where members of said phage display library can be internalized if bound to an internalizing marker; v) identifying internalized members of the phage display library if members of the phage display library are internalized into one or more of said target cells; vi) contacting the same or different target cells with the identified internalized members of step (v) or members propagated therefrom, whereby the members bind to the surface of said same or different target cells. The method can further involve isolating a component of the same or different target cells to which the members bind. In some methods the “identifying” step involves recovering internalized phage and repeating steps (i) through (v) to further select for internalizing binding moieties.

The contacting, washing, culturing, and identifying steps are preferably performed as described herein and the target and subtractive cells include the cells described herein.

In still another embodiment, this invention provides a multivalent antibody phage display library. The library preferably comprises a plurality of phage wherein the phage display, on average, at least two copies of a single-chain antibody and the library comprises a plurality of species of single-chain antibody. In preferred embodiments, the phage display, on average, at least 3, at least 4, or at least 5 copies of a single chain antibody per phage particle. Particularly preferred libraries comprise, on average, at least 10⁵, preferably at least 10⁶, more preferably at least 10⁷, and most preferably at least 10⁸ different species of single chain antibody. In a most preferred embodiment, the antibodies are encoded by a nucleic acids that are phage (not phagemid) vectors.