WO2005027845A2 - Method of identifying cell surface molecules involved in cancer metastasis - Google Patents

Abstract

Methods for identifying antibodies to cell surafce molecules common to cells which migrate through the human lymph system which utilize alternating panning on two different types of cells known to migrate through the human lymph system.

Description

METHOD OF IDENTIFYING CELL SURFACE MOLECULES INVOLVED IN CANCER METASTASIS

RELATED APPLICATIONS This application claims priority to U. S. Provisional Application No.

60/503,770 filed September 18, 2003, the disclosure of which is incorporated herein by reference.

Technical Field This disclosure relates to the identification of cell surface molecules which are found on cells migrating through the human lymph system, including metastatic cancer cells.

Background Tumor cell migration and metastasis are two key steps in cancer development ultimately leading to mortality. The occurrence of cancer cells in the draining lymph node is considered one of the first steps in cancer metastasis, yet it remains to be fully elucidated what causes cancer cells to migrate to lymph nodes and throughout the lymph system in humans. Dendritic cells are the most important antigen presenting cells in the body. Dendritic cells are derived from bone marrow progenitor cells and monocytes.

Immature dendritic cells (iDCs) are found under the skin and mucous membranes where they engulf microorganisms and antigenic molecules through phagocytosis and pinocytosis. The immature dendritic cells have the unique ability to migrate to the follicles of secondary lymphoid organs such as lymph nodules, lymph nodes and the spleen after taking up and processing antigens in the peripheral immune system. During the migration process, iDCs mature into mature dendritic cells (mDCs), a class of professional APCs. Some of the molecules involved in migration of dendritic cells have been identified, such as CCR7. (Saeki, H. et al., "Cutting edge: secondary lymphoid- tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes," J. Immunol. 162:2472 (1999)). Interestingly, over-expression of CCR7 mRNA has also been found in non-small cell lung cancer and a correlation has been found with lymph node metastasis. (Takanami, "Overexpression of CCR7 mRNA in nonsmall cell lung cancer: correlation with lymph node metastasis, "Int. J. Cancer 105:186 (2003)). While common upregulated genes in immature dendritic cells and various cancers can be identified by comparing available microarray data, protein expression can be different from mRNA expression thus making it difficult to correlate genes with the ability of a cell to migrate through the lymph system. (Dietz, et al. "Maturation of human monocyte-derived dendritic cells studied by microarray hybridization," Biochem. Biophys. Res. Commun. 275:731 (2000); Aim, et al. "Identification of the genes differentially expressed in human dendritic cell subsets by cDNA subtraction and microarray analysis", Blood 100:1742 (2002)). Panning of antibody phage display libraries on cell lines, or tissues, is a well established method to identify molecules on the surfaces of cells. Using a panning approach, markers unique to cancer cells as well as to dendritic cells can be identified. It would be desirable to provide improved methods for identifying metastatic cancer cells on the basis of cell surface molecules, as well as antibodies or other compounds targeting such cells via cell surface molecules.

SUMMARY The present disclosure relates to methods of identifying antibodies which bind to cell surface molecules found on cells which migrate through the human lymph system. The methods include immunizing a mammal with at least one type of migrating human cells (such as, for example, dendritic cells or metastatic cancer cells) and once an immune response is elicited obtaining, from the immunized mammal, a library of nucleic acid sequences encoding antibodies produced by the immunized mammal. The library of nucleic acid sequences are then cloned into display vectors and the library is panned on a first type of migrating human cells. The members of the library that bind to the first type of migrating cells are then panned on a second, different type of migrating human cells. The antibodies which bind to both sets of migrating human cells are thus identified and can be used to to identify cell surface molecules present on cells which migrate through the lymph system. In one embodiment, immature dendritic cells and metastatic cancer cells are used as the two different types of migrating cells. In other embodiments, antibodies to the cell surface molecules are produced by the methods of the present disclosure are utilized in applications to bind to metastatic cancer cells and interfere with their migration through the human lymph system. In other embodiments, antibodies to the cell surface molecules are produced by the methods of the present disclosure are utilized in diagnostic applications to detect the presence of cancer cells or to evalute compounds for their ability to bind to cancer cells.

DETAILED DESCRIPTION The present disclosure relates to methods for identifying antibodies to cell surface molecules found on cells having the physiologic property of migrating to lymph nodes and through the human lymph system. Such cells are referred to herein from time to time as "migrating cells". The antibodies, or fragments thereof, which target these cell surface molecules can then be used therapeutically or diagnostically. For example, the antibodies specific to cell surface molecules on migrating cells can be used to identify and target migrating cancer cells. Using the antibodies, the cell surface molecules themselves can be identifie . Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present teachings pertain, unless otherwise defined herein. Reference is made herein to various methodologies known to those of skill in the art. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Practice of the methods described herein will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such conventional techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and 11 (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, (1984)); Nucleic Acid Hybridization (B.D. Haines & S.J. Higgins eds. (1984)); the series, Methods in Enzymology (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.); PCR-A Practical Approach (McPherson, Quirke, and Taylor, eds., (1991)); Immunology, 2d Edition, (1989), Roitt et al., C.V. Mosby Company, and New York; Advanced Immunology, 2d Edition, (1991), Male et al., Grower Medical Publishing, New York.; DNA Cloning: A Practical Approach, Volumes 1 and 11 , (1985) (D.N. Glover ed.); Oligonucleotide Synthesis, (1984), (M.L. Gait ed); Transcription and Translation, (1984) (Harnes and Higgins eds.); Animal Cell Culture, (1986) (R.I. Freshney ed.); Immobilized Cells and Enzymes, (1986) (IRL Press); Perbal, (1984), A Practical Guide to Molecular Cloning; and Gene Transfer Vectors for Mammalian Cells, (1987) (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); W097/083220; US. Patent Nos. 5,427,908; 5,885,793; 5,969,108; 5,565,332; 5,837,500; 5,223,409; 5,403,484; 5,643,756; 5,723,287; 5,952,474; Knappik et al., (2000), J. Mol. Biol. 296:57-86; Barbas et al., (1991), Proc. Natl.

Acad. Sci. USA 88:7978-7982; Schaffitzel et al. (1999), J. Immunol. Meth.

10:119-1 35; Kitamura, (1998), Int. J. Hematol., 67:351-359; Georgiou et al.,

(1997), Nat. Biotechnol. 15:29-34; Little, et al., (1995), J. Biotech. 41 :187-195; Chauthaiwale et al., (1992), Microbiol. Rev., 56:577-591 ; Aruffo, (1991), Curr.

Opin. Biotechnot. 2:735-741 ; McCafferty (Editor) et al., (1996), Antibody

Engineering: A Practical Approach, the contents of which are incorporated herein by reference. Any suitable materials and/or methods known to those skilled in the art can be utilized in carrying out the methods described herein; however, preferred materials and/or methods are described. Materials, reagents and the like to which reference may be made in the following description and examples are obtainable from commercial sources, unless otherwise noted. It should be understood that the terms "including", "included", "includes" and "include" are used in their broadest sense, i.e., they are open ended and mean, e.g., including but not limited to, included but limited to, includes but not limited to, and include but not limited to. As used herein, "antibody" refers to an entire immunoglobulin molecule or molecules that contain immunologically active portions of whole immunoglobulin molecules and includes Fab, F(ab')2, scFv, Fv, heavy chain variable regions and light chain variable regions. The terms immunoglobulin and antibody are used interchangeably herein. In order to identify the cell surface molecules related to cell migration, and produce antibodies thereto, libraries of antibodies to the cell surface molecules are constructed. As used herein, a "library" or "libraries" means a collection of nucleic acid molecules representing the immune repertoire of an organism, which includes molecules encoding for antibodies and fragments directed toward the cell surface molecules/receptors of cells which migrate through the lymph system, such as immature dendritic cells and metastatic cells. Libraries are typically presented as nucleic acid molecules cloned in a vector, e.g. a surface display vector. In accordance with the present disclosure, the first step in producing a library is immunizing a host animal, preferably a mammal, with at least one type of cells that are capable of migrating through the body, especially the human lymph system. A suitable mammalian host animal for immunization is preferably a rodent, such as a mouse, rat or hamster. More preferably, the host is a mouse, and can be a Swiss, Balb/c or NIH mouse. Different individual host animals, or groups of animals, can be immunized for different types of cells, for later comparison. The host animal is immunized with a first set of human cells of a type which migrate through the human lymph system. The immunization protocol may be selected by one skilled in the art without undue experimentation. Preferably, immature dendritic cells are used in host immunization because they migrate to the follicles of secondary lymphoid organs, such as lymph nodes, lymph nodules, and the spleen, where they mature into dendritic cells. Immunization with other types of migrating cells, particularly metastatic cancer cells (such as, for example, colon cancer cell line KM12L4a), is also contemplated. It is also contemplated that a host animal can be immunized with two or more different types of migrating cells to enhance the immune response to cell surface molecules common to the multiple types of cells. Thus, for example, a host animal can be immunized with both immature dendritic cells and metastatic cancer cells, either simultaneously or sequentially. Where immature dendritic cells are used, they may be obtained from bone marrow, blood, the spleen, or any other source. In one embodiment, monocytes are obtained from a suitable source, such as bone marrow, and the monocytes are matured in vitro to produce immature dendritic cells. In such a case, the monocytes can be matured in the presence of a cytokine to help orchestrate the immune response in the host animal. As used herein, the term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Examples of cytokines include, but are not limited to IL-1 , IL-2, IL- 4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF), interferon-γ (y - INF), tumor necrosis factor (TNF), TNF-α, TGF-β, Flt-3 ligand, and CD40 ligand. In one embodiment a cytokine such as GM-CSF, IL-4 and combinations thereof are added to the dendritic cells. In another embodiment, metastatic cancer cells are utilized as the first set of human cells known to migrate through the human lymph system and injected into the host animal for the generation of antibodies to cell surface molecules thereon . Non-limiting examples of metastatic cancer cells that can be used include colon cancer cell line KM12L4a, breast cancer cell line MDAMB435, and prostate cancer cell line PC3. Metastatic cancer cells can be collected from patients or are available for research purposes from a variety of sources, such as, for example, American Type Culture Collection ("ATCC"), Manasas, VA, USA and the National Cancer Institute ("NCI"), Bethesda, MD, USA. Once obtained, the migrating cells (e.g., immature dendritic cells, metastatic cancer cells or both) are introduced into the host mammal, preferably by injection. After a time sufficient for the formation of antibodies, which can range from about 2 to about 90 days, preferably from about 40 to about 60 days, a blood sample is taken to confirm the presence of antibodies. Once antibodies are detected, antibody producing cells are harvested from the host animal. Once the cells are collected, RNA is isolated therefrom using techniques known to those skilled in the art and a combinatorial antibody library is prepared. In general, techniques for preparing a combinatorial antibody library involve amplifying target sequences encoding antibodies or portions thereof, such as, for example the light and/or heavy chains using the isolated RNA of an antibody. Thus, for example, starting with a sample of antibody mRNA that is naturally diverse, first strand cDNA can be produced to provide a template. Conventional PCR or other amplification techniques can then be employed to generate the library. A variety of techniques are known for display of antibody libraries including phage display, phagemid display, ribosomal display and cell surface display. As one example, phage libraries expressing antibody Fab fragments (kappa or lambda light chains complexed to the IgG heavy chain fragment (Fd) can be constructed in plasmid vectors using the methods described in U.S. Application No. 10/251 ,085 (the disclosure of which is incorporated herein in its entirety by this reference). The constructed library is then panned against two types of migrating cells. That is, the library is panned with the first type of human cell which migrates through the human lymph system, followed by panning with a second, different type of human cells which migrate through the human lymph system. Preferably, the first type of migrating cells are the same cells utilized to immunize the host animal. In one embodiment, the first type of migrating cells used to pan the library is immature dendritic cells and the second type of migrating cells is metastatic cancer cells. In an alternative embodiment, the first type of migrating cells used to pan the library are metastatic cancer cells and the second type of cells is immature dendritic cells. Methods for panning antibody libraries are well known to those skilled in the art and include, for example the relatively simple panning methods described in: Scott et al., Science, 249:386-290, (1990); Devlin et al., Science, 249:404-406, (1990); Cwirla et al., Proc. Natl. Acad. Sci. U.S.A, 87:6378-6382, (1990); McCafferty et al., Nature, 348:552-554, (1990); Lowman et al., Biochemistry, 30:10832-10838, (1992); and Kang et al., Proc. Natl. Acad. Sci. U.S.A., 88:4363-4366, (1991). It may be advantageous in some embodiments, to perform negative selection panning to eliminate antibodies that bind to very common, unneccessary or unwanted molecules. The library can be panned on commonly found cells to subtract binders to very common surface molecules. By way of example, those antibodies that bind to HLA or certain chemokine receptors can be eliminated. Where such a process is used, the common, unneccessary or unwanted molecule is bound to a plate followed by addition of the phage. After appropriate incubation, unbound phage are washed from the plates and collected for amplification. In addition, if desired, the library can be panned to remove those members that bind to cells outside the target population of cells, i.e., cells other than metastatic cancer cells. Specifically, for example, the library can be panned against non-metastatic cancer cells. Fluorescence activated cell sorting (FACS), magnetic cell sorting, cell ELISA or expression ELISA can also be used for screening to confirm that the relevant binding antibodies have been identified. Methods for FACS sorting of surface displayed molecules include Georgiou, G. et al., Nature Biotechnology 15: 29-34, 1996, the disclosure of which is incorporated herein by reference. In addition, migration assays can also be utilized to determine the potential effect of the identified antibodies, including fragments thereof, on cell migration. Preferably, the effects of the antibodies, or antibody fragments, are tested on the same type of cells as those for host immunization, above. Migration assays are predominantly performed with one of three types of devices: a flow through device, complex fabricated microcell device, or an immunosensor device. In migration assays, a membrane is impregnated with the required reagents, an analyte detection zone is provided in which labeled analyte is bound, and the assay indicia is read. See, for example, Tom et al., U.S. Pat. No. 4,366,241 , and Zuk, EP-A 0 143 574. Migration assay devices usually incorporate reagents which have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances. See, for example, Bernstein, U.S. Pat. No. 4,770,853; May et al., WO 88/08534; and Ching et al., EP-A O 299 428. The sensitivity of migration assays can be reduced by, and must be protected from, the presence of undesirable solid components which block the passage of labeled analyte to the detection zone. Assay sensitivity can also be reduced by the presence of too much liquid sample. The migration assay can be performed by any method known to those skilled in the art, including, for example, those delineated in U.S. Patent Nos. 5,800,999; 6,451 ,531 ; Falk, W., et al. "A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration", J. Immunol. Methods 33:239.(1980); Bacon, et al. Br. J. Pharmacol. 95:966-974 (1988); Seppa et al. J. Cell Biol. 92, 584-588 (1982);

Postlewaith et al. J. Exp. Med. 144, 1188-1203 (1976); the disclosures of each of which are incorporated herein by reference. Using the above approach, markers common to both to cancer and dendritic cells can be identified. To identify molecules involved in migration to lymph nodes, alternate panning for several rounds on immature dendritic cells and metastatic cancer cells specifically enriches for antibodies recognizing molecules involved in cell migration. Such antibodies are useful for cancer diagnostic or therapy as well as for therapeutic applications in autoimmune diseases. Identification of cell surface molecules common to cells which migrate through the lymph system thus allows antibodies to be raised to those molecules. Quantities of those antibodies, or fragments thereof, which target cell surface molecules common to migrating cells can then be produced. Antibodies can be synthesized by those of ordinary skill in the art, using known techniques, in organisms such as bacteria, yeast, mammalian cells, insect cells, and plant cells (Carlson, J. R. and Weissman, I. L., Mol. Cell. Biol., 8:2647-2650, 1988; Trill, J. J., Shatzman, A. R., Ganguly, S. Curr. Opin. Biotechnol. 6:553-560, 1995; Hiatt, A., Cafferkey, R. Bowdish, K. Nature 342: 76-78, (1989)). The antibodies can be tested as described above for their effectiveness in preventing, or inhibiting, migration of cells through the lymph system, in vitro and in vivo. The antibodies thus identified may then be utilized in therapeutic and diagnostic applications. The antibodies may be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulation and administration of the antibodies are known to those skilled in the art and can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. Therapeutic compositions can be administered intravenously or through the nose or lung, preferably as a liquid or powder aerosol (lyophilized). The composition may also be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are well known to those skilled in the art. Dosage formulations are to be determined by the judgment of the medical staff according to the status of the patient's health. Administration of antibodies would be accomplished according to known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical or by sustained release systems. The antibodies would be administered in either a local or systemic manner. For therapeutic uses, agonist antibodies could be used to treat patients suffering from cancer involving migrating cancer cells by targeting, interfering with, and/or ultimately destroying the cells, thereby inhibiting the spread of metastatic cancers throughout the body. By way of example, an antibody identified in accordance with the processes described herein can be chemically linked to a therapeutic agent to provide targeted delivery of the therapeutic agent to metastatic cancer cells or other migrating cells. The therapeutic agent can advantageously be a cancer treatment agent of a type known to those skilled in t e art. Methods for producing such antibody-therapeutic compounds for cancer treatment can be found, for example, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet," Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein (1991) Nature 349:293-299. Suitable toxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin and phospholipase enzymes. See, generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985). The antibodies could also be used for preventive care, where the antibodies would be administered to a patient with a previous history of cancer, or where a patient's family history would indicate a possibility of cancer occuring. The antibodies, or fragments thereof, can also be utilized in diagnostic applications. For example, the antibody or fragment thereof can be conjugated to detectable markers or used as primary antibodies with secondary antibodies that are conjugated to detectable markers. These antibodies would be useful as diagnostic tools to detect the presence, and quantity, of cells that migrate through the human lymph system, including metastatic cancer cells. Detectable markers, include radioactive and non-radioactive labels and are well-known to those with skill in the art. Common non-radioactive labels include detectable enzymes such as horseradish peroxidase, alkaline phosphatase and fluorescent molecules. All of these markers would be acceptable for in vivo use. Any suitable technique can be used to identify the cell surface molecules common to multiple types of migrating cells with which the antibody interacts. In accordance with one known technique, the antibodies (or scFvs thereof) are used to immunoprecipitate the cell surface molecules from lysates prepared from the microsomal fraction of the migrating cells. The immunoprecipitated antigens are purified (for example by SDS-PAGE) and identified using known techniques (including, for example, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) or microcapillary reverse-phase HPLC nano- electrospray tandem mass spectrometry (μLC/MS/MS)). Parameters for performing these various techniques are within the purview of one skilled in the art. The following non-limiting examples are provided to illustrate the methods described herein.

EXAMPLE 1 Library Construction Human monocytes are matured in vitro for 5 days using GM-CSF and IL-4 and conventional techniques. Mice are immunized with the immature dendritic cells by injecting between 1 ,000,000 and 10,000,000 cells. The mice are tested periodically for the production of antibodies to the migrating cells. Standard FACS staining is used to detect binding. Once antibodies are detected the spleen is harvested. Fab phage display libraries are then constructed by isolating splenic RNA from the mice immunized with migrating cells. The Fab phase display libraries are constructed using a method of Patent No. 5,766,905, the disclosure of which is incorporated herein by reference.

EXAMPLE 2 Panning Phagemid particles displaying Fab are panned for several rounds as follows:

1. Phage are incubated on immature dendritic cells plated on 75 cm² flasks for 2 hours at 37°C followed by washes with phosphate buffers saline ("PBS"). The captured phage particles are then eluted by addition of 3 ml of acid elution buffer (0.1 N HCI, pH adjusted to 2.2 with glycine, plus 1 μg/ml bovine serum albumin ("BSA")). After a 10 minute incubation period at 25°C, the buffer is neutralized with 80 μl of 2M Tris base, at a pH of 10.5. The eluted phage are then amplified in E. coli using known techniques for the next round of panning. 2. Phage are incubated on a metastatic cancer cell line following the protocol described above in Step 1.

3. Binders to common antigens are subtracted by panning. The phage are incubated on negative adsorber cells for 2 hours at 37°C. followed by washes with phosphate buffers saline ("PBS"). The unbound phage found in the supernatant are removed and amplified in E. coli using known techniques. EXAMPLE 3

Fluorescence Activated Cell Sorting ("FACS") Clones, obtained after several rounds of panning, are tested for binding to both immature dendritic cells and the metastatic cancer cell line. These clones are also tested for the absence of binding to negative adsorber cells. The clones are grown overnight and the culture supernatant is used. Optionally, the antibodies can be purified using an anti-Fab column. Approximately 0.5-1x10⁶ cells are incubated with the supernatant for 20 minutes in FACS buffer (PBS containing 0.1% BSA and 0.01% NaN3). The cells are washed twice in the FACS buffer, then incubated for 15 minutes with a fluorochrome-conjugated anti- mouse Fab. The bound antibody is detected using a FACS Calibur (Becton, Dickinson and Company, San Jose, CA, USA) . The clones are tested to verify binding to both immature dendritic cells and metastatic cancer cells.

EXAMPLE 4 Migration Assay The potential effect of the identified Fabs on cell migration is tested based on the method described by Falk, W. et al., "A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration", J. Immunol. Methods 33:239 (1980), the disclosure of which is incorporated herein by reference. To test for an effect of the antibody on dendritic cell migration, immature dendritic cells are at a concentration of one million per ml in chemotaxis medium (RPMI supplemented with 2mM glutamine, 20 mM HEPES (N-2-Hydroxyethyl-piperazine-N'-2-ethanesulfonic acid) and 0.5% BSA). 25 μl of the cell suspension is placed in a 96-well chemotaxis chamber with a 5 μm pore polycarbonate membrane (Neuroprobe, Gaithersburg, MD) and 30 μl of medium in the presence, or absence, of varying concentrations of purified antibody. The plates are incubated for 90 minutes at 37°C in a humidified incubator, and cells that remain on the topside of the membrane are removed by gentle washing. The migrating cells attached to the bottom of the filter are fixed in 2.5% glutaraldehyde and stained with 0.1% toluidine blue. The migrating cells are quantified using a light microscope. Similarly, the effect of antibody on migration of metastatic cancer cells is tested. For these assays, a fibroblast conditioned medium is placed in the lower chamber as a chemoattractant. The fibroblast conditioned medium is obtained by a 24 hour incubation of NIH-3T3 cells with 50 μg/ml ascorbic acid in serum- free DME media. 25 μl of the cell suspension is placed in a 96-well chemotaxis chamber with a 5 μm pore polycarbonate membrane (Neuroprobe) and 30 μl of medium in the presence, or absence, of varying concentrations of purified antibody. The plates are incubated for 90 minutes at 37°C in a humidified incubator, and cells that remain on the topside of the membrane are removed by gentle washing. The migrating cells attached to the bottom of the filter are fixed in 2.5% glutaraldehyde and stained with 0.1% toluidine blue. The migrating cells are quantified using a light microscope. While the above description contains many specific details of methods in accordance with this disclosure, these specific details should not be construed as limitations on the scope of the disclosure, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that all within the scope and spirit of the disclosure as defined by the claims appended hereto.

Claims

WHAT IS CLAIMED IS: 1. A method comprising: creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system; and identifying members of the library which bind to two different types of human cells which migrate through the human lymph system.

2. A method as in claim 1 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with immature dendritic cells, metastatic cancer cells, or both.

3. A method as in claim 1 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with both immature dendritic cells and metastatic cancer cells.

4. A method as in claim 1 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with metastatic cancer cells.

5. A method as in claim 1 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with metastatic cancer cells selected from the group consisting of KM12L4a colon cancer cells, MDAMB435 breast cancer cells, and PC3 prostate cancer cells.

6. A method as in claim 1 , wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises panning members of the library against human dendritic cells.

7. A method as in claim 1 , wherein the step of identifying members of

the library which bind to two different types of human cells which migrate through

the human lymph system comprises panning members of the library against metastatic cancer cells selected from the group consisting of KM12L4a colon cancer cells, MDAMB435 breast cancer cells, and PC3 prostate cancer cells.

8. A method as in claim 1 , wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises panning members of the library against metastatic cancer cells.

9. A method as in claim 1 , wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises at least one round of panning the members of the library against human dendritic cells followed by at least one round of panning the members of the library which bind to human dendritic cells against metastatic cancer cells.

10. A method as in claim 1 , wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises at least one round of panning the members of the library against metastatic cancer cells followed by at least one round of panning the members of the library which bind to metastatic cancer cells against human dendritic cells.

1 1. A method as in claim 1 further comprising the step of performing

negative selection panning to eliminate antibodies that bind to receptors that are

present on cells which do not migrate through the human lymph system.

12. An antibody identified in accordance with the method of claim 1 which binds cell surface molecules found on cells which migrate through the human lymph system.

13. A method for preventing migration of metastatic cancer cells by administering antibodies identified in accordance with the method of claim 1 to a subject.

14. A method for diagnosing the presence of metastatic cancer cells by administering antibodies identified in accordance with the method of claim 1 in conjunction with a detectable marker.

15. An antibody identified in accordance with the method of claim 1 which binds ceil surface molecules found on metastatic cancer cells.

16. A method of identifying cell surface molecules on human cells which migrate through the human lymph system comprising: creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system; identifying members of the library which bind to two different types of human cells which migrate through the human lymph system; and determining the cell surface molecules to which the identified antibodies bind.

17. A method as in claim 16 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with immature dendritic cells, metastatic cancer cells, or both.

18. A method as in claim 16 wherein the step of creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with at least one type of human cells which migrate through the human lymph system comprises creating a library of nucleic acid sequences encoding antibodies produced by a mammal immunized with metastatic cancer cells selected from the group consisting of KM12L4a colon cancer cells, MDAMB435 breast cancer cells, and PC3 prostate cancer cells.

19. A method as in claim 16, wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises panning members of the library against human dendritic cells, metastatic cnacer cells or both.

20. A method as in claim 16, wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises panning members of the library against human dendritic cells followed by panning members of the library which bind to human dendritic cells against metastatic cancer cells.

21. A method as in claim 1 , wherein the step of identifying members of the library which bind to two different types of human cells which migrate through the human lymph system comprises panning members of the library against metastatic cancer cells followed by panning members of the library which bind to metastatic cancer cells against human dendritic cells.

22. A method for treating cancer cells by administering one or more antibodies identified in accordance with the method of claim 1 to a subject.

23. A method as in claim 22 wherein a cancer treating therapeutic is conjugated to the antibody.