WO2007022424A2 - Laminin receptor targeting method for delivering a toxic agent inside a cell - Google Patents

Abstract

The present invention is based, at least in part, on the discovery that laminin binding protein p67 (LBP/p67) is internalized, and thus able to deliver toxic agents into cells, e.g. t cancer cells which express LBP/p67, to thereby induce cytotoxicity. In another embodiment, a cell expressing LBP/p67 is exposed to a complex comprising a toxic agent and an LBP/p67 targeting agent, e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody, or a fragment thereof. In one embodiment, the toxic agent is conjugated to the LBP/p67 targeting agent. The LBP/p67 targeting agent directs the toxin to the cancer cell expressing LBP/p67 and the complex is internalized by the cell, resulting in selective cytotoxicity of the cancer cell.

Description

LAMININ RECEPTOR TARGETING METHOD FOR DELIVERING A TOXIC AGENT INSIDE A CELL

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application Serial No. 60/709,178 filed August 17, 2005, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Immunoconjugates are chimeric molecules in which a molecule with an intended function, termed the "effector molecule," is coupled to an antibody which targets the conjugate, e.g., to a cell expressing a particular receptor or antigen recognized by the targeting molecule. Immunotoxins are a form of immμnoconjugate in which a toxin, usually truncated, serves as the effector molecule, and is fused to an antibody, or a portion of the antibody that serves as the targeting moiety.

Immunotoxins are often specific for a cell surface receptor. If the receptor is preferentially expressed on the surface of tumor cells, the immunotoxin can serve as an efficient cancer therapeutic. Immunotoxins are a potentially powerful approach for targeting a population of cells bearing a specific antigen. Examples in which growth factors, cytokines, hormones or antibody domains have been fused to toxins have been described and bacterially expressed molecules have shown anti-tumor effects in vitro and in vivo. Since many tumors are not sensitive to the existing immunotoxins, the search continues for new antigens with tumor specific or tumor enhanced expression and new conjugates that target these antigens.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that laminin binding protein p67 (LBP/p67) is internalized, and thus able to deliver toxic agents into cells, e.g. _t cancer cells which express LBP/p67, to thereby induce cytotoxicity upon internalization. In one aspect of the invention, a cell expressing LBP/p67 is exposed to a complex comprising a toxic agent and an LBP/p67 targeting agent, e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody, or a fragment thereof. In one embodiment, the toxic agent is conjugated to the LBP/p67 targeting agent. The LBP/p67 targeting agent directs the toxin to the cancer cell expressing LBP/p67 and the complex is internalized by the cell, resulting in selective cytotoxicity of the cancer cell. Internalization of the toxin conjugate or complex may be detected by any method known in the art, e.g., via detection of labeled complex or conjugate or by carrying out a cell death assay.

A cell expressing LBP/p67 may be targeted indirectly or directly by the toxic agent. For example, if a cell expressing LBP/p67 is indirectly targeted, the cell is exposed to a complex comprising an LBP/p67 targeting agent, e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody or fragment thereof, and a conjugate comprising (i) a toxic agent and (ii) a secondary binding agent, wherein the secondary binding agent is specific for the LBP/p67 targeting agent, e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody, or fragment thereof, under conditions that allow LBP/p67- mediated internalization of the complex or conjugate .

The invention also provides methods for inducing cancer cell death by exposing a cancer cell to a complex comprising (i) a toxic agent and (ii) an LBP/p67 targeting agent, or a fragment thereof, wherein the complex becomes internalized by the cancer cell. In another aspect, the invention provides methods for treating cancer in a subject, e.g., a mammal, by administering to the subject (i) a toxic agent and (ii) an LBP/p67 targeting agent, or a fragment thereof, that is specific for LBP/p67, wherein the complex becomes internalized by the cancer cell.

In one embodiment, the targeting agent includes LBP/p67 specific antibodies and LBP/p67 specific ligands. The antibodies used in the methods of the invention, include, but are not limited to, human antibodies, humanized antibodies, chimeric antibodies, and non-human antibodies. Any antibody fragment that is capable of binding LBP/p67 is understood to be included in the methods of the invention. For example, V_h, V₁, Fab, F(ab')₂, Fabc, Fd, scFv, dsFv, and Fv antibody fragments may be vised in the present invention. In one embodiment, the murine antibody MLuC5 is used in the methods of the invention. In another embodiment, an LBP/ρ67 targeting agent includes, but is not limited to, a synthetic peptide that includes AA 2091-2108 of the laminin A chain that binds to LBP/p67 (CSRARKQAASIKVAVSADR SEQ ID NO: 1), a nine amino acid sequence CDPGYIGSR (peptide 11 ; SEQ ID NO: 2) within the beta chain of laminin 1 that binds to LBP/p67, the YIGSR pentapeptide LBP/p67 binding sequence from the beta chain of laminin 1 (SEQ ID NO: 3), cellular prion protein (PrP), cellular prion protein together with heparin sulfate proteoglycan, synthetic peptides comprising PrPLRPbdl (amino acids 144-179 of PrP; DYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDC SEQ ID NO: 4) and/or PrPLRPbd2 (amino acids 53-93 of PrP; GGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG SEQ ID NO: 5), PrPLRPbd2 together with heparin-sulfate, ribosomal S21 protein, and Sindbis virus.

In a specific embodiment, the toxin complex is a recombinant fusion protein. In another embodiment, the toxic agent and the LBP/p67 targeting agent e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody, are linked by a linker, e.g., they are covalently linked. In another embodiment, the toxin and the targeting agent are directly linked, e.g., without the use of a linker. The linker may be a cleavable linker that becomes cleaved upon internalization into the cell. Cleavable linkers include, but are not limited to, disulfide linkers, acid cleavable linkers, photolabile linkers, and peptide linkers. The toxin component of the complexes of the invention may be a radioisotope or a chemotherapeutic, e.g., a chemical toxin, that is toxic to a cell upon internalization. Chemical toxins may be derived from, for example, bacteria, plants, or fungi. Bacterial toxins that may be targeted to cancer cells include Pseudomonas exotoxin and diphtheria toxin, which are well suited to forming recombinant single-chain or double-chain fusion toxins or derivatives thereof. Derivatives include truncated toxins and toxins with additional targeting sequence, e.g., the KDEL amino acid sequence (SEQ ID NO: 6). Additional bacterial toxins include, for example, botulinum. Plant toxins include, for example, ribosome inactivating proteins (RIPs), e.g., saporin, ricin, abrin, pokeweed antiviral protein, gelonin, momordin, andbryodin. Fungal toxins, i.e., mycotoxins include clavin or trichothecene. The toxins used in the invention also include hybrid toxins, e.g., the ricin A chain-diptheria toxin A chain hybrid. Protoxins may also be used in the invention.

The above features and many other attendant advantages of the invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE IA-D. LBP/p67 expression on various breast carcinoma cell lines. Serial dilutions of MLuC5 antibody (solid lines) or IgM isotype control (dotted lines) were reacted with MCF-7, MDA-MB-231, or SK-BR-3 cells in (A) CELISA or (B) flow cytometry. (C) Overlay of flow cytometry histograms for selected MLuC5 dilutions and a single concentration of mouse IgM isotype control (1.8e-9 M) on MCF-7 cells. (D) Overlay of flow cytometry histograms at 1.8e-9M MLuC5 or IgM isotype control on all 3 breast carcinoma cell lines. A smoothing factor of 5 was applied to the raw data using FCS Express v2 in Panels (C) and (D). FIGURE 2A-C. ErbB2 expression on various breast carcinoma cell lines. Serial dilutions of mouse IgG monoclonal antibodies (A) Nl 2 or (B) N24 were reacted with MCF-7, MDA-MB-231, or SK-BR-3 cells in (A & B) CELISA. (C) Serial dilutions of Nl 2 antibody were tested using flow cytometry.

FIGURE 3A-B. MUC-I expression on various breast carcinoma cell lines. Serial dilutions of mouse IgM monoclonal antibody 1.B.763 were reacted with MCF-7, MDA-MB-231, or SK-BR-3 cells in (A) CELISA and (B) flow cytometry. Mouse IgM isotype control (not shown) was used as in Figure IB.

FIGURE 4A-D. Treatment of various breast carcinoma cell lines with MLuC5 and anti-IgM:saporin in vitro. (A) MCF-7, (B) MDA-MB-231, and (C) SK-BR-3 cells were treated in an indirect toxin-dependent cytotoxicity assay with MLuC5 antibody and anti-IgM:saporin incubated together; or with either MLuC5 or anti-IgM:saporin alone as controls. The percent remaining viability was assessed with the chemiluminescent, ATP-reactive substrate, TiterGlo after 4d culture. In (D) remaining viability for each of the 3 cell lines treated with MLuC5 + anti-IgM:saporin is plotted together on a log scale to highlight differences in apparent dose-response. FIGURE 5A-C. Indirect toxin-dependent cytotoxicity as a function of primary MAb and secondary (immunotoxin) concentrations. (A&B) SK-BR-3 or (C) MDA- MB-231 cells were co-incubated with varying concentrations of (A) anti-ErbB2 (N24) IgG MAb + anti-IgG:saporin or (B&C) anti-LBP/p67 (MLuC5) + anti-IgM:saporin for 5d. Viability was assessed using TiterGlo.

FIGURE 6A-D. Treatment of various breast carcinoma cell lines with anti- MUC-I (IgM) and anti-IgM:saporin in vitro. (A) MCF-7, (B) MDA-MB-231, (C) SK- BR-3, and (D) FHs74Int embryonic colon epithelial cells (non-cancer control) were treated in an indirect toxin-dependent cytotoxicity assay with 1.B.763 antibody and anti- IgM:saporin incubated together; or with 1.B.763 alone. Viability was assessed with the chemiluminescent, ATP-reactive substrate, TiterGlo after 4d culture.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that cell surface laminin binding protein p67 (LBP/p67) can be internalized and, thus, by targeting LBP/p67, toxic agents may be delivered into the cancer cells to induce cytotoxicity in these cells.

It has been shown that LBP/p67 is expressed at higher levels on certain cancer cells. Accordingly, targeting cancer cells through antibody-mediated cytotoxicity has been suggested. Therefore, the present invention provides compositions and methods for the selective targeting of cancer cells using complexes comprising an LBP/p67 binding agent and a toxin that is active upon internalization into the cell.

The present invention also provides methods and compositions for inducing cell death of a cancer cell and for the treatment of cancer in a subject using complexes comprising an LBP/p67 specific binding agent and a toxin. Any cancer in which the cancer cells overexpress LBP-p67 may be treated by the methods of the invention. For example, cancers that overexpress LBP-p67 include, but are not limited to, breast cancer, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, gastric cancer, laryngeal squamous cell cancer, lymphoma, ovarian cancer, pancreatic endocrine cancer, prostate cancer, thyroid cancer, and urothelial cancer. Accordingly, the present invention is directed to a method for delivering a toxic agent inside a cancer cell expressing LBP/p67 comprising exposing a cell expressing LBP/p67 to a complex comprising a toxic agent and an LBP/p67 targeting agent, e.g., an LBP/p67 specific ligand or an LBP/p67 specific antibody, or a fragment thereof, under conditions that allow for LBP/p67-mediated internalization of the complex. In a specific embodiment, the toxic agent is conjugated to the LBP/p67 targeting agent, e.g., either chemically conjugated or conjugated through recombinant means. The LBP/p67 targeting agent directs the toxin to the cancer cell expressing LBP/p67 and the complex is internalized by the cell, resulting in selective cytotoxicity of the cancer cell. Internalization of the toxin complex may be detected by any method known in the art, e.g., via detection of labeled complex or by carrying out a cell death or cell viability assay. In one embodiment, a receptor-mediated endocytosis assay is used to detect internalization. For example, endocytosis may be assessed by quantifying the uptake of Tf or aggregated human IgG as described in Tse, S. M., W. Furuya, et al. (2003J J Biol Chem 278(5): 3331-8, the contents of which are expressly incorporated herein by reference. Additional methods for detecting internalization are described in, for example, GeIi, M. I. and H. Riezman (1998) J Cell Sci Hl (Pt 8): 1031-7; Keyel, P. A. and L. M. Traub (2004) Dev Cell 7(3): 283-4; Liu, J. and J. I. Shapiro (2003) Biol Res Nurs 5(2): 117-28; Nichols, B. J. and J. Lippincott-Schwartz (2001) Trends Cell Biol 11(10): 406-12; Schmid, S. L. (1992) Bioessavs 14(9): 589-96; and Smythe, E. (2003) Biochem Soc Trans 31(Pt 3): 736-9, the contents of which are expressly incorporated herein by reference. Cells may be targeted directly by the complex comprising an LBP/p67 targeting agent and a toxin, or indirectly. ^"Where the cell is targeted indirectly, a complex comprising a primary targeting agent, e.g., an LBP/p67 specific antibody or an LBP/p67 specific ligand, is conjugated to a toxin and a secondary binding agent. The secondary binding agent binds to the primary targeting agent and the primary targeting agent delivers the secondary binding agent-toxin conjugate to the cell. Once the cell is targeted, internalization of the complex results in cytotoxicity. While administration of ∑ direct immunotoxin is a one step procedure, indirect immunotoxins allow a wide range of unmodified primary targeting agents to be evaluated, e.g., in a high throughput manner. For example, a candidate targeting agent may be evaluated for the ability to be internalized by LBP/p67. Examples of LBP/p67 targeting agents include any protein or composition that binds to LBP/p67. In one embodiment, the LBP/p67 targeting agent is an LBP/p67 specific antibody. In another embodiment, the LBP/ρ67 targeting agent is an LBP/p67 specific ligand. In a particular embodiment, the LBP/p67 targeting agent is a humanized or a fully human antibody. Fragments of anti-LBP/p67 antibodies may also be used, e.g., a Fab or an ScFv may be conjugated to a toxin.

Any toxic agent that has a toxic effect on a cell through internalization may be used in the invention. For example, chemical toxins may be derived from plants, bacteria, fungi, or may be chemically synthesized, as described in detail herein. Toxic agents also include radionuclides. In one embodiment, a chemical toxin and a radionuclide may administered simultaneously. The same LBP/p67 targeting agent can be armed with both radionuclide and toxin, or separate LBP/p67 targeting agents can be armed with radionuclide and toxin. Where separate LBP/p67 ligands armed with radionuclide and toxin are used, these may be administered simultaneously or sequentially.

In any treatment regimen, the toxic complexes of the invention may be administered to a subject either singly or in a cocktail containing additional therapeutic agents, compositions, or the like, including, but not limited to, immunosuppressive agents, tolerance-inducing agents, potentiators and side-effect relieving agents. General Definitions

The following definitions are provided for clarity and illustrative purposes only, and are not intended to limit the scope of the invention.

The term "LBP/p67" or "laminin binding protein p67," include variants, isoforms and species homologs of human LBP/p67. The complete nucleotide and amino acid sequences of human LBP/pό7 are included in Genbank accession numbers GI: 186840, GI:250126, and GL19483807, the contents of which are incorporated herein by reference. LBP/p67 is also referred to as Laminin Receptor 1, Laminin Receptor, LAMRl, LAMBR, Ribosomal Protein SA (RPSA), 67'KD, and 67LR, and is located at gene map locus 3p21.3. The term "internalization" refers to the delivery of a molecule inside of a cell. Assays for determining internalization of a toxin complex of the invention are well- known in the art. For example, internalization of a toxin complex can be determined by assessing cell death. The term "toxin" or "toxic agent," as used interchangeably herein, includes any agent that is toxic to a cell upon internalization. The term "toxic" refers to any adverse effect caused by a toxic agent including, e.g., cell death. For example, toxic agents include, but are not limited to chemotherapeutic agents, e.g., chemical toxins, and radionuclides. Chemotherapeutic agents include those that are derived from, for example, plants, bacteria, or fungi. The invention includes derivatives of these toxins, including modified and mutated forms of toxins that retain cytotoxic properties when targeted to cells of interest.

Chemotherapeutic agents derived from plants include, for example, ribosome inactivating proteins (RIPs), e.g., saporins. RIPs are potent inhibitors of eukaryotic protein synthesis that have been isolated from plants and some bacteria. The N- glycosidic bond of a specific adenine base is hydrolytically cleaved by RIPs in a highly conserved loop region of the 28S rRNA of eukaryotic ribosomes, thereby inactivating translation and resulting in apoptotic cell death (Porowska, H, et al. Neoplasma, 2002; 49(2); 104-109; Bennett, R, J Histochem Cytochem, 2001; 49(1): 67-77; Gauczynski, S, et al. Embo J, 2001; 20(21): 5863-5875). Plant RIPs have been divided into two types (see Stirpe et al., (1986) FEBS Lett., 195(1, 2):l-8). Type I proteins each consist of a single peptide chain having ribosome-inactivating activity, while Type II proteins each consist of an A-chain, essentially equivalent to a Type I protein, disulfide-linked to a B- chain having cell-binding properties. Gelonin, dodecandrin, tricosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP), barley ribosome-inactivating protein (BRIP), pokeweed antiviral proteins (PAPs), saporins, luffins, and momordins are examples of Type I RIPS; whereas ricin and abrin are examples of Type II RIPs. Ribosome inactivating proteins (RIPs) are described in, for example, U.S. Patent Application Publication No. 20050054835, the contents of which are incorporated herein by reference. Chemotherapeutic agents derived from bacteria include, for example, Diptheria toxin and Pseudomonas exotoxin and derivatives thereof, e.g., derivatives wherein the C- terminal end contains the native sequence of residues 609-613 (REDLK) (SEQ ID NO: 7), derivatives containing additional targeting sequence, e.g., KDEL (SEQ IDNO: 6) and REDL (SEQ ID NO: 8), and repeats of these sequences, and derivatives comprising modifications which may include, but are not limited to, elimination of domain Ia, various amino acid deletions in domains Ib, II and m, and single amino acid substitutions. See, e.g., U.S. Patent Application Publication No. 20040018203, U.S. Pat. Nos. 5,854,044; 5,821,238; 5,602,095, PCT Publication No. WO 99/51643, and Siegall, et al., J. Biol, Chem. 264:14256-14261(1989)). An additional bacterial toxin includes botulinum. Fungal toxins, i.e., mycotoxins, include, for example, clavin and trichothecene. The invention also includes hybrid toxins including, for example, ricin A chain-diptheria toxin A chain toxins. Various toxins are described in, for example, Fracasso, G., et al. Mini Rev Med Chem 4.5 (2004): 545-62 and Bondy, G. S., and J. J. Pestka. J Toxicol Environ Health B Crit Rev 3.2 (2000): 109-43.

Toxins that may be used in complexes of the invention also include protoxins, e.g., toxins that become active once inside the cell, e.g., by certain chemical or enzymatic modifications of their structure. For example, a protoxin includes a toxin containing a disulfide bonded loop with a trypsin-sensitive site (O'Hare, M., et al. (1990) FEBS Lett 273.1-2: 200-4) or a factor Xa cleavage site (Westby, M., et al. (1992) Bioconjug Chem 3.5: 375-81).

Examples of toxic agents also include chemical toxins that reduce or inhibit the growth or spread of cancer cells and tumors. Examples of chemotherapeutic agents are known in the art and include, but are not limited to, taxane derivatives, anthracycline derivatives and topoisomerase inhibitors, busulphan, cisplatin, cyclophosphamide, methotrexate, daunorubicin, doxorubicin, melphalan, vincristine, vinblastine and chlorambucil and those described in, for example, U.S. Patent Application Publication No. 20050048070.

The term "radioisotope" or "radionuclide," as used herein, is an unstable isotope of an element that decays or disintegrates spontaneously, emitting radiation. Examples of radionuclides include ²¹²Bi, ¹³¹1, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re. Therapeutic radionuclides are described in Goldsmith SJ, Kostakoglu LA, Somrov S, Palestro CJ. (2004) Thorac Surg Clin. 2004 Feb;14(l):95-112; Kassis AI, Adelstein SJ. (2005) JNucl Med. Jan;46 Suppll:4S-12S; and Srivastava S, Dadachova E. (2001) SeminNucl Med. Oct;31(4):330-41, the contents of which are incorporated herein by reference. The term "direct targeting" with respect to the targeting of a cell with an

LBP/p67 targeting agent and a toxin, refers to exposing the cell to a complex comprising an LBP/p67 targeting agent and a toxin. In one embodiment, the targeting agent and toxin are conjugated, either through chemical means or recombinant means. Internalization of the toxin results in targeted cytotoxicity. The term "indirect targeting," with respect to the targeting of a cell with an

LBP/p67 targeting agent and a toxin, requires exposing the cell to a complex comprising a primary targeting agent, e.g., an LBP/p67 targeting agent, and a conjugate comprising (i) a toxic agent and (ii) a secondary binding agent. The secondary binding agent is specific for the primary targeting agent, e.g., the LBP/p67 targeting agent (see, for example, Winthrop, MD, DeNardo, SJ, Albrecht, H, et al. Clin Cancer Res, 2003; 9(10 Pt 2): 3845S-3853S and Ardini, E, et al. Cancer Res, 2002; 62(5): 1321-1325). A secondary binding agent can be, for example, anti-IgM. Again, internalization of the toxin results in targeted cytotoxicity.

In the context of the present invention, the term "complex" includes any association between a toxin and an LBP-p67 targeting agent. The toxin and LBP -p67 targeting agent may, in one embodiment, be conjugated.

In the context of the present invention, the term "conjugated" include reference to joining of an LBP-p67 targeting agent to a toxin. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the LBP-p67 targeting agent and the toxin such that there is a covalent or non-covalent linkage formed between the two molecules. In a specific embodiment, "chemical means" refers to reactions by which a linker molecule is covalently bound on one end to a targeting molecule and on the other end to a toxin molecule (see, for example, Kazmin, DA, et al. Biochem Biophys Res Commun, 2003; 300(1): 161-166). The term "conjugated" also includes a direct linkage, i.e., without the use of a linker. Conjugation of an LBP-p67 targeting agent to a toxin may also be through recombinant means to produce a recombinant fusion protein comprising an LBP-p67 targeting agent to a toxin.

The terms "immunotoxin conjugate" or "immunotoxin" include reference to a covalent or non-covalent linkage of a toxin to an antibody. The toxin may be linked directly to the antibody, or indirectly through, for example, a linker molecule.

The terms "recombinant DNA," "recombinant nucleic acid" or "recombinantly produced DNA" refer to DNA which has been isolated from its native or endogenous source and modified either chemically or enzymatically by adding, deleting or altering naturally-occurring flanking or internal nucleotides. Flanking nucleotides are those nucleotides which are either upstream or downstream from the described sequence or sub-sequence of nucleotides, while internal nucleotides are those nucleotides which occur within the described sequence or subsequence.

The term "recombinant means" refers to techniques where nucleotide sequences encoding proteins are isolated, the cDNA sequence encoding the protein identified and inserted into an expression vector. The vector is then introduced into a cell and the cell expresses the protein. Recombinant means also encompasses the ligation of coding or promoter DNA from different sources into one vector for expression of a chimeric protein, constitutive expression of a protein, or inducible expression of a protein.

The terms "recombinant fusion protein" and "recombinantly produced protein" refer to a peptide or protein produced using non-native cells that do not have an endogenous copy of DNA able to express the protein. The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein. Methods of producing recombinant fusion proteins are well known to those of skill in the art. For example, Chaudhary et al., (1989) Nature 339:394; Batra et al., (1990) J. Biol. Chem. 265:15198; Batra et al., (1989) Proc. Natl. Acad. Sci. USA 86:8545; and Chaudhary et al., (1990) Proc. Natl. Acad Sci. USA 87:1066 describe the preparation of various single chain antibody-toxin fusion proteins. The term "linker", as used herein, is a molecule that is used to join two other molecules, e.g., a toxin and a LBP-p67 targeting agent. The linker is capable of forming covalent bonds or high-affinity non-covalent bonds to both molecules. Typically, a linker has no specific biological activity other than to join the molecules or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a linker may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule. Linkers are well-known in the art and are described in detail herein.

The phrase "disulfide bond" or "cysteine-cysteine disulfide bond" refers to a covalent interaction between two cysteines in which the sulfur atoms of the cysteines are oxidized to form a disulfide bond. The average bond energy of a disulfide bond is about 60 kcal/mol compared to 1-2 kcal/mol for a hydrogen bond.

The term "LBP/p67 targeting agent" includes any protein or composition that binds to LBP/ρ67. For example, LBP/p67 targeting agent includes LBP/p67 specific antibodies and LBP/p67 specific ligands, e.g., peptides. In one embodiment, an

LBP/p67 specific antibody may comprise an entire antibody heavy chain and an entire antibody light chain. In another embodiment, the antibody comprises fragments or portions of the antibody heavy and/or light chain genes, as described below. The antibody heavy and light chain genes can be from any type of antibody including, but not limited to, IgG, IgM, IgE, IgA, etc. Thus, the term antibody, as used herein, includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. Furthermore, the term " LBP/p67 specific antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies).

Non-limiting examples of LBP/p67 specific antibodies include MLuC5, MLuC6 (Martignone, S., et al. Clin Exp Metastasis 10.6 (1992): 379-86) (available from Abeam, Inc.), an anti-36kD antibody developed against polypeptides purified on laminin, as described in (Salas, P. J., et al. Biol Cell 75.3 (1992): 197-210), an anti-laminin receptor IgM 8A5.3C3 produced against synthetic laminin receptor peptide LR-I (Leu-23-His), as described in Rahman, A., et al. J Natl Cancer Inst 81.23 (1989): 1794-800; an antibody to LBP/p67 C terminal domain that blocks Sindbis virus binding to mammalian cells, as described in Wang et al., J Virol 66.8 (1992): 4992-5001, an IgM antibody against Staphylococcus aureus laminin-binding protein that crossreacts with LBP/p67, as described in Mota, G. F., et al. Infect Immun 56.6 (1988): 1580-4, and a rabbit polyclonal antibody to 67kD Laminin Receptor Synthetic peptide: SGALD VLQ-MAP (SEQ ID NO: 9) MAP = 'multiple antigen presentation' of peptides coupled to a lysine 'tree'), corresponding to amino acids 2-9 of Human 67kD Laminin Receptor (available from Abeam, Inc.).

When the ligand is an LBP/p67 specific peptide, the ligand may contain the entire peptide that binds to the desired receptor, or may contain only a portion of the peptide. For example, it may be desirable to remove a portion of the peptide that has an undesirable biological activity, or it may be .desirable to remove a portion of the peptide to enable attachment of the toxic agent. The only requirement when a portion of the peptide is used is that the LBP/p67 binding activity of the peptide is retained. The terms "portion" and "fragment" are used herein interchangeably. In addition, a ligand may contain substitutions (including but not limited to conservative and semi-conservative substitutions) as well as additions and insertions to the native ligand's sequence that do not substantially affect the ligand's receptor binding activity.

Non-limiting examples of LBP/p67 specific ligands include, but are not limited to, peptides which include amino acids 2091-2108 of the laminin A chain that binds to LBP/p67 (CSRARKQAASIKVAVSADR SEQ ID NO: 1), as described in Ferrarini et al. J Natl Cancer Inst 88.7 (1996): 436-41, a nine amino acid sequence CDPGYIGSR (peptide 11 ; SEQ ID NO: 2) within the beta chain of laminin 1 that binds to LBP/p67, as described in Landowski, Uthayakumar and Starkey, Clin Exp Metastasis 13.5 (1995): 357-72, the YIGSR pentapeptide LBP/p67 binding sequence from the beta chain of laminin 1 (SEQ ID NO: 3), as described in Iwamoto et al. Science 238.4830 (1987): 1132-4, the cellular prion protein (PrP), as described in Gauczynski et al. Embo J 20.21 (2001): 5863-75, the cellular prion protein together with heparin sulfate proteoglycan, as described in Hundt et al. Embo J 20.21 (2001): 5876-86, synthetic peptides comprising PrPLRPbdl (amino acids 144- 179 of PrP;

DYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDC SEQ ID NO: 4) and/or PrPLRPbd2 (amino acids 53-93 of PrP;

GGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG SEQ ID NO: 5), PrPLRPbd2 together with heparin-sulfate, the ribosomal S21 protein, as described in Sato et al. Biochem Biophys Res Commun 256.2 (1999): 385-90, and the Sindbis virus, as described in Strauss et al. Arch Virol Suppl 9 (1994): 473-84; Tseng et al. Cancer Res 64.18 (2004): 6684-92; and Wang et al. J Virol 66.8 (1992): 4992-5001).

As used herein, "polypeptide", "peptide" and "protein" are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the protein remains functional.

The term "residue" or "amino acid residue" or "amino acid" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "peptide").

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region may be comprised of three or more domains, (e.g., C_HI, C_H2 and Cm)- Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, e.g., LBP/ρ67. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), that consists of a V_H domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883/ The term "antigen-binding portion" of an antibody also encompasses such single chain antibodies. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities {e.g., an isolated antibody that specifically binds LBP/p67 is substantially free of antibodies that specifically bind antigens other than LBP/p67). An isolated antibody that specifically binds LBP/p67 may, however, have cross-reactivity to other antigens, such as LBP/p67 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In that case, the term "humanized" applies.

The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity that have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma derived from a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from_, the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_H and V_L sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

The terms "cancer" and "cancerous" refer to or describe the physiological condition that is typically characterized by unregulated cell growth and/or proliferation. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Cancers that may be treated by the methods of the invention include, but are not limited to, any cancer where LBP/p67 is expressed on cancer cells, including breast cancer, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, gastric cancer, laryngeal squamous cell cancer, lymphoma, ovarian cancer, pancreatic endocrine cancer, prostate cancer, thyroid cancer, and urothelial cancer. The terms "treat", "treatment", and the like refer to curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting a disease or disorder, the symptoms of a disease or disorder, or the predisposition toward a disease or disorder, in a subject. The term "subject" as used herein refers to a mammal (e.g., a rodent such as a mouse or a rat, a pig, a primate, or companion animal (e.g., dog or cat, etc.)). In particular, the term refers to humans.

The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" is implicit and in this context means within an acceptable error range for the particular value.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (herein "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994; among others. Linker Molecules Suitable linkers for use in the conjugates of the invention are well known to those of ordinary skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, acid cleavable linkers, photo-labile linkers, and peptide linkers. A linker may be joined to constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). Linkers may also be pegylated, as described in U.S. Patent Application Publication No. 20040018203, the contents of which are incorporated herein by reference.

Linkers used in the methods of the invention include cleavable linkers, e.g., linkers which are susceptible to cleavage under conditions found in vivo or under certain biological conditions. Readily cleavable linkages can be, for example, linkages that are cleaved by an enzyme having a specific activity (e.g., an esterase, protease, phosphatase, peptidase, and the like) or by hydrolysis. For example, linkers containing carboxylic acid esters and disulfide bonds may be used, where the former groups are hydrolyzed enzymatically or chemically, and the latter are severed by disulfide exchange, e.g., in the presence of glutathione (see U.S. Patent No. 6,730,293).

Other linkers are described in, for example, Groot et al, J. Med. Chem. 42, 5277 (1999); de Groot et al J. Org. Chem. 43, 3093 (2000); de Groot et al, J. Med. Chem. 66, 8815, (2001); WO 02/083180; Carl et al, J. Med. Chem. Lett. 24, 479, (1981); Dubowchik et al, Bioorg & Med. Chem. Lett. 8, 3347 (1998). These linkers include aminobenzyl ether spacers, elongated electronic cascade and cyclization spacer systems, cyclisation eliminations spacers, such as w-amino aminocarbonyls, and a p aminobenzy oxycarbonyl linker.

A linker may include an intermediate self-immolative spacer moiety which spaces and covalently links together the drug moiety, e.g., the toxin and the targeting agent. A self-immolative spacer may be defined as a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a normally stable tripartate molecule, releasing one of said spaced chemical moieties from the tripartate molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the molecule to release the other of said spaced chemical moieties. In one embodiment, the self-immolative spacer is covalently linked at one of its ends to the targeting agent and covalently linked at its other end to the chemical reactive site of the drug moiety, e.g., the toxin, whose derivatization inhibits pharmacological activity, so as to space and covalently link together the protein peptide moiety and the drug moiety into a tripartate molecule which is stable and pharmacologically inactive in the absence of a target enzyme, but which is enzymatically cleavable by such target enzyme at the bond covalently linking the spacer moiety and the protein peptide moiety to thereby effect release of the protein peptide moiety from the tripartate molecule. Such enzymatic cleavage, in turn, will activate the self-immolating character of the spacer moiety and initiate spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety (see U.S. Patent No. 6,214,345). Additional linkers are described in, for example, U.S. Patent Nos. 6,759,509, 6,730,293, and 6,512,101.

If the linker is a peptide, the linker can be from 1 to 150 residues in length, 3 to 100 residues in length, about 3 to 50 or fewer residues in length, about 4 to about 20 amino acid residues in length, about 6 to about 17 residues in length, or about 6 to 10 residues in length. In general, a peptide can be used as long as it does not interfere with the proper folding or activity of the LBP -p67 targeting agent or of the toxin of the conjugate. The effect of the linker on the activity of a toxin or LBP-p67 targeting agent can be readily determined by assaying the binding of the LBP-p67 targeting agent to its target and by assaying the cytotoxicity of the toxin on cells targeted by the targeting agent. Assays for determining the binding capabilities of numerous antibodies and ligands are known in the art. Internalization of the toxin leads to cell death. Assays for determining internalization of the complex are well-known in the art.

Assays for determining tumor penetration by toxin-LBP-p67 targeting agent conjugates are well known in the art, and can be performed, for example, by radiolabeling the toxin or LBP-p67 targeting agent, introducing toxin-LBP-p67 targeting agent conjugate into an animal having a solid tumor (such as a nude mouse into which cells of a human solid tumor have been introduced) and then sectioning and autoradiographing the tumor to determine how far into the tumor the conjugate has penetrated. Conjugation of LBP-p67 Targeting Agent and Toxin The toxin and the LBP-p67 targeting agent may be conjugated by chemical or by recombinant means (see, Rybak et al., (1995) Tumor Targeting 1 : 141). In one embodiment, the targeting and toxin moieties are connected through a linker molecule, as described herein. Chemical modifications include, for example, derivitization for the purpose of linking the moieties to the linker, by methods that are well known in the art of protein chemistry. Suitable means of linking the toxic moiety and the targeting agent to the linker comprise a heterobifunctional coupling reagent which ultimately contributes to formation of an intermolecular disulfide bond between the moieties and the linker. Other types of coupling reagents that are useful in this capacity for the present invention are described, for example, in U.S. Pat. No. 4,545,985. Alternatively, an intermolecular disulfide bond may conveniently be formed between cysteines in each moiety which occur naturally or which are inserted by genetic engineering. The means of linking moieties may also use thioether linkages between heterobifunctional crosslinking reagents or specific low pH cleavable crosslinkers or specific protease cleavable linkers or other cleavable or noncleavable chemical linkages. The means of linking may also comprise a peptidyl bond formed between moieties which are separately synthesized by standard peptide synthesis chemistry or recombinant means.

Possible genetic engineering modifications of the different proteins or portions of the conjugates include combination of the relevant functional domains of each into a single chain multi-functional biosynthetic protein expressed from a single gene derived by recombinant DNA techniques (see, for example, PCT published application WO/88/09344). Furthermore, recombinant DNA techniques can be used to link the recombinant toxin and the targeting agent to the linker. Accordingly, the conjugates can comprise a fused protein beginning at one end with the toxin and ending with the targeting agent at the other.

The procedure for attaching a toxin to an LBP-p67 targeting agent will vary according to the chemical structure of the toxin. For example, LBP-p67 antibodies contain a variety of functional groups; e.g., sulfhydryl (--S), carboxylic acid (COOH) or free amine (--NH₂) groups, which are available for reaction with a suitable functional group on a toxin. Additionally, or alternatively, the targeting agent or toxin can be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules.

A bifunctional linker having one functional group reactive with a group on the toxin, and another group reactive with an LBP-p67 targeting agent, can be used to form a desired conjugate. Alternatively, derivatization may involve chemical treatment of the toxin or targeting agent, e.g., glycol oxidation of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups. The free aldehyde groups on the targeting agent may be reacted with free amine or hydrazine groups on the toxin to bind the toxin thereto (See U.S. Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groups on antibodies or antibody fragments, are also known (See U.S. Pat. No. 4,659,839).

Where the LBP-p67 targeting agent and the toxin molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). However, in specific embodiments, the linkers will be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.

Many procedures and linker molecules for attachment of various compounds including toxins are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987), which are incorporated herein by reference. In particular, production of various immunotoxin conjugates is well-known within the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann, Science, 252: 1657 (1991), Youle and Nevelle, Proc. Natl. Acad. Sci., 77(9):5483-5486 (1980), and U.S. Pat. Nos. 4,545,985 and 4,894,443, which are incorporated herein by reference. See also, e.g., Birch and Lennox, Monoclonal Antibodies: Principles and Applications, Chapter 4, Wiley-Liss, New York, N. Y. (1995); U.S. Pat. Nos. 5,218,112, 5,090,914; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996); see, for example, Wong, S. S., Ed., Chemistry of Protein Conjugation and Cross- Linking, CRC Press, Inc., Boca Raton, FIa. (1991). Methods of producing recombinant fusion proteins are well known to those of skill in the art. For example, Chaudhary et al., (1989) Nature 339:394; Batra et al., (1990) J. Biol. Chem. 265:15198; Batra et al., (1989) Proc. Natl. Acad. Sci. USA 86:8545; and Chaudhary et al., (1990) Proc. Natl. Acad Sci. USA 87:1066 describe the preparation of various single chain antibody-toxin fusion proteins.

In general, producing fusion proteins involves separately preparing the DNA encoding the targeting agent, e.g., the antibody, antibody fragment (e.g., the Fv light and heavy chains), or ligand, the DNA encoding the linker, and the DNA encoding the toxin to be used. The sequences are then combined in a plasmid or other vector to form a construct encoding the particular desired chimeric protein. Another approach involves inserting the DNA encoding the particular targeting agent, e.g., the antibody fragment (e.g., the Fv region) or ligand into a construct already encoding the desired toxin and the linker.

Nucleic acid sequences encoding conjugates of the present invention can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al., Tetra. Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862 (1981), e.g., using an automated synthesizer as described in, for example, Needham- VanDevanter, et al. Nucl. Acids Res. 12:6159-6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Nucleic acid sequences encoding conjugates can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VoIs. 1-3, Cold Spring Harbor Laboratory (1989)), Berger and Kimmel (eds.), GUIDE TO MOLECULAR CLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)), or Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, NY (1987). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, NJ.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill. Mammalian cells have been used to express and secrete hybrid molecules such as antibody-cytokines (Hoogenboom et al., (1991) Biochem. Biophys. Acta 1096:345; Hoogenboom et al., (1991) MoI. Immunol. 28:1027) and antibody-enzyme (Casadei et al., (1990) Proc. Natl. Acad. Sci. USA 87:2047; Williams et al., (1986) Gene 43:319). In part, immunogenicity of foreign proteins is due to incorrect glycosylation patterns present on recombinant proteins. Therefore, eukaryotic cell lines are preferred over prokaryotic cells as the expressed proteins are glycosylated. Human-derived cell lines are particularly preferred in that these cells incorporate a sialic acid as the terminal glycoside. Cell lines such as the hamster CHO and BHK, as well as the HEK-293 human fibroblast line have been used to express recombinant human proteins. Other genetic engineering modifications include deletions of functionally unnecessary domains to reduce the size of the protein or to modify other parameters which facilitate production or utility, such as sequence changes to affect the solubility (e.g., cysteine to serine) or glycosylation sites. One skilled in the art would appreciate that many additional well known chemical and genetic modifications of proteins may be advantageously applied to any protein. Once expressed, the recombinant fusion proteins of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer- Verlag, N. Y. (1982)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies of this invention. See, Buchner, et al., Anal. Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 (1991); Huse, et al., Science 246: 1275 (1989) and Ward, et al., Nature 341:544 (1989), all incorporated by reference herein. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of complexes or conjugates of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) complexes or conjugates of the invention. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the complexes or conjugates of the invention may be used in combination with a additional therapeutic agents such as, for example, immunosuppressants, tolerance-inducing agents, potentiators, and side-effect relieving agents.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, pptassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N₉N'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. For intravenous administration, typical pharmaceutical compositions are about

0.01 to 100 mg per patient per day. Dosages from 0.1 up to about 1000 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a tumor or an organ within which a tumor resides.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A "therapeutically effective dosage" of a ligand of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, a composition of the present invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. PatentNos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, the complexes or conjugates of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery {see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et at, (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); pi 20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; MX. Laukkanen (1994) FEBS Lett. 346:123; JJ. Killion; IJ. Fidler (1994J Immunomethods 4:273.

The present invention is further illustrated by the following examples which should not be construed as limiting.

EXAMPLES

Example 1: Internalization of Cell Surface LBP/p67 Materials and Methods CELL LINES, CULTURE, AND PROPAGATION CONDITIONS: Human breast cancer cell lines SK-BR-3 (ATCC # HTB-30), MCF-7 (ATCC # HTB-22), and MDA-MB-231 (ATCC # HTB-26) were obtained from the American Type Culture collection (ATCC, Manassas, VA). MCF-7 cells were grown in Minimum essential medium Eagle with 2 mM L- glutamine and Earle's BSS (cat.# 10-010-CV, Mediatech Inc., Herndon, VA) supplemented with 10% Fetal Bovine Serum (FBS) (cat.# 35-010-CV, Mediatech Inc., Herndon, VA), IX non-essential amino acids (cat.# 25-025-CI, Mediatech Inc., Herndon, VA), ImM Sodium Pyruvate (cat.# 25-000-CI, Mediatech Inc., Herndon, VA), and lOng/ml insulin (cat.# Sigma 1-0516, Sigma- Aldrich, St. Louis, MO) at 37⁰C in 5% CO₂. Cells were split 1:4-1:6 from flasks that were 95% confluent.

MDA-MB-231 cells were grown in RPMI 1640 without L-glutamine (15-040- CV, Mediatech Inc., Herndon, VA) supplemented with 10% Fetal Bovine Serum (FBS) (cat.# 35-010-CV, Mediatech Inc., Herndon, VA) and 2 mM L-glutamine (25-005-CI, Mediatech Inc., Herndon, VA) at 37°C in 5% CO₂. Cells were split 1:8-1:10 from flasks that were 95% confluent.

SK-BR-3 cells were grown in McCoy's 5A medium (10-050-CV) supplemented with 10% Fetal Bovine Serum (FBS) (cat.# 35-010-CV, Mediatech Inc., Herndon, VA) and 1.5 mM L-glutamine (25-005-CI, Mediatech Inc., Herndon, VA) at 37⁰C in 5% CO₂. Cells were split 1 :2-l :3 from flasks that were 90% confluent.

Propagation: Cells were passaged by aspirating the culture medium and using 2 ml of 0.25% trypsin, 2.2ImM EDTA solution (cat. # 25-053-CI, Mediatech Inc., Herndon, VA) to rinse cells briefly. The solution was aspirated and 2 ml of trypsin- EDTA was added to the cells. The flask was placed flat at room temperature until cells detached (about 1-2 minutes for MCF-7 and MDA-MD-231 cells; about 3 to 5 minutes for SK-BR-3 cells). Detached cells were added to fresh medium at appropriate split ratios. Medium was renewed 2 or 3 times a week.

PRIMARY ANTIBODIES: Anti-Laminin Receptor mouse monoclonal IgM antibody (Clone MLuC5, cat.#MS-259-PABX); and anti-c-erbB-2/HER-2/neu mouse monoclonal IgG antibodies (Clone Nl 2, cat.#MS-301-PABX and Clone N24, cat.#MS- 302-PABX) were obtained from Labvision Corporation (Fremont, CA). Anti-Human Epithelial Membrane Antigen (Clone 1.B.763, cat.# E3414-17) was obtained from United States Biological (Swampscott, MA). CELISA: Target cells were plated at 5,000 cells per well in clear 96-well tissue culture plates (Microtest 96, cat.# 353072, Becton Dickinson Labware, Franklin Lakes, NJ) and cultured for 72 hours at 37⁰C in 5% CO₂. The plates were aspirated on a plate washer (ELX405; BIO-TEK Instruments Inc., Winooski, VT) and 100 μl IX Dulbecco's Phosphate Buffered Saline (IX DPBS) with Ca²⁺ and Mg²⁺ (cat.# 21-030-CV, Mediatech Inc., Herndon, VA) was added by hand and the plate aspirated again. The cells were fixed with 50 μl 4% formaldehyde (cat.# BP-531-500, Fisher Biotech, Fair Lawn, NJ) in IX DPBS with Ca²⁺ and Mg²⁺ for 15 minutes at room temperature. The plates were washed IX with 250 μl IX DPBS without Ca²⁺ or Mg²⁺ (cat.# D5652, Sigma-Aldrich, St. Louis, MO)/0.01% Tween-20 (cat# BP337-500, Fisher Biotech, Fair Lawn, NJ) on a plate washer. 100 μl ELISA buffer (IXDPBS; 4 mg/ml BSA, Fraction V (cat # 2930, EMD Chemicals Inc., Gibbstown, NJ); 0.01% Tween-20) containing 0.1 M glycine was added using a multichannel pipettor and the plate was incubated for 10 minutes. The plates were washed IX with 250 μl lXDPBS/0.01% Tween-20 on a plate washer. Serial dilutions of antibodies were added at 50 μl/well to the corresponding wells of the fixed cell plates. Plates were incubated at room temperature for 2 hours without shaking. The plates were washed 2X with 250 μl lXDPBS/0.01% Tween-20 on a plate washer. 50 μl of a mixture of 1:4000 dilutions of HRP-conjugated goat anti-human kappa and lambda chains (cat.# 2060-05 and 2070-05, Southern Biotechnology Associates Inc., Birmingham, AL) in 4 mg/ml BSA/lXDPBS/0.05% Tween-20 was added to all wells. The plates were incubated for 45 minutes at room temperature. The plates were washed twice with 250 μl lXDPBS/0.01% Tween-20 on a plate washer, the plates were flipped and washed twice again. 50 μl of 3, 3', 5, 5'-tetramethylbenzidine (TMB) ultra sensitive substrate (cat.# TMBUS-1000, Moss Inc., Pasadena, MD) were added to all wells and the plates allowed to develop for 2-5 minutes. The reaction was stopped with 100 μl of 0.18 M sulfuric acid. Plates were read at 450nm on a Wallac Victor2 1420 multilabel counter (Perkin Elmer Life and Analytical Sciences, Boston, MA).

FLOW CYTOMETRY: Target cell culture media was aspirated from adherent target cells that had been grown to 80-90 % confluence in 150 cm² sterile tissue culture flask(s) (cat.#430825, Corning Inc., Coming, NY). Cells were rinsed with 3 ml Cell

Stripper reagent (Cat. #25-056-CI, Mediatech Inc., Herndon, VA). 2 ml of Cell Stripper reagent was added to cells and incubated 2-5 minutes at 37⁰C. Cells were detached by tapping the flask. Cells were resuspended in 8 ml of target cell culture media. Cells were centrifuged at 4⁰C for 3 minutes at 600 x g in an Allegra 6R centrifuge (Beckman) and media was aspirated. Cells were resuspended in 25 ml of blocking buffer (IX Dulbecco's Phosphate Buffered Saline (IX DPBS) without Ca²⁺ or Mg²⁺) (cat.#21-031- CV, Mediatech Inc., Herndon, VA) + 4 mg/ml BSA, Fraction V (cat # 2930, EMD Chemicals Inc., Gibbstown, NJ) for 30 minutes, at 4°C. Cells were centrifuged at 4⁰C for 3 minutes at 600 x g. Media was aspirated and cells were resuspended to 500,000 cells/ml in blocking buffer at 4°C. Cells were then plated into 96-well round-bottom plates (cat.#3797, Corning Inc., Corning, NY) at 50 μl/well resulting in 25,000 cells/well. 50 μl/well of antibody was added at appropriate dilutions. The plate was covered and incubated for 1 hour at 4°C. Plates were centrifuged at 4°C for 3 minutes at 600 x g to pellet cells. Supernatant was carefully removed from the wells using a multichannel pipettor. 150 μl/well of cold blocking buffer was added and cells were resuspended. Plates were centrifuged at 4⁰C for 3 minutes at 600 x g to pellet cells. Supernatant was carefully removed from the wells using a multichannel pipettor. 100 μl/well of blocking buffer containing a 1:1000 dilution of secondary antibody (R- Phycoerythrin conjugated AffmiPure Whole IgG Goat Anti-Human IgA , alpha chain specific (cat.# 109-115-011); R-Phycoerythrin conjugated AffiniPure F(ab')₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (cat.# 109-116-098); and R-Phycoerytimn conjugated AffmiPureF(ab')₂ Fragment Donkey Anti-Human IgM, Fc5μ fragment specific (cat.# 709-116-073); Jackson ImmunoResearch, West Grove, PA) and a 1 :500 dilution of Guava Express 7- aminoactinomycin D (7-AAD) (cat.# 4600-0061, Guava Technologies Inc., Hayward, CA) was added to wells. Cells were incubated 30 minutes at 4°C. Plates were then centrifuged at 4°C for 3 minutes at 600 x g to pellet cells. Supernatant was carefully removed from the wells using a multichannel pipettor. 150 μl/well of cold blocking buffer was added and cells were resuspended. Plates were centrifuged at 4⁰C for 3 minutes at 600 x g to pellet cells. Supernatant was carefully removed from the wells using a multichannel pipettor. Cells were resuspended in 120 μl/well of blocking buffer. Plates were read on the Guava PCA-96 (Guava Technologies Inc., Hayward, CA) flow cytometer using the Guava Express or MultiCaspase program.

SAPORIN IMMUNOTOXIN-DEPENDENT CYTOTOXICITY: Target cells were plated into white 96-well microplates with Clear Bottom (ViewPlate-96, cat.# 6005181 Perkin Elmer Life and Analytical Sciences, Boston, MA) at 2500-5000 cells/well in 50 μl of appropriate target cell culture media. Cells were allowed to attach and grow for 24 hours at 37°C in 5% CO₂. Indicated concentrations of primary antibody were added to the appropriate wells. Secondary Ab-saporin conjugates Mab-ZAP (affinity-purified goat anti-mouse IgG-saporin, cat.# IT-04) or Anti-M Zap (affinity- purified goat anti-mouse IgM-saporin, cat.# IT-30) (Advanced Targeting Systems, Inc., San Diego, CA) were then added together with the primary antibodies in a final total volume of 100 μl/well. Cells were grown for an additional for 3-4 days at 37⁰C in 5% CO₂. Cell viability assay was performed using the Cell Titer GIo kit (Invitrogen, Carlsbad, CA) as per manufacturer's instructions. Luminescence was measured using a Wallac Victor2 1420 multilabel counter (Perkin Elmer Life and Analytical Sciences, Boston, MA) using Program 96 Luminescence.

Results and Discussion

Laminin-binding protein/p40 (LBP/p40) precursor is both intracellular, comprising part of the 4OS ribosomal subunit, and extracellular, abundantly distributed on tumor cell surfaces as laminin binding protein p67 (LBP/p67). Using the MLuC5 mouse IgM monoclonal antibody, tumor cell surface expression of LBP/p67 was found to be equivalent (within 2.5-fold) for 3 breast carcinoma cell lines (MCF-7, MDA-MB- 231, and SK-BR-3) via CELISA and flow cytometry (see Figure 1). In contrast, differences in surface expression of both ErbB2 and MUC-I (EMA, CD227, CA 15-3) were observed. ErbB2 was approximately 10-fold higher on SK-BR-3 than on MCF-7 and MDA-MB-231 cells (see Figure 2). MUC-I was approximately 10-fold greater on MCF-7 than on SK-BR-3 and absent on MDA-MB-231 (see Figure 3).

In an indirect toxin-dependent cytotoxicity (TDC) in vitro assay using saporin- conjugated antiimmunoglobulin, it was found that MLuC5 antibody incubated together with anti-IgM:saporin immunotoxin (10^"9M) resulted in greater than 75% cytoxicity of SK-BR-3 cells after 4 days. TDC was much less pronounced on MCF-7 (approximately 30% induced cytoxicity) and intermediate on MDA-MB-231 cells (with a different apparent dose-response curve), despite approximately equivalent surface expression of LBP/p67 (see Figure 4). As expected, indirect TDC targeting of ErbB2 provided efficient dose-responsive killing of SK-BR-3 cells (see Figure 5), whereas none of the 3 cell lines were killed by anti-MUC-1 plus anti-IgM:saporin (see Figure 6). These results provide evidence that cell surface LBP/p67 can be internalized.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, procedures, GenBank Accession Numbers, and publications cited throughout this application are incorporated herein by reference in their entireties.

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Claims

WHAT IS CLAIMED IS:

1. A method for delivering a toxic agent inside a cancer cell expressing laminin binding protein p67 (LBP/p67), which method comprises exposing the cell to a complex comprising (i) a toxic agent and (ii) an LBP/p67 targeting agent, under conditions that allow LBP/p67 -mediated internalization of the complex.

2. The method of claim 1, wherein the toxic agent is conjugated to the LBP/p67 targeting agent.

3. The method of claim 1, wherein the complex comprises a conjugate comprising (i) the toxic agent and (ii) a secondary binding agent, wherein the secondary binding agent is specific for the LBP/p67 targeting agent.

4. The method of claim 1, wherein the toxic agent becomes toxic upon entry into the cell.

5. The method of claim 4, wherein the toxic agent is saporin.

6. The method of claim 4, wherein the toxic agent is a protoxin which becomes toxic upon a reaction inside the cell.

7. The method of claim 6, wherein the protoxin is contains a disulfide bonded loop with trypsin-sensitive site.

8. The method of claim 6, wherein the protoxin contains a factor Xa cleavage site.

9. The method of claim 1, wherein the LBP/p67 targeting agent is an

LBP/p67-specific antibody, or an LBP/p67 specific binding fragment thereof.

10. The method of claim 9, wherein the antibody is selected from the group consisting of: a human antibody and a humanized antibody, or an LBP/p67 specific binding fragment thereof.

11. The method of claim 9, wherein the antibody, or fragment thereof, is selected from the group consisting of: MLuC5, MLuC6, anti-laminin receptor IgM 8A5.3C3, an antibody to LBP/p67 C terminal domain that blocks Sindbis virus binding to mammalian cells, an IgM antibody against Staphylococcus aureus laminin-binding protein that crossreacts with LBP/p67, and a rabbit polyclonal to 67kD Laminin Receptor Synthetic peptide: SGALDVLQ-MAP (SEQ ID NO: 9).

12. The method of claim 9, wherein the antibody is MLuC5.

13. The method of claim 1, wherein the LBP/p67 targeting agent is an LBP/p67-sρecific ligand, or fragment thereof.

14. The method of claim 13, wherein the ligand is selected from the group consisting of: a synthetic peptide that includes AA 2091-2108 of the laminin A chain that binds to LBP/p67 (CSRARKQAASIKVAVSADR SEQ ID NO: 1), a nine amino acid sequence CDPGYIGSR (peptide 11; SEQ ID NO: 2) within the beta chain of laminin 1 that binds to LBP/p67, the YIGSR pentapeptide LBP/p67 binding sequence from the beta chain of laminin 1 (SEQ ID NO: 3), cellular prion protein (PrP), cellular prion protein together with heparin sulfate proteoglycan, synthetic peptides comprising PrPLRPbdl (amino acids 144-179 of PrP;

DYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDC SEQ IDNO: 4) and/or PrPLRPbd2 (amino acids 53-93 of PrP; GGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGG SEQ ID NO: 5), PrPLRPbd2 together with heparin-sulfate, ribosomal S21 protein, and Sindbis virus.

15. The method of claim 1, wherein the toxic agent is a radioisotope.

16. The method of claim 1, wherein the toxic agent is a chemotherapeutic.

17. The method of claim 16, wherein the chemotherapeutic is a chemical toxin.

18. The method of claim 17, wherein the chemical toxin is derived from bacteria.

19. The method of claim 18, wherein the chemical toxin is selected from the group consisting of: Pseudomonas exotoxin and diphtheria toxin or derivatives thereof.

20. The method of claim 19 , wherein said derivative is truncated.

21. The method of claim 19, wherein said derivative comprises an additional targeting sequence.

22. The method of claim 21, wherein said additional targeting sequence is KDEL (SEQ ID NO: 6).

23. The method of claim 17, wherein the chemical toxin is a mycotoxin.

24. The method of claim 23 , wherein the mycotoxin is clavin or trichothecene

25. The method of claim 16, wherein the chemotherapeutic is a hybrid toxin.

26. The method of claim 25, wherein said hybrid toxin is a Ricin A chain - Diptheria toxin A chain hybrid.

27. The method of claim 17, wherein the chemical toxin is derived from a plant.

28. The method of claim 27, wherein the chemical toxin is a ribosome inactivating protein (RIP).

29. The method of claim 28, wherein the RIP is selected from the group consisting of: ricin, abrin, pokeweed antiviral protein, gelonin, saporin, momordin, and bryodin.

30. A method for treating a cancer in a mammal comprising administering to the mammal a complex comprising (i) a toxic agent and (ii) an LBP/p67 targeting agent, wherein the complex is capable of becoming internalized by a cancer cell.

31. The method of claim 30, wherein the mammal is human.

32. A method for inducing cell death of a cancer cell comprising exposing the cancer cell to a complex comprising (i) a toxic agent and (ii) an LBP/p67 targeting agent, wherein the complex is capable of becoming internalized by the cancer cell.

33. The method of claim 1, 30, or 32, wherein the complex comprises a recombinant fusion protein.

34. The method of claim 1, 30, or 32, wherein the toxic agent and the LBP/p67 targeting agent are linked by linker.

35. The method of claim 34, wherein the toxic agent and the LBP/p67 targeting agent are covalently linked.

36. The method of claim 34, wherein the linker is a cleavable linker which becomes cleaved upon internalization into the cell.

37. The method of claim 36, wherein the cleavable linker is selected from the group consisting of: a disulfide linker, an acid cleavable linker, a photolabile linker, and a peptide linker.

38. The method of claim 30, wherein the cancer is selected from the group consisting of: breast cancer, colorectal cancer, lung cancer, cervical, endometrial, gastric, laryngeal squamous cell, lymphoma, ovarian, pancreatic endocrine, prostate, thyroid, and urothelial.

39. A pharmaceutical composition comprising a toxic agent and an LBP/p67 targeting agent, wherein the toxic agent and the LBP/p67 targeting agent form a complex, and wherein the complex is capable of being internalized by a cancer cell expressing LBP/p67.

40. The pharmaceutical composition of claim 39, wherein the toxic agent is conjugated to the targeting agent.

41. The pharmaceutical composition of claim 40, wherein the toxic agent is conjugated to the LBP/p67 targeting agent via a linker.

42. The pharmaceutical composition of claim 41, wherein the linker is a cleavable linker which becomes cleaved upon internalization.

43. The pharmaceutical composition of claim 42, wherein the cleavable linker is selected from the group consisting of: a disulfide linker, an acid cleavable linker, a photo-labile linker, and a peptide linker.

44. The pharmaceutical composition of claim 39, wherein the LBP/p67 targeting agent is an LBP/p67-specific antibody, or an LBP/p67 specific binding fragment thereof.

45. The pharmaceutical composition of claim 39, wherein the LBP/p67 targeting agent is an LBP/p67-specifϊc ligand, or fragment thereof.

46. The pharmaceutical composition of claim 39, wherein the toxic agent is a protoxin which becomes toxic upon a reaction inside the cell.

47. The pharmaceutical composition of claim 39, wherein the targeting agent is capable of binding to LBP/p67 with sufficient specificity and affinity to be internalized by a cancer cell expressing LBP/p67.

48. The composition of claim 1, wherein internalization of the complex is detectable in vitro via detection of labeled toxin or LBP/p67 targeting agent or a cell death assay.