DEIMMUNIZED LINKER AND METHODS OF USE

Cross-reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/164,446, filed May 20, 2015, which is hereby incorporated by reference in its entirety for all purposes.

Statement Regarding the Sequence Listing

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is VIVE-033_01WO_ST25.txt. The text file is about 266 KB, was created on May 19, 2016, and is being submitted electronically via EFS-Web.

Field of the Invention

[0003] The present invention is directed to novel deimmunized furin protease- sensitive peptide linkers that have been specifically engineered to lack T-cell epitopes and therefore elicit reduced immune response. The present disclosure also relates to immunotoxins comprising such linkers and methods of use of said immunotoxins comprising such linkers.

Background of the Invention

[0004] In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive, or because of recurrence due to regrowth at the original site and/or metastases. Even in cancers such as breast cancer where the mortality rate has decreased, successful intervention relies on early detection of the cancerous cells. The etiology, diagnosis and ablation of cancer remain a central focus for medical research and development.

[0005] Current methods of cancer treatment are relatively non-selective. Surgery removes the diseased tissue, radiotherapy shrinks solid tumors and chemotherapy kills rapidly dividing cells. Chemotherapy, in particular, results in numerous side effects, in some cases so severe to preclude the use of potentially effective drugs. Moreover, cancers often develop resistance to chemotherapeutic drugs.

[0006] Numerous efforts are being made to enhance the specificity of cancer therapy. For review, see Kohn and Liotta (1995) Cancer Res. 55: 1856-1862. In particular, identification of cell surface antigens expressed exclusively or preferentially on certain tumors allows the formulation of more selective treatment strategies. Antibodies specific to cell surface proteins can be used as a vehicle to carry and specifically deliver an effector such as a cytotoxin to cells. A recombinant antibody carrying a cytotoxin is referred to as an immunotoxin. Typically, peptide linkers are required between the effector and the antibody moiety to allow for the release of the effector from the antibody or ligand. Such peptide linkers are usually cleaved by intracellular proteases.

[0007] The choice of linker for an immunotoxin has been typically dictated by the type of enzymes that are either present in most cells, known to be upregulated in target cells, or selected from different regions of the cell (Fuchs et al, 2006) (Heisler et al, 2003) (Keller et al, 2001). Some proteases are ubiquitously expressed. Others are linked to particular cell types or areas within or around the cells. A furin-sensitive linker is commonly used because furin is a ubiquitous enzyme known to be present in both the endosome and the Golgi apparatus of most cells and is thus expected to cleave a conjugate in any cell it enters.

[0008] There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. The key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC, so-called "T-cell epitopes."

[0009] T-cell epitope identification is the first step to epitope elimination. T-cell epitopes may be removed by the use of judicious amino acid substitution within the protein of interest. There are also in vitro methods for measuring the ability of synthetic peptides to bind MHC class II molecules. However, such techniques are not adapted for the screening of multiple potential epitopes to a wide diversity of MHC allotypes, nor can they confirm the ability of a binding peptide to function as a T-cell epitope.

[0010] A therapeutic protein such as an immunotoxin has a number of regions that are potential sources of unwanted immune reaction, for example, the antibody or antibody fragment, the toxin and the linker connecting the two.

[0011] Thus there is considerable need for the development of tumor-specific therapies with effective tumor killing and reduced propensity to elicit an immune reaction in patients. Brief Summary of the Invention

[0012] The present invention relates to novel immunotoxins, which are both effective in tumor targeting and killing and has reduced propensity to elicit an immune reaction, and methods for treating or preventing cancer by administering, to a patient in need thereof, an effective amount of said recombinant immunotoxin that specifically binds to (and therefore is "targeted to") a protein on the surface of cancer cells. Where desired, the immunotoxin may be co-administered, concurrently administered, and/or sequentially administered with one or more other anti-cancer agents, and/or in conjunction with radiation or surgery.

[0013] In one aspect, the invention contemplates a modified furin protease sensitive peptide linker, wherein said modified peptide linker has a reduced propensity to activate an immune response. In one embodiment, said peptide linker has a reduced propensity to activate T-cells and is modified at one or more amino acid residues in a T-cell epitope. In another embodiment, the peptide linker is modified at one or more amino acid residues in a T-cell epitope represented by SEQ ID NO: 17. In another embodiment, the peptide linker is modified at amino acid position 12 of SEQ ID NO: 17. In another embodiment, the amino acid sequence of the modified peptide linker shares at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17.

[0014] In one embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 32. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 33. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 34. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 35. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 36. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 119. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 124.

[0015] In another aspect, the invention contemplates an immunotoxin comprising: (a) a binding protein and; (b) a toxin, wherein the binding protein and toxin are linked by a modified furin protease sensitive peptide linker. In one embodiment, the modified peptide linker has a reduced propensity to activate an immune response. In one embodiment, the peptide linker has a reduced propensity to activate T-cells and is modified at one or more amino acid residues in a T-cell epitope. In another embodiment, the peptide linker is modified at one or more amino acid residues in a T-cell epitope represented by SEQ ID NO: 17. In another embodiment, the peptide linker is modified at amino acid position 12 of SEQ ID NO: 17. In one embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 32. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 33. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 34. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 35. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 36. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 119. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 124.

[0016] In one embodiment, the binding protein of the immunotoxin comprises an antibody or antibody fragment. In another embodiment, the antibody or antibody fragment comprises an anti-epithelial cell adhesion molecule (EpCAM) antibody or an anti-EpCAM antibody fragment. In a further embodiment, the anti-EpCAM antibody or antibody fragment comprises a heavy chain having an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, and SEQ ID NO: 57, and a light chain having an amino acid sequence selected from SEQ ID NO: 51 and SEQ ID NO: 55. In one embodiment, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 49, and the light chain has an amino acid sequence as shown in SEQ ID NO: 51. In another embodiment, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 53, and the light chain has an amino acid sequence as shown in SEQ ID NO: 55. In another embodiment, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 56, and the light chain has an amino acid sequence as shown in SEQ ID NO: 55. In a further embodiment,the heavy chain has an amino acid sequence as shown in SEQ ID NO: 57, and the light chain has an amino acid sequence as shown in SEQ ID NO: 55. In one embodiment, the anti-EpCAM antibody or antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, immunoglobulin scaffolds, multimers, and any combination thereof. In a particular embodiment, the anti-EpCAM antibody fragment is an Fab.

[0017] In one embodiment, the antibody or antibody fragment comprises an anti- HER2/neu antibody or an anti-HER2/neu antibody fragment. In another embodiment, the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10. In one embodiment, the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a heavy chain variable region. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a light chain variable region. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 4. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the anti-HER2/neu antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds- scFv, dimers, minibodies, diabodies, bispecific antibody fragments, immunoglobulin scaffolds, multimers, and any combination thereof. In a particular embodiment, the anti- HER2/neu antibody fragment is a diabody. In another embodiment, the diabody is comprised of a heavy chain variable region and a light chain variable region. In a further embodiment, the heavy chain variable region and the light chain variable region are linked by a linker. In yet another embodiment, the linker is encoded by an amino acid sequence shown in SEQ ID NO: 15. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by the amino acid sequence of SEQ ID NO: 131. In another embodiment, the anti-HER2/neu antibody fragment is a scFv. In yet another embodiment, the anti-HER2/neu antibody fragment is an Fab.

[0018] In one embodiment, the antibody or antibody fragment comprises an Hl l antibody or Hl l antibody fragment. In another embodiment, the Hl l antibody or antibody fragment comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 105-110. In one embodiment, the Hl l antibody or antibody fragment comprises a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the Hl l antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In one embodiment, the HI 1 antibody or antibody fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, immunoglobulin scaffolds, multimers, and any combination thereof. In a particular embodiment, the Hl l antibody fragment is an Fab.

[0019] In one embodiment, the toxin portion of the immunotoxin is deimmunized bouganin. In another embodiment, the deimmunized bouganin toxin is encoded by an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61. In another embodiment, the deimmunized bouganin toxin is linked to the binding protein by a modified furin protease sensitive linker encoded by an amino acid sequence chosen from SEQ ID NOs: 32-36, 119 and 124.

[0020] In one embodiment, the toxin portion of the immunotoxin is deimmunized diptheria toxin.

[0021] The invention also relates to a method of treating or preventing cancer comprising administering an effective amount of an immunotoxin to a subject in need thereof, wherein said immunotoxin comprises: (a) a binding protein and; (b) a toxin, wherein the binding protein and toxin are linked by a modified furin protease sensitive peptide linker of the present invention. In one embodiment, the binding protein comprises an anti-EpCAM antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an anti-HER2/neu antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an Hl l antibody or antibody fragment of the present invention. In one embodiment, the toxin portion of the immunotoxin is deimmunized bouganin. In another embodiment, the toxin portion of the immunotoxin is deimmunized diphtheria toxin.

[0022] In one embodiment, the cancer is selected from the group consisting of lung cancer, gastric cancer, renal cancer, thyroid cancer, breast cancer, bladder cancer, ovarian cancer, colorectal cancer, head and neck cancer, hepatocellular carcinoma, esophageal, pancreas, and prostate cancer. In another embodiment, the cancer is of neuro-ectodermal origin. In a further embodiment, the cancer is neuroblastoma, glioblastoma, melanoma or sarcoma.

[0023] In one embodiment, the immunotoxin is administered directly to the cancer site. In another embodiment, the direct administration is intratumoral, intravesicular or peritumoral. In another embodiment, the direct administration is systemic. In another embodiment, the direct administration is intravenous. In a further embodiment, the direct administration is intracranial infusion.

[0024] The invention also relates to additionally comprising the administration of one or more further cancer therapeutics for simultaneous, separate or sequential treatment or prevention of cancer.

[0025] The invention also relates to a method for enhancing the activity of an anticancer agent comprising administering to a subject in need thereof an anti-cancer agent and an effective amount of an immunotoxin, wherein said immunotoxin comprises: (a) a binding protein and; (b) a toxin, wherein the binding protein and toxin are linked by a modified furin protease sensitive peptide linker of the present invention. In one embodiment, the binding protein comprises an anti-EpCAM antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an anti-HER2/neu antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an HI 1 antibody or antibody fragment of the present invention. In one embodiment, the toxin portion of the immunotoxin is deimmunized bouganin. In another embodiment, the toxin portion of the immunotoxin is deimmunized diphtheria toxin. [0026] The invention also relates to a kit for treating or preventing cancer comprising an effective amount of an immunotoxin comprising: (a) a binding protein; (b) a toxin, wherein the binding protein and toxin are linked by a modified furin protease sensitive peptide linker of the present invention, and; (c) directions for the use thereof to treat the cancer. In one embodiment, the binding protein comprises an anti-EpCAM antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an anti-HER2/neu antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an Hl l antibody or antibody fragment of the present invention. In one embodiment, the toxin portion of the immunotoxin is deimmunized bouganin. In another embodiment, the toxin portion of the immunotoxin is deimmunized diphtheria toxin.

[0027] The invention also related to an expression vector comprising an immunotoxin comprising: (a) a binding protein and; (b) a toxin, wherein the binding protein and toxin are linked by a modified furin protease sensitive peptide linker of the present invention. In one embodiment, the binding protein comprises an anti-EpCAM antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an anti- HER2/neu antibody or antibody fragment of the present invention. In another embodiment, the binding protein comprises an HI 1 antibody or antibody fragment of the present invention. In one embodiment, the toxin portion of the immunotoxin is deimmunized bouganin. In another embodiment, the toxin portion of the immunotoxin is deimmunized diphtheria toxin.

[0028] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Description of Drawings

[0029] FIG. 1 shows an analysis of the junction comprised of the C-terminal end of the light chain Fab fragment, furin linker and N-terminus end of deBouganin (highlighted red in the "Sequence" row using iTope™). Peptides spanning the entire sequence were tested as 9mer peptides in one amino acid increments. The position of pi anchor residues comprises the first residue of a MHC class II ligand (core 9mer) and is highlighted as a yellow box if the binding score was 0.55-0.6 and as a red box if the binding score was >0.6. Regions containing potentially immunogenic peptides are indicated in the "Promiscuous High" and "Promiscuous Moderate" rows; red indicates promiscuous high affinity MHC class II binding peptides, yellow indicates promiscuous moderate affinity MHC class II binding peptides. In the "TCED" row, regions representing closely homologous peptides from the T cell epitope database are shown in green.

[0030] FIG. 2 shows Western blot analysis of VB6-901-DI linker variants. Two independent induced VB6-901-DI linker H (lanes 3 and 4), linker K (lanes 5 and 6), linker N (lanes 7 and 8), linker P (lanes 9 and 10) and linker S (lanes 11 and 12) supernatants were loaded under non-reducing conditions on an SDS-PAGE gel and immunoblotted with an anti- human kappa-HRP antibody and compared to VB6-901-DI induced supernatant (lane 1). Lane 2 corresponds to the ladder. The arrow indicates the full-length proteins.

[0031] FIG. 3A-3D shows MTS curves and serum stability. Representative example of FIG. 3A) OVCAR3 and FIG. 3B) CAL27 cytotoxicity curves obtained after 5 days treatment with VB6-901-DI linker H (black circle), linker K (white circle), linker N (black triangle), linker S (white triangle) and VB6-901-WT (black square). FIG. 3C-3D) Time course analysis (0, 24, 48 and 72 hours) of VB6-901-DI linker K (lanes 1 to 4), VB6-901-DI linker N (lane 5 to 8) and VB6-901-WT (lane 9 to 12) incubated in FIG. 3C) mouse and FIG. 3D) human serum by Western blot. Lane C corresponds to serum only, lane L to ladder and lane 13 to 200ng loaded of purified VB6-901-WT.

[0032] FIG. 4 shows an analysis of the linker sequence (highlighted red in the "Sequence" row using iTope™). Peptides spanning the entire sequence were tested as 9mer peptides in one amino acid increments. The position of pi anchor residues comprises the first residue of a MHC class II ligand (core 9mer) and is highlighted as a yellow box if the binding score was 0.55-0.6 and as a red box if the binding score was >0.6. Regions containing potentially immunogenic peptides are indicated in the "Promiscuous High" and "Promiscuous Moderate" rows; red indicates promiscuous high affinity MHC class II binding peptides, yellow indicates promiscuous moderate affinity MHC class II binding peptides. In the "TCED" row, regions representing closely homologous peptides from the T cell epitope database are shown in green.

[0033] FIG. 5A-5B shows Western blot analysis of deBouganin-F-AvP07-17 linker variants. FIG. 5 A) Two independent induced deBouganin-F(E)-AvP07-17-His (lanes 2 and 3, SEQ ID NO: 125), deBouganin-F(P)-AvP07-17-His (lanes 4 and 5, SEQ ID NO: 129), deBouganin-F(T)-AvP07-17-His (lanes 6 and 7, SEQ ID NO: 127) supernatants were loaded under non-reducing conditions on a SDS-PAGE gel and immunoblotted with a rabbit anti- deBouganin antibody followed by an anti-rabbit-HRP antibody and compared to deBouganin- F-AvP07-17-His induced supernatant (lane 1). FIG. 5B) Two independent induced deBouganin-F(E)-AvP07-17 (lanes 2 and 3, SEQ ID NO: 131) and deBouganin-F(P)-AvP07- 17 (lanes 4 and 5, SEQ ID NO: 135) supernatants were analyzed as described previously and the expression level compared to deBouganin-F-AvP07-17 (lane 5, amino acids 23-529 of SEQ ID NO: 25). Lane L corresponds to the ladder. The full arrows indicate the full-length protein.

[0034] FIG. 6A-6B shows MTS curves. Representative example of BT-474 (FIG. 6A) and SkBR3 (FIG. 6B) cytotoxicity curves obtained after 5 days treatment with deBouganin-F(E)-AvP07-17 (black squares, SEQ ID NO: 131), deBouganin-F(P)-AvP07-17 (green circles, SEQ ID NO: 135) and deBouganin-F-AvP07-17(red inverted triangles, amino acids 23-529 of SEQ ID NO: 25).

[0035] FIG. 7A-7B shows serum stability. deBouganin-F(E)-AvP07-17 (E), deBouganin-F(P)-AvP07-17 (P) and deBouganin-F-AvP07-17 (WT) were incubated in mouse (FIG. 7A) and human serum (FIG. 7B). After 0 hr (black), 24 hr (light grey) and 48 hr (dark grey), flow cytometry using an anti-deBouganin antibody was used as a surrogate for the measurement of the full-length fusion protein.

[0036] FIG. 8 shows immunogenicity risk scores for the first 100 amino acids of furin-deBouganin (top panel) and furin(K)-deBouganin (bottom panel).

Detailed Description

Definitions

[0037] This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0038] Singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an "antioxidant" is a reference to one or more antioxidants and equivalents thereof known to those skilled in the art, and so forth.

[0039] The term "aC" represents any antibody, or antigen binding fragment thereof, either monoclonal, polyclonal or derivative thereof that recognizes specifically the chondroitin sulfate A (CSA) antigen and distinguishes between cancer and noncancer cells. aC includes Hl l .

[0040] The term "about" means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.

[0041] The term "animal," "patient, " or "subject" as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals.

[0042] "Antibody fragments" that may be used include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof, and fragments from recombinant sources and/or produced in transgenic animals. The antibody or fragment may be from any species including mice, rats, rabbits, hamsters and humans. Chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, humanized antibodies which comprise the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies. It is expected that chimeric antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody. The humanized antibodies can be further stabilized for example as described in WO 00/61635 and is incorporated by reference in its entirety.

[0043] "Anticancer agents" or "cancer therapeutics" refers to compounds or treatments that are effective in treating or preventing cancer including, without limitation, chemical agents, other immunotherapeutics, cancer vaccines, anti-angiogenic compounds, certain cytokines, certain hormones, gene therapy, radiotherapy, surgery, and dietary therapy.

[0044] The term "binding protein" refers to proteins that specifically bind to another substance such as an antigen. In an embodiment, binding proteins are antibodies or antibody fragments. In another embodiment, binding proteins are engineered non-immunoglobulin proteins. In another embodiment, binding proteins can be scaffolds.

[0045] The term "CSA" refers to the glycosaminoglycan or antigen recognized by an Hl l antibody, an Hl l antibody fragment or an Hl l scaffold on a cancer cell. "CSA positive cancer cells" refer to cancer cells that express CSA on their cell surface. [0046] A "cell line" or "cell culture" denotes bacterial, plant, insect or higher eukaryotic cells grown or maintained in vitro. The descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. A monoclonal antibody may be produced by a hybridoma or other cell. Methods of making hybridomas, both murine and human, are known in the art.

[0047] "De-immunized" refers to a molecule that lacks or elicits reduced immune response when compared to the wild type counterpart.

[0048] "De-immunized antibodies" or "de-immunized antibody fragments" refers to antibodies and antibody fragments that lack one or more T-cell epitopes and elicit reduced immune response when compared to the wild type counterpart.

[0049] The terms "deimmunized bouganin toxin", "deimmunized bouganin protein", "deBouganin", "modified bouganin toxin" and "modified bouganin protein" refer to a bouganin toxin that has been modified by nucleotide or amino acid substitution, deletions, additions, or truncations of the protein in order to have a reduced propensity to elicit an immune response, preferably a T-cell response, as compared to a non-deimmunized or non- modified bouganin toxin. The deimmunized or modified bouganin toxin can be a modified full length sequence or a modified fragment of the non-deimmunized or non-modified bouganin toxin. The deimmunized or modified bouganin toxin may also contain other changes as compared to the wild-type bouganin sequence which do not alter immunogenicity of the peptide. The deimmunized or modified bouganin toxin will preferably have the same biological activity as the non-deimmunized or non-modified bouganin toxin.

[0050] The terms "deimmunized diphtheria toxin", "deimmunized diphtheria protein", "deimmunized DT", "deDT", "modified diphtheria toxin", "modified diphtheria protein" and "modified DT" refer to a diphtheria toxin that has been modified by nucleotide or amino acid substitution, deletions, additions, or truncations of the protein in order to have a reduced propensity to elicit an immune response, preferably a T-cell response, as compared to a non- deimmunized or non-modified diphtheria toxin. The deimmunized or modified diphtheria toxin can be a modified full length sequence or a modified fragment of the non-deimmunized or non-modified diphtheria toxin. The deimmunized or modified diphtheria toxin may also contain other changes as compared to the wild-type diphtheria sequence which do not alter immunogenicity of the peptide. The deimmunized or modified diphtheria toxin will preferably have the same biological activity as the non-deimmunized or non-modified diphtheria toxin. [0051] The terms "deimmunized furin linker", "modified furin linker" and "mutated furin linker" refer to a furin protease sensitive linker that has been modified by nucleotide or amino acid substitution, deletions, additions, or truncations of the linker in order to have a reduced propensity to elicit an immune response, preferably a T-cell response, as compared to a non-deimmunized or non-modified furin protease sensitive linker. The deimmunized or modified furin protease sensitive linker can be a modified full length sequence or a modified fragment of the non-deimmunized or non-modified furin protease sensitive linker. The deimmunized or modified furin protease sensitive linker may also contain other changes as compared to the wild-type furin protease sensitive linker which do not alter immunogenicity of the linker. The deimmunized or modified furin protease sensitive linker will preferably have the same biological activity as the non-deimmunized or non-modified furin protease sensitive linker. In one embodiment, the deimmunized furin protease sensitive linker comprises a sequence selected from SEQ ID NOs: 32-36, 119 and 124.

[0052] "De-immunized VB5-845" refers to Fab fragment of anti-EpCAM antibody wherein the putative T-cell epitopes on the VH domain and the VL domain are mutated and might result in eliciting a reduced immune response when compared to the non-deimmunized Fab fragment VB5-845 (VB5-845-WT). De-immunized VB5-845 Fab fragment (VB5-845- DI) comprises a de-immunized VH-CH domain (SEQ ID NO: 49) and a de-immunized VL-CL domain (SEQ ID NO: 51). The non-deimmunized VB5-845 Fab fragment (VB5-845-WT) comprises a wild type VH-CH domain (SEQ ID NO: 63) and a wild type VL-CL domain (SEQ ID NO: 65). De-immunized anti-EpCAM antibodies and antibody fragments are described in PCT/CA2014/050950, which is incorporated herein in its entirety.

[0053] "Effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts of an immunotoxin may vary according to factors such as the disease state, age, sex, weight of the animal. Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0054] The term "fusion polypeptide" is a polypeptide comprising regions in a different position in the sequence than occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide; they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide; or they may be synthetically arranged. In one embodiment, as described below, the invention encompasses recombinant proteins that are comprised of a functional portion of a HER2/neu binding protein and a toxin. In another embodiment, the invention encompasses recombinant proteins that are comprised of a functional portion of an EpCAM binding protein and a toxin. Methods of making these fusion proteins are known in the art and are described for instance in WO93/07286.

[0055] As used herein, the term "functionally equivalent fragment" of, for instance, a polypeptide or polynucleotide, varies from the native sequence by any combination of additions, deletions, or substitutions while preserving at least one functional property of the fragment relevant to the context in which it is being used. In one embodiment, a functionally equivalent fragment of a polynucleotide encoding a binding protein for HER2/neu either encodes a polypeptide that is functionally equivalent to a HER2/neu binding protein when produced by an expression system, or has similar hybridization specificity as a polynucleotide encoding a HER2/neu binding protein when used in a hybridization assay. A functionally equivalent fragment of a HER2/neu binding protein typically has one or more of the following properties: ability to bind a human epidermal growth factor receptor 2; ability to bind at least one type of cancer cell in a specific manner; and an ability to elicit an immune response with a similar antigen specificity as that elicited by a HER2/neu binding protein. In another embodiment, a functionally equivalent fragment of a polynucleotide encoding a binding protein for EpCAM either encodes a polypeptide that is functionally equivalent to an EpCAM binding protein when produced by an expression system, or has similar hybridization specificity as a polynucleotide encoding an EpCAM binding protein when used in a hybridization assay. A functionally equivalent fragment of an EpCAM binding protein typically has one or more of the following properties: ability to bind epithelial cell adhesion molecule; ability to bind at least one type of cancer cell in a specific manner; and an ability to elicit an immune response with a similar antigen specificity as that elicited by an EpCAM binding protein.

[0056] The term "Hl l antibody" means an antibody or antibody fragment that recognizes cancer cells from a wide variety of cancers but does not recognize normal, noncancerous cells. By "does not recognize" is meant that noncancer cells are either not specifically bound to by Hl l or are only poorly recognized by the antibody. "Hl l " is an antibody obtained from the fusion of peripheral blood lymphocytes of a 64 year old male with a low grade glioma and fused to a human myeloma cell line to produce a hybridoma designated NBGM1/H1 1. The antibodies are designated aC and include Hl l and any antibody with the "immunologic specificity" of Hl l , that is, recognizing the antigen recognized by Hl l, and that is specific for at least one type of cancer cell but does not recognize normal cells. These antigen binding fragments include, but are not limited to, whole native antibodies, exemplified by Hl l; bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), diabodies, fusion polypeptides and Hl l scaffolds. An Hl l antibody is described in US Patent Nos. 6,207,153 and 7,166,286, each of which is incorporated herein by reference in its entirety. The term "Oi l VH-CH-F(K)- deBouganin" refers to an antibody fragment comprised of, starting at the N-terminus: an anti- chondroitin sulfate antigen Oi l heavy chain (VH-CH) (SEQ ID NO: 99), a de-immunized furin protease sensitive peptide linker (SEQ ID NO: 33), and a deBouganin toxin (SEQ ID NO: 12). The term "VB6-011-C _H -deBouganin" refers to an Fab comprised of Oi l VH-CH- F(K)-deBouganin and an 011 light chain (V _L -C _L ) (SEQ ID NO: 120). The term "deBouganin- F(E)-VH-CH Oi l " refers to an antibody fragment comprised of, starting at the N-terminus: a deBouganin toxin (SEQ ID NO: 12), a de-immunized furin protease sensitive peptide linker (SEQ ID NO: 119), and an anti-chondroitin sulfate antigen Oi l heavy chain (VH-CH) (SEQ ID NO: 99). The term " VB6-011-NV _H -deBouganin" refers to an Fab comprised of deBouganin-F(E)-VH-CH Oi l and an Oi l light chain (V _L -C _L ) (SEQ ID NO: 120). The term "VB6-011 " refers to an Fab of an anti-chondroitin sulfate antigen 011 antibody.

[0057] As used herein, the term "HER2/neu," "HER2/neu polypeptide," or "HER2/neu protein," refer to a human epidermal growth factor receptor 2. "HER2/neu" can also be known as erbB2.

[0058] As used herein, the term "anti-HER2/neu binding protein", "anti-HER2/neu antibody" or "anti-HER2/neu antibody fragment" means a binding protein, an antibody or antibody fragment, respectively, that recognizes a human epidermal growth factor receptor 2 expressed on cancer cells. The antibodies or antibody fragments include, but are not limited to, whole native antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain

V region fragments (scFv), diabodies, fusion polypeptides and HER2/neu scaffolds. In one embodiment, the anti-HER2/neu antibody fragment is a diabody engineered with the C6.5 anti-HER2 scFv (in V _H -V _L orientation) with a short G ₄ S linker (SEQ ID NO: 15) between the

V domains and comprise the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10. In another embodiment, "HER2/neu antibody" is an antibody or antibody fragment obtained from the humanization of the murine monoclonal antibody 4D5 (mumAb4D5). The antibodies are designated humAb4D5 and include any antibody with the "immunologic specificity" of a humAb4D5, that is, recognizing the antigen recognized by humAb4D5, and that is specific for at least one type of cancer cell. HER2/neu antibodies are described in US Patent Nos. 5677171 ; 5821337; 6054297; 6165464; 6339142; 6407213; 6639055; 6719971; 6800738; 7074404, each of which is herein incorporated by reference in its entirety and in the following literature: Coussens et al. (1985) Science 230: 1132-1139; Slamon et al. (1989) Science 244:707-712; Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289; Slamon et al. (2001) New Engl. J. Med. 344:783-792, each of which is herein incorporated by reference in its entirety.

[0059] As used herein, the term "PelB-DeBouganin-F-AvP07-17-(V _H -V _L -C6.5)-His" refers to an antibody fragment comprised of, starting at the N-terminus: a PelB leader sequence, deBouganin toxin, wild-type furin linker (SEQ ID NO: 17), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by SEQ ID NO: 22 (nucleotide sequence) and SEQ ID NO: 23 (amino acid sequence). The terms "deBouganin-AvP07-17- His" and "deBouganin-AvP07-17-(V _H -VL)-His" refer to an antibody fragment comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23. The term "PelB- DeBouganin-F-AvP07-17-(VH-VL-C6.5)" refers to an antibody fragment comprised of, starting at the N-terminus: a PelB leader sequence, deBouganin toxin, wild-type furin linker (SEQ ID NO: 17), an anti-HER2/neu heavy chain variable region (VH) linked to an anti- HER2/neu light chain variable region (VL), and which is represented by SEQ ID NO: 24 (nucleotide sequence) and SEQ ID NO: 25 (amino acid sequence). The terms "deBouganin- AVP07-17-(VH-V _l )", "deBouganin-AvP07-17" and "VB7-756" refer to an antibody fragment comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25. The term "PelB-AvP07-17-(VH-VL-C6.5)-F-deBouganin-His" refers to an antibody fragment comprised of, starting at the N-terminus: a PelB leader sequence, an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), wild- type furin linker (SEQ ID NO: 17), deBouganin toxin, and a His tag at the C-terminus, and which is represented by SEQ ID NO: 26 (nucleotide sequence) and SEQ ID NO: 27 (amino acid sequence). The terms "AvP07-17-deBouganin-His" and "AVP07-17(VH-VL)- deBouganin-His" refer to an antibody fragment comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 27. The term "PelB-AvP07-17-(V _H -V _L -C6.5)-F- deBouganin" refers to an antibody fragment comprised of, starting at the N-terminus: a PelB leader sequence, an anti-HER2/neu heavy chain variable region (VH) linked to an anti- HER2/neu light chain variable region (VL), wild-type furin linker (SEQ ID NO: 17) and deBouganin toxin, and which is represented by SEQ ID NO: 28 (nucleotide sequence) and SEQ ID NO: 29 (amino acid sequence). The terms "AvP07-17-deBouganin" and "AvP07- 17(VH-VL)-deBouganin" refer to an antibody fragment comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 29. The term "PelB-deBouganin-F-AvP07- 17-(VL-VH-C6.5)" refers to an antibody fragment comprised of, starting at the N-terminus: a PelB leader sequence, deBouganin toxin, wild-type furin linker (SEQ ID NO: 17), an anti- HER2/neu light chain variable region (VL) linked to an anti-HER2/neu heavy chain variable region (VH), and which is represented by SEQ ID NO: 30 (nucleotide sequence) and SEQ ID NO: 31 (amino acid sequence). The terms "deBouganin-AvP07-17(VL-VH)" and " deBouganin- VL-VH AVP07-17" refer to an antibody fragment comprised of amino acids 23- 529 of the amino acid sequence shown in SEQ ID NO: 31. The term "His-AvP07-17- deBouganin" refers to an antibody fragment comprised of, starting at the N-terminus: a His tag, an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), wild-type furin linker (SEQ ID NO: 17) and deBouganin toxin. The term "deBouganin-F(E)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 119), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 126 (nucleotide sequence) and by SEQ ID NO: 125 (amino acid sequence). The term "deBouganin-F(T)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 124), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 128 (nucleotide sequence) and by SEQ ID NO: 127 (amino acid sequence). The term "deBouganin-F(P)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 35), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 130 (nucleotide sequence) and by SEQ ID NO: 129 (amino acid sequence). The term "deBouganin-F(E)-AvP07-17" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 119), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 132 (nucleotide sequence) and by SEQ ID NO: 131 (amino acid sequence). The term "deBouganin-F(T)-AvP07-17" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 124), an anti-HER2/neu heavy chain variable region (V _H ) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 134 (nucleotide sequence) and by SEQ ID NO: 133 (amino acid sequence). The term "deBouganin-F(P)-AvP07-17" refers to an antibody fragment comprised of, starting at the N- terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 35), an anti-HER2/neu heavy chain variable region (V _H ) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 136 (nucleotide sequence) and by SEQ ID NO: 135 (amino acid sequence). The PelB leader sequence, which directs an immunotoxin to the periplasm, is cleaved off after localization of the immunotoxin to the periplasm.

[0060] As used herein, the terms "trastuzumab-deBouganin", "Herceptin- deBouganin", "T-deB", and "Herc-deB" refer to the humanized anti-Her2/neu antibody (described in US Patent Nos. 5677171 ; 5821337; 6054297; 6165464; 6339142; 6407213; 6639055; 6719971 ; 6800738; 7074404, which are incorporated herein by reference in their entirety, and in Coussens et al. (1985) Science 230: 1132-1139; Slamon et al. (1989) Science 244:707-712; Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89: 4285-4289; Slamon et al. (2001) New Engl. J. Med. 344:783-792, also herein incorporated by reference in their entirety) chemically conjugated to deimmunized Bouganin. As used herein, the term "T- DM1 " refers to trastuzumab conjugated to maytansinoid, a microtubule-disrupting agent.

[0061] As used herein, the term "heavy chain variable region" refers to the variable region of a heavy chain of an antibody molecule. The heavy chain variable region has three complementarity determining regions (CDRs) termed heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR- H2) and heavy chain complementarity determining region 3 (CDR-H3) from the amino terminus to carboxy terminus. In one embodiment, the heavy chain CDRs comprise SEQ ID NOs: 5-7. In another embodiment, the heavy chain CDRs comprise SEQ ID NOs: 105-107.

[0062] As used herein, the term "heterologous" means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

[0063] As used herein, the terms "homologous sequences" or "homologs" are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. The degree of sequence identity may vary, but in one embodiment, is at least 50% (when using standard sequence alignment programs known in the art), at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA).

[0064] As used herein, the term "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell, and to the progeny thereof.

[0065] "Humanized antibody or antibody fragment" means that the antibody or fragment comprises human framework regions.

[0066] The term "immunologic activity" of HER2/neu binding protein or EpCAM binding protein refers to the ability to specifically bind a human epidermal growth factor receptor 2 or an epithelial cell adhesion molecule, respectively. Such binding may or may not elicit an immune response. A specific immune response may comprise antibody, B cells, T cells, and any combination thereof, and effector functions resulting therefrom. Included are the antibody-mediated functions ADCC and complement-mediated cytolysis (CDC). The T cell response includes T helper cell function, cytotoxic T cell function, inflammation/inducer T cell function, and T cell mediated suppression. A compound able to elicit a specific immune response according to any of these criteria is referred to as "immunogenic."

[0067] The term "immune response" includes both cellular and humoral immune responses. In a preferred embodiment, a deimmunized bouganin toxin has a reduced propensity to activate T-cells. In another embodiment, a deimmunized furin linker has a reduced propensity to activate T-cells. [0068] "Immunoconjugate" refers to a binding protein conjugated to an effector molecule. In one embodiment, the binding protein is an antibody. In another embodiment, the antibody may be full length antibody or antibody fragments, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof, and fragments from recombinant sources and/or produced in transgenic animals. In some embodiments, the antibody may be a synthetic protein, a binding protein or a polypeptide. In some embodiments, the effector molecule may be a toxin, a radionucleotide, a radiopharmaceutical, a labeling agent, a drug, a cytotoxic agent, a peptide, a protein and the like. These effector molecules may be capable of killing, lysing or labeling or inducing other effects when the antibody binds to an antigen.

[0069] The term "immunotoxin" comprises (1) a binding protein attached to (2) a toxin. The terms "immunotoxin" and "immunoconjugate" are used interchangeably herein.

[0070] The phrase "the immunotoxin is administered directly to the cancer site" refers to direct or substantially direct introduction including, without limitation, single or multiple injections of the immunotoxin directly into the tumor or peritumorally, continuous or discontinuous perfusion into the tumor or peritumorally, introduction of a reservoir into the tumor or peritumorally, introduction of a slow-release apparatus into the tumor or peritumorally, introduction of a slow-release formulation into the tumor or peritumorally, direct application onto the tumor, direct injection into an artery that substantially directly feeds the area of the tumor, direct injection into a lymphatic vessel that substantially drains into the area of the tumor, direct or substantially direct introduction in a substantially enclosed cavity (e.g., pleural cavity) or lumen (e.g., intravesicular). "Peritumoral" is a term that describes a region, within about 10 cm, preferably within 5 cm, more preferably within 1 cm, of what is regarded as the tumor boundary, such as, but not limited to, a palpable tumor border. "Direct administration" in the context of prevention of occurrence or prevention of recurrence is defined as administration directly into a site at risk for development or recurrence of a cancer. In one embodiment, direct administration is by systemic delivery.

[0071] A "leader sequence" is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmic reticulum in eukaryotic cells, and either the inner membrane or both inner and outer membranes of bacteria. Leader sequences are typically at the N-terminal portion of a polypeptide and are typically removed enzymatically between biosynthesis and secretion of the polypeptide from the cell. The leader sequence is not present in the secreted protein, only during protein production. In one embodiment, the leader sequence comprises PelB (pectate lyase B) shown in SEQ ID NO: 21.

[0072] The phrase "ligand that binds to a protein on the cancer cell" includes any molecule that can selectively target the immunotoxin to the cancer cell by binding to a protein on the cancer cells. The targeted protein on the cancer cell is preferably a tumor associated antigen that is expressed at higher levels on the cancer cell as compared to normal cells.

[0073] The term "light chain variable region" refers to the variable region of a light chain of an antibody molecule. Light chain variable regions have three complementarity determining regions (CDRs) termed light chain complementarity determining region 1 (CDR- Ll), light chain complementarity determining region 2 (CDR-L2) and light chain complementarity determining region 3 (CDR-L3) from the amino terminus to the carboxy terminus. In one embodiment, the light chain CDRs comprise SEQ ID NOs: 8-10. In another embodiment, the light chain CDRs comprise SEQ ID NOs: 108-110.

[0074] The term "linker" or "peptide linker" refers to a short peptide sequence that occurs between protein domains. In one embodiment, linkers are composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another. In another embodiment, linkers are rigid and function to prohibit unwanted interactions between discrete protein domains. Fusion proteins or polypeptides can use linkers to connect the regions that do not naturally occur together in nature. In a particular embodiment, a furin protease sensitive peptide linker connects, links, joins or fuses a toxin to a binding protein that recognizes one or more tumor associated antigens on the surface of cancer cells. A "furin protease sensitive peptide linker", "furin protease sensitive linker" or "furin linker" comprises a furin cleavage site that is recognized and cleaved by furin, an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. This encoded protein is a calcium- dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. In one embodiment, a furin protease sensitive peptide linker fuses a binding protein portion to a toxin portion in an immunotoxin. The toxin is cleaved from the binding protein of the immunotoxin by a furin enzyme once the immunotoxin is internalized in a cancer cell, allowing the free toxin to exert its cytotoxic effect. [0075] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0076] The term "polynucleotide" is a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and analogs in any combination analogs. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes double-, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form of either the DNA, RNA or hybrid molecules.

[0077] The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

[0078] The term "recombinant" polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.

[0079] "Pharmaceutically acceptable" refers to general clinical use and/or approval by a regulatory agency of the Federal or state government, listing in the United States Pharmacopoeia, or general acceptance by those skilled in the relevant art. [0080] "Physiologic conditions" for antibody binding reflect but do not necessarily exactly duplicate the conditions in which, for instance, an EpCAM-binding polypeptide would encounter an EpCAM molecule in vivo. Binding under physiologic conditions should be reasonably predictive that binding in vivo will occur.

[0081] "Preventing cancer" refers to prevention of cancer occurrence. In certain instances, the preventative treatment reduces the recurrence of the cancer. In other instances, preventative treatment decreases the risk of a patient from developing a cancer, or inhibits progression of a pre-cancerous state (e.g. a colon polyp) to actual malignancy.

[0082] "Reduced dose" refers to a dose that is below the normally administered and/or recommended dose. The normally administered dose of an anticancer agent can be found in reference materials well known in the art such as, for example, the latest edition of the Physician's Desk Reference.

[0083] "Reduced propensity to elicit an immune response" as used herein means that the modified anti-EpCAM antibody or the antibody fragment is less immunogenic than non- modified anti-EpCAM antibody. In another embodiment, a modified furin protease sensitive linker is less immunogenic than a non-modified furin protease sensitive linker.

[0084] The term "reduced propensity to activate human T-cells" as used herein means the modified furin protease sensitive peptide linker has a reduced propensity to activate human T-cells as compared to the non-modified furin protease sensitive peptide linker. One of skill in the art can test whether or not a modified furin protease sensitive peptide linker has a reduced propensity to activate T-cells using assays known in the art including assessing the stimulation index of the peptide.

[0085] The term "scaffold" refers to at least one engineered protein, polypeptide or protein domain that yields specificity and affinity for a particular antigen or antigens. The scaffolds can include a diverse group of compact and stably folded proteins differing in size, structure and origin that serve as novel binding reagents. The scaffolds can be generated by rational design and molecular evolution procedures, often involving creating a random library by mutagenesis. The random library consists of a collection of amino acid sequences focused at a loop region or at an otherwise permissible surface area, and selection of variant amino acid sequences against a given target biomolecule or antigen can be by known molecular display methods such as phage display, yeast display, ribosome/mRNA display or other techniques. In addition to target specificity and affinity, scaffolds can also possess other desirable molecular properties, such as stability, better tissue penetration, solubility, and pharmacokinetic behavior. In one embodiment, a HER2/neu scaffold has specificity and affinity for a human epidermal growth factor receptor 2. In another embodiment, an EpCAM scaffold has specificity and affinity for an epithelial cell adhesion molecule. Examples of a protein and/or protein domain that is engineered as a scaffold include an Affibody ^® , a Kunitz protease inhibitor domain, a fibronectin domain, a lipocalin domain, a designed ankyrin repeat domain, a thioredoxin, a cell surface receptor A domain, and/or a cysteine-rich knottin peptide. The present invention also contemplates scaffolds that incorporate only non- immunoglobulin components or both non-immunoglobulin and immunoglobulin components.

[0086] The term "stimulation index" as used herein refers to the measure of the ability of the modified or non-modified furin protease sensitive peptide linker to activate human T cells. For example, the modified or non-modified furin protease sensitive peptide linkers thereof, can be tested for their ability to evoke a proliferative response in human T-cells cultured in vitro. Where this type of approach is conducted using naive human T-cells taken from healthy donors, the inventors have established that in the operation of such an assay, a stimulation index equal to or greater than 2.0 is a useful measure of induced proliferation. The stimulation index is conventionally derived by division of the proliferation score (e.g. counts per minute of radioactivity if using H-thymidine incorporation) measured to the test peptide by the score measured in cells not contacted with a test peptide.

[0087] The term "T-cell epitope" means an amino acid sequence which is able to bind a major histocompatibility complex (MHC), able to stimulate T-cells and/or also able to bind (without necessarily measurably activating) T-cells in complex with MHC.

[0088] "Therapeutic" means an agent utilized to discourage, combat, ameliorate, prevent or improve an unwanted condition, disease or symptom of a patient.

[0089] The term "toxin" refers to any anticellular agent, and includes, but is not limited to, cytotoxins and/or any combination of anticellular agents. In certain aspects, the toxin is, for example, a plant toxin, a fungal toxin, a bacterial toxin, a ribosome inactivating protein (RIP) or a combination thereof. Toxins include, but are not limited to, Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Shiga- like toxin, Gelonin, Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barley toxin, Bouganin and various other toxins known in the art. Modified bouganin proteins are described in WO 2005/090579, which is incorporated herein by reference in its entirety.

[0090] "Treating cancer" refers to inhibition of cancer cell replication, apoptosis, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction of cancer cell number or tumor growth, decrease in the malignant grade of a cancer (e.g., increased differentiation), or improved cancer-related symptoms. [0091] The term "V region" or "V domain" of an antibody or antibody fragment refers to the variable region or domain of a light chain or the variable region or domain of a heavy chain, either alone or in combination. These V regions are contained in, for example, SEQ ID NOs: 2, 4, 49, 51, 53, 55, 56, 57, 99-101, 103, 120-123, 125, 127, 129, 131, 133, 135.

[0092] "Variant" refers to any pharmaceutically acceptable derivative, analogue, or fragment of an immunotoxin, an antibody or antibody fragment, a toxin (e.g., Pseudomonas toxin), or an effector molecule described herein. A variant also encompasses one or more components of a multimer, multimers comprising an individual component, multimers comprising multiples of an individual component (e.g., multimers of a reference molecule), a chemical breakdown product, and a biological breakdown product. In particular, non- limiting embodiments, an immunotoxin may be a "variant" relative to a reference immunotoxin by virtue of alteration(s) in the EpCAM-binding portion and/or the toxin portion of the reference immunotoxin. For example, a variant immunotoxin may contain multimers of the antibody portion and/or the toxin portion. A variant of the toxin portion of the molecule retains toxicity of at least 10%, at least 30%, at least 50%, at least 80%, at least 90%, in a standard assay used to measure toxicity of a preparation of the reference toxin. In some embodiments, a variant may also refer to polypeptides having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 95% sequence identity to the immunotoxin of the present invention. In some embodiments, a variant antibody may refer to polypeptides or proteins having at least 30%, at least 60%, at least 70%, at least 80%, at least 90%, or 95% sequence identity of the antibody of the present invention. In some embodiments, a variant antibody or the immnunoconjugate may refer to polypeptides or proteins having at least 30%, at least 60%, at least 70%, at least 80%, at least 90%, or 95% binding affinity of the antibody of the present invention when measured by a competitive binding assay.

[0093] A variant immunotoxin having a variation of the EpCAM-binding portion of the reference immunotoxin competes with the binding of an anti-EpCAM reference antibody, under physiologic conditions, by at least 10 percent and preferably at least 30 percent (and see infra). Competition by 10 percent means that, in an assay where a saturating concentration of anti-EpCAM reference antibody is bound to EpCAM, 10 percent of these bound reference antibodies is displaced when equilibrium is reached with an equivalent concentration of the variant anti-EpCAM immunotoxin. As a non-limiting example, competition between antibodies, or between an antibody and an immunotoxin, is measured by binding labeled anti-EpCAM reference antibody to EpCAM on the surface of cells or to an EpCAM-coated solid substrate, such that virtually all EpCAM sites are bound by the antibody, contacting these antibody-antigen complexes with unlabeled test anti-EpCAM antibody or unlabeled test immunotoxin, and measuring the amount of labeled antibody displaced from EpCAM binding sites, wherein the amount of freed, labeled antibody indicates the amount of competition that has occurred.

[0094] The term "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered into a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of therapy, and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors for the replication of a polynucleotide, and expression vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors, which comprise a polynucleotide encapsidated or enveloped in a viral particle.

[0095] "VB5-845" refers to a Fab fragment of EpCAM antibody without toxin conjugate.

[0096] "VB6-845" or "VB6-901" are used interchangeably herein and refer to a Fab fragment of anti-EpCAM antibody genetically linked to a modified bouganin protein (deBouganin) toxin. VB6-845 has been described in U.S. Patent No. 7,339,031 and is incorporated herein by reference in its entirety.

[0097] "4D5MOC-B" or "4D5" means the humanized scFv MOC31 antibody grafted onto the artificial human consensus framework of scFv 4D5 as described in WO 00/61635 which is incorporated herein by reference in its entirety. 4D5MOC-B is represented by SEQ ID NO: 66. "MOC-31 antibody" means the murine anti-EpCAM or anti-EGP-2 antibody and is available from commercial sources such as BioGenex, Cat No. MU316-UC, Zymed Laboratories Inc., Cat No. 18-0270 or United States Biological, Cat No. M4165.

Therapeutic proteins

[0098] Immunotherapy has emerged as a powerful tool to combat cancer. Murine and humanized/chimeric antibodies, and their respective antibody fragments, directed against tumor-associated antigens ("TAAs") have been used for diagnosis and therapy of certain human cancers. Unconjugated, toxin-conjugated, and radiolabeled forms of these antibodies have been used in such therapies.

[0099] One tumor associated antigen of interest for immunotherapy is Epithelial Cell Adhesion Molecule (EpCAM) which is also known as 17-1 A, KSA, EGP-2 and GA733-2. EpCAM is a transmembrane protein that is highly expressed in many solid tumors, including carcinomas of the lung, breast, ovary, colorectal, and squamous cell carcinoma of the head and neck, but weakly expressed in most normal epithelial tissues. Its expression correlates with the rate of cellular proliferation. EpCAM-specific antibodies have been used to image and detect primary tumors and metastases in patients with small cell lung cancer and non- small cell lung cancer.

[0100] Another tumor associated antigen of interest for immunotherapy is human epidermal growth factor receptor 2 (HER2/neu), a transmembrane glycoprotein with tyrosine kinase activity, which is also known as erbB2. HER2/neu is highly expressed in breast cancer cells. The amplification of the HER2/neu gene occurs in 20-30% of human breast cancers and is associated with aggressive tumor growth and poor clinical outcome. A murine monoclonal antibody, 4D5, effective at stopping growth of HER2-overexpressing cell lines and xenografts, was humanized (Molina et al., 2001, Trastuzumab (Herceptin ^® ), a humanized anti-HER2 receptor monoclonal antibody, inhibits basal and activated HER2 ectodomain cleavage in breast cancer cells, Cancer Research 61 : 4744-4749). The resulting humanized anti-HER2 antibody of the IgGl isotype, trastuzumab (Herceptin ^® ), has been approved for treatment of HER2 overexpressing metastatic breast cancer.

[0101] Another tumor associated antigen of interest for immunotherapy is chondroitin sulfate A (CSA), a glycosaminoglycan found on the surface of a wide variety of cancer cells. An Hl l antibody or antibody fragment recognizes CSA.

[0102] There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings, but in certain cases, have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response. For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response. These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant "humanized" antibodies have, in several cases, still elicited an immune response in patients.

[0103] The key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC molecules, so-called "T-cell epitopes". Such T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC molecules. Implicitly, a "T-cell epitope" means an epitope, which when bound to MHC molecules, can be recognized by a T- cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.

[0104] Each part of an immunotoxin, such as the binding protein portion, the toxin, and the linker that joins the two can potentially harbor peptides that can trigger an immune response. Thus, it is desirable to identify and to remove T-cell epitopes from therapeutic proteins such as immunotoxins so that they elicit a reduced immune response.

Peptide linkers

[0105] Disclosed herein are de-immunized peptide linkers that can be used to link the binding protein portion and the toxin portion of an immunotoxin.

[0106] In one embodiment, the deimmunized peptide linker is a modified furin protease sensitive peptide linker. A furin protease sensitive peptide linker comprises a furin cleavage site that is recognized and cleaved by furin, an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. This encoded protein is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. When the furin protease sensitive peptide linker is used to link a toxin and a binding protein in an immunotoxin, the linker is cleaved when the immunotoxin is taken up by a cancer cell. The toxin released from the immunotoxin by cleavage can then kill the cancer cell by, for example, interfering with translation by ribosomes as in the case of the bouganin toxin.

[0107] In one embodiment, the modified furin protease sensitive peptide linker has a reduced propensity to activate an immune response. In another embodiment, the peptide linker has a reduced propensity to activate T-cells compared to a non-modified furin protease sensitive peptide linker and has a biological function that is comparable to non- modified furin protease sensitive peptide linker.

[0108] In some embodiments, the modified furin protease sensitive peptide linker is modified at one or more T-cell epitopes in the peptide linker sequence.

[0109] In one embodiment, a method that can be used to generate the modified furin protease sensitive peptide linkers with modified T-cell epitopes comprises the following steps: (i) determining the amino acid sequence of the peptide linker; (ii) identifying one or more potential T-cell epitopes within the amino acid sequence of the peptide linker by methods such as determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (iii) designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptide linkers to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in rum, modified in such a way to substantially reduce or eliminate the activity of the T- cell epitope; (iv) constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties according to well-known recombinant techniques; and (v) optionally repeating steps (ii) to (iv). In an example, step (iii) is carried out by substitution, addition or deletion of amino acid residues in any of the T-cell epitopes in the non-modified furin protease sensitive peptide linker. In another example, the method to make the modified furin protease sensitive peptide linker is made with reference to the homologous peptide sequence and/or in silico modeling.

[0110] In an embodiment of the invention, the modified furin protease sensitive peptide linker has at least one T-cell epitope removed. In another embodiment the modified furin protease sensitive peptide linker has biological function, such as the linking of the binding protein portion and the toxin portion of an immunotoxin.

[0111] For the elimination of T-cell epitopes, amino acid substitutions are made at appropriate points within the peptide sequence predicted to achieve substantial reduction or elimination of the activity of the T-cell epitope. In practice an appropriate point will in one embodiment equate to an amino acid residue binding within one of the pockets provided within the MHC class II binding groove.

[0112] In one embodiment, the epitopes are compromised by mutation to result in sequences no longer able to function as T-cell epitopes. It is possible to use recombinant DNA methods to achieve directed mutagenesis of the target sequences and many such techniques are available and well known in the art. In practice a number of modified furin protease sensitive peptide linkers will be produced and tested for the desired immune and functional characteristic. Mutations to the peptide linker to eliminate T-cell epitopes are also dependent upon the sequence context of the peptide linker in an immunotoxin construct, for example, what sequence residues exist at the amino-terminal and carboxy-terminal junctions of the peptide linker. It is particularly important when conducting modifications to the peptide sequence that the contemplated changes do not introduce new immunogenic epitopes. This event is avoided in practice by re-testing the contemplated sequence for the presence of epitopes and/or of MHC class II ligands by any suitable means. [0113] In one embodiment, the peptide linker is modified at one or more amino acid residues in a T-cell epitope represented by SEQ ID NO: 17. In another embodiment, the peptide linker is modified at amino acid position 12 of SEQ ID NO: 17.

[0114] In one embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 32. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 33. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 34. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 35. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 36. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 119. In another embodiment, the amino acid sequence of the modified peptide linker comprises SEQ ID NO: 124. In certain embodiments, the amino acid sequence of the modified peptide linker shares at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 17.

Binding proteins

[0115] Disclosed herein are immunotoxins comprising: (a) a binding protein and; (b) a toxin, wherein the binding protein and toxin are linked by a deimmunized peptide linker. In one embodiment, the binding protein comprises an antibody or antibody fragment. In another embodiment, the antibody or antibody fragment comprises an anti-epithelial cell adhesion molecule (EpCAM) antibody or an anti-EpCAM antibody fragment. In some embodiments, the antibody may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain with an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. The antibody may be full length antibody or antibody fragments, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof.

[0116] In some embodiments, the anti-EpCAM antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 55.

[0117] In one embodiment, the antibody or antibody fragment comprises an anti- HER2/neu antibody or an anti-HER2/neu antibody fragment. In some embodiments, the anti- HER2/neu antibody or the anti-HER2/neu antibody fragment comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10. The antibody may be full length antibody or antibody fragments, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof. In one embodiment, the anti-HER2/neu antibody fragment is a diabody. In another embodiment, the diabody is comprised of a heavy chain variable region and a light chain variable region. In a further embodiment, the heavy chain variable region and the light chain variable region are linked by a linker. In yet another embodiment, the linker is encoded by an amino acid sequence shown in SEQ ID NO: 15. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by an amino acid sequence set forth in SEQ ID NO: 131.

[0118] In some embodiments, the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a heavy chain variable region encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the anti-HER2/neu antibody or the anti-HER2/neu antibody fragment comprises a light chain variable region encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 4.

[0119] In one embodiment, the antibody or antibody fragment comprises an Hl l antibody or Hl l antibody fragment. In some embodiments, the Hl l antibody or Hl l antibody fragment comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 105-110. The antibody may be full length antibody or antibody fragments, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof.

[0120] In one embodiment, the HI 1 antibody or antibody fragment comprises a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the Hl l antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0121] The antibody or fragment may be from any species including mice, rats, rabbits, hamsters and humans. Chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, humanized antibodies which comprise the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81 : 6851 (1985); Takeda et al, Nature 314: 452 (1985), Cabilly et al, U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al, European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom patent GB 2177096B).

[0122] In some embodiments, the sequences of the light chain and the heavy chain fragments may be modified or replaced with other amino acids such that the antibody elicits reduced immune response in humans. For making humanized antibody or antibody fragments, any method known in the art may be used. For example, human antibody fragments can be obtained by screening human antibody libraries. Another solution is to transplant the specificity of a non-human monoclonal antibody by grafting the CDR regions onto a human framework. In an improvement of said technique, humanized antibodies or antibody fragments with improved binding behavior can be produced by incorporating additional residues derived from said non-human antibody. In addition to achieving humanization, techniques to "repair" antibody fragments with suboptimal stability and/or folding or yield may be used by grafting the CDRs of a scFv fragment with the desired binding affinity and specificity onto the framework of a different, better behaved scFv. Such methods for making humanized antibodies or antibody fragments are well known in the art and include, by way of example, production in SCID mice, and in vitro immunization. [0123] Specific antibodies, or antibody fragments, reactive to proteins on cancer cells may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules encoding the proteins. For example, complete Fab fragments, VH regions and Fv regions can be expressed in bacteria using phage expression libraries (See for example Ward et al, Nature 341 : 544-546: (1989); Huse et al, Science 246: 1275-1281 (1989); and McCafferty et al. Nature 348: 552-554 (1990)). Alternatively, a SCID-hu mouse, for example the model developed by Genpharm, can be used to produce antibodies or fragments thereof.

[0124] In some embodiments, the antibody fragment may be Fab, and the light chain and the heavy chain are linked by a covalent bond. In some embodiments, the covalent linkage may be disulfide bond. In some embodiments, the covalent linkage may be through chemical crosslinkers, such as dimethyl adipimidate, dimethyl suberimidate, and the like. In some embodiments, amino acid crosslinkers, such as (Gly ₄ -Ser) _n may be used. The sequences of the light chain and the heavy chain described herein may be used to derive scFv, diabodies, tribodies, tetrabodies, and the like. Various protein linking strategies may be used to produce bivalent or bispecific Fab and scFvs, as well as bifunctional Fab and scFv fusions.

[0125] In one embodiment, the immunotoxin comprises an anti-EpCAM antibody fragment. In another embodiment, the anti-EpCAM antibody fragment is an Fab. In another embodiment, the Fab is comprised of a heavy chain with an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain with an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In another embodiment, the anti- EpCAM Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti- EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-EpCAM Fab described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 51.

[0126] In one embodiment, the immunotoxin comprises an anti-HER2/neu antibody fragment. In another embodiment, the anti-HER2/neu antibody fragment is a diabody. In one embodiment, the anti-HER2/neu diabody comprises a heavy chain variable region and a light chain variable region. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments the HER2/neu heavy chain variable region and light chain variable region are encoded by an amino acid sequence set forth in SEQ ID NO: 131. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the heavy chain variable region and the light chain variable region are linked by a linker. In another embodiment, the linker is encoded by an amino acid sequence shown in SEQ ID NO: 15.

[0127] In one embodiment, the immunotoxin comprises an HI 1 antibody fragment. In another embodiment, the Hl l antibody fragment is an Fab. In another embodiment, the Fab is comprised of a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the Hl l antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0128] The antibody fragments described herein may be cloned and expressed in E. coli in a biologically functional form. Antibodies and antibody fragments may also be produced by recombinant DNA technology using either bacterial or mammalian cells.

[0129] In some embodiments, affinity maturation process may be used whereby the binding specificity, affinity or avidity of the antibody described herein can be modified. A number of laboratory techniques have been devised whereby amino acid sequence diversity is created by the application of various mutation strategies, either on the entire antibody fragment or on selected regions such as the CDRs.

[0130] In some embodiments, the variant amino acid sequences of the heavy chain and the light chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 49 and 51, respectively. In other embodiments, the variant amino acid sequences of the heavy chain and the light chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 53 and 55, respectively. [0131] In some embodiments, the variant amino acid sequences of the heavy chain variable region have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NO: 2. In other embodiments, the variant amino acid sequences of the light chain variable region have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NO: 4. In other embodiments, the variant amino acid sequences of the heavy chain variable region and the light chain variable region have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 125, 127, 129, 131, 133 and 135. In other embodiments, the variant amino acid sequences of the heavy chain variable region and the light chain variable region have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NO: 131.

[0132] In some embodiments, the variant amino acid sequences of the heavy chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOs: 99 and 101. In other embodiments, the variant amino acid sequences of the light chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 100 and 103. In certain embodiments, the variant amino acid sequences of the light chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 120 and 123. In other embodiments, the variant amino acid sequences of the heavy chain and the light chain have at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably 95% sequence identity to SEQ ID NOS: 99 and 120, respectively.

[0133] The binding protein portion of the immunotoxin may be immunoglobulin derived, i.e., can be traced to a starting molecule that is an immunoglobulin (or antibody). For example, the ligand may be produced by modification of an immunoglobulin scaffold using standard techniques known in the art. In another, non-limiting example, immunoglobulin domains (e.g., variable heavy and/or light chains) may be linked to a non-immunoglobulin scaffold. Non-immunoglobulin scaffolds can include an Affibody ^® , a Kunitz protease inhibitor domain, a fibronectin domain, a lipocalin domain, a designed ankyrin repeat domain, a thioredoxin, a cell surface receptor A domain, and/or a cysteine-rich knottin peptide. Further, the ligand may be developed by, without limitation, chemical reaction or genetic design. Accordingly, in a non-limiting example, an immunotoxin may comprise (1) an immunoglobulin-derived polypeptide (e.g., an antibody selected from an antibody library), or variant thereof, that specifically binds to cancer cells, and (2) a deimmunized bouganin toxin or variant thereof. Such immunoglobulin polypeptide ligands can be re-designed to affect their binding characteristics to a target tumor associated molecule, or to improve their physical characteristics, for example.

Immunotoxins

[0134] In some embodiments, immunotoxins are provided. In one embodiment, the immunotoxin disclosed herein may be an antibody or antibody fragment attached to an effector molecule by a deimmunized furin linker. In one embodiment, the antibody or antibody fragment comprises a human kappa light chain. In another embodiment, the effector molecule is deimmunized bouganin encoded by an amino acid sequence selected from SEQ ID NOs: 12, 58, 59, 60 and 61. In another embodiment, the antibody fragment may be an Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof. In yet another embodiment, the immunotoxins can comprise SEQ ID NOs: 67, 69, 71, 73, 75-89, 92, 94-98, 99-104, 120-123, 125, 127, 129, 131, 133 and 135.

[0135] In some embodiments, the immunotoxin disclosed herein may be an antibody attached to an effector molecule, wherein the antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In some embodiments, the antibody may be an antibody fragment, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof. The antibody or the antibody fragment may be any of the de-immunized antibodies or de-immunized antibody fragments disclosed herein.

[0136] In some embodiments, the antibody in the immunotoxin may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibody in the immunotoxin may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody in the immunotoxin may have a heavy chain with an amino acid sequence of SEQ ID NO: 49, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody in the immunotoxin may have a heavy chain with an amino acid sequence of SEQ ID NO: 53, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 56, and a light chain with an amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibodies described herein may have a heavy chain with an amino acid sequence of SEQ ID NO: 57, and a light chain with an amino acid sequence of SEQ ID NO: 55.

[0137] In some embodiments, the immunotoxin disclosed herein may be an antibody attached to an effector molecule, wherein the antibody comprises a heavy chain variable region encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the antibody comprises a light chain variable region encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the antibody comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10. In some embodiments, the antibody may be an antibody fragment, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof. In one embodiment, the antibody fragment is a diabody. In another embodiment, the diabody is comprised of a heavy chain variable region and a light chain variable region. In a further embodiment, the heavy chain variable region and the light chain variable region are linked by a linker. In yet another embodiment, the linker is encoded by an amino acid sequence shown in SEQ ID NO: 15. In some embodiments the heavy chain variable region and light chain variable region are encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments the heavy chain variable region and light chain variable region are encoded by an amino acid sequence set forth in SEQ ID NO: 131.

[0138] In some embodiments, the immunotoxin disclosed herein may be an antibody attached to an effector molecule, wherein the antibody comprises a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the Hl l antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In some embodiments, the antibody comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 105- 110. In some embodiments, the antibody may be an antibody fragment, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof.

[0139] In the embodiments described herein, the effector molecule may be radioisotopes, antineoplastic agents, immunomodulators, biological response modifiers, lectins, toxins, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof. In some embodiments, the effector molecule may be a toxin, such as abrin, modeccin, viscumin, gelonin, bouganin, modified or de-immunized bouganin protein (deBouganin), saporin, ricin, ricin A chain, bryodin, luffin, momordin, restrictocin, Pseudomonas exotoxin A, pertussis toxin, tetanus toxin, botulinum toxin, Shigella toxin, cholera toxin, diphtheria toxin and any combination thereof. In embodiments, the toxin may be deBouganin as shown in SEQ ID NO: 12, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61. In another embodiment, the toxin may be deimmunized diphtheria toxin.

[0140] In other non-limiting embodiments, the toxin comprises an agent that acts to disrupt DNA. Thus, toxins may be, without limitation, enediynes (e.g., calicheamicin and esperamicin) and non-enediyne small molecule agents (e.g., bleomycin, methidiumpropyl- EDTA-Fe(II)). Other toxins useful in accordance with the invention include, without limitation, daunorubicin, doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins, duocarmycin/CC-1065, and bleomycin/pepleomycin. In other non-limiting embodiments, the toxin comprises an agent that acts to disrupt tubulin. Such toxins may comprise, without limitation, rhizoxin/maytansine, paclitaxel, vincristine and vinblastine, colchicine, auristatin, dolastatin 10, peloruside A, alkylating agents, antimitotic agents, topoisomerase I inhibitors, and camptothecin derivatives.

[0141] In other nonlimiting embodiments, the toxin comprises an agent that acts to disrupt tubulin. Such toxins may comprise, without limitation, rhizoxin/maytansine, paclitaxel, vincristine and vinblastine, colchicine, auristatin, dolastatin 10, peloruside A, alkylating agents, antimitotic agents, topoisomerase I inhibitors, and camptothecin derivatives.

[0142] In other non-limiting embodiments, the toxin portion of the immunotoxin may be an alkylating agent including, without limitation, busulfan, carboxyphthalatoplatinum, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan, mitomycin C, mitozolamide, nitrogen mustard, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, triethylenemelamine, and the like.

[0143] In other non-limiting embodiments, the toxin portion of the immunotoxin of the invention may be an antimitotic agent including, without limitation, allocolchicine, halichondrin B, colchicine, colchicine derivative, maytansine, rhizoxin, taxol, taxol derivative, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate.

[0144] In other nonlimiting embodiments, the toxin portion of an immunotoxin of the invention may comprise an topoisomerase I inhibitor including, without limitation, camptothecin NSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC 603071, camptothecin derivative NSC 95382, camptothecin derivative NSC 107124, camptothecin derivative NSC 643833, camptothecin derivative NSC 629971, camptothecin derivative NSC 295500, camptothecin derivative NSC 249910, camptothecin derivative NSC 606985, camptothecin derivative NSC 374028, camptothecin derivative NSC 176323, camptothecin derivative NSC 295501, camptothecin derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin derivative NSC 610458, camptothecin derivative NSC 618939, camptothecin derivative NSC 610457, camptothecin derivative NSC 610459, camptothecin derivative NSC 606499, camptothecin derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin derivative NSC 606497, and morpholinodoxorubicin NSC 354646.

[0145] In other non-limiting embodiments, the toxin portion of an immunotoxin of the invention may comprise a topoisomerase II inhibitor including, without limitation, doxorubicin, amonafide, anthrapyrazole derivative, pyrazoloacridine, bisantrene HC1, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, Ν,Ν-dibenzyl daunomycin, oxanthrazole, and rubidazone.

[0146] In other non-limiting embodiments, the toxin portion of the immunotoxin may be an RNA or DNA antimetabolite including, without limitation 5-azacytidine, 5- fluorouracil, acivicin, aminopterin, aminopterin derivative, 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivative, N-(phosphonoacetyl)-L-aspartate, pyrazofurin, trimetrexate, 2'-deoxy-5-fluorouridine, aphidicolin glycinate, 5-aza-2'-deoxycytidine, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, and thiopurine.

[0147] Furthermore, a cytotoxin may be altered to decrease or inhibit binding outside of the context of the immunotoxin, or to reduce specific types of toxicity. For example, the cytotoxin may be altered to adjust the isoelectric point to approximately 7.0 such that liver toxicity is reduced.

[0148] In some embodiments, the immunotoxin is a humanized antibody fragment that binds to the extracellular domain of human EpCAM linked to a modified bouganin. In particular, the immunotoxin may be a recombinant stabilized and humanized Fab fragment of EpCAM antibody that has been fused to a modified bouganin by a deimmunized furin protease sensitive peptide linker. This immunotoxin may bind to EpCAM expressed on cancer cells. In another embodiment, the immunotoxin is a humanized antibody fragment that binds to human epidermal growth factor receptor 2 (HER2/neu) and is fused to a modified bouganin by a deimmunized furin protease sensitive peptide linker. In another embodiment, the immunotoxin is an Hl l antibody fragment that binds to CSA. Once bound, the immunotoxin is internalized and the toxin kills cells or blocks the protein synthesis, thereby leading to cell death. Importantly, since most normal normal cells do not widely express tumor-associated antigens, and therefore cannot internalize the immunotoxin, they are protected from the potential side-effects of the toxin.

[0149] In some embodiments, the immunotoxin may be a Fab attached to a modified bouganin by a deimmunized furin protease sensitive peptide linker. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 49. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 51. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 53. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 55. In some embodiments, the Fab may have a heavy chain with an amino acid sequence selected from SEQ ID NOs: 63, 92, 95 and 96 and a light chain with an amino acid sequence selected from SEQ ID NOs: 65, 94, 97 and 98.

[0150] In some embodiments, the immunotoxin may be a diabody attached to modified bouganin by a deimmunized furin protease sensitive peptide linker. In some embodiments, the diabody may have a heavy chain variable region with an amino acid sequence of SEQ ID NO: 2 and a light chain variable region with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 2. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 4. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 4. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 2.

[0151] In some embodiments, the immunotoxin may be an anti-HER2/neu diabody attached to modified bouganin encoded by amino acid sequences depicted by SEQ ID NOs: 23, 25, 27, 29, 31 (amino acid sequences) and SEQ ID NOs: 22, 24, 26, 28, 30 (nucleotide sequences). In another embodiment, the immunotoxin comprises amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23. In another embodiment, the immunotoxin comprises amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25. In another embodiment, the immunotoxin comprises amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 27. In another embodiment, the immunotoxin comprises amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 29. In another embodiment, the immunotoxin comprises amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the immunotoxin may be an anti- HER2/neu diabody attached to modified bouganin encoded by amino acid sequences depicted by SEQ ID NOs: 125, 127, 129, 131, 133 and 135 (amino acid sequences) and SEQ ID NOs: 126, 128, 130, 132, 134 and 136 (nucleotide sequences).

[0152] In some embodiments, the immunotoxin may be a Fab attached to a modified bouganin by a deimmunized furin protease sensitive peptide linker. In some embodiments, the Fab may have a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the HI 1 antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In some embodiments, the Fab may have a heavy chain with an amino acid sequence selected from SEQ ID NOs: 121 and 122 and a light chain with an amino acid sequence selected from SEQ ID NOs: 100 and 120. In certain embodiments, the Fab may have a heavy chain with an amino acid sequence shown in SEQ ID NO: 121 and a light chain with an amino acid sequence shown in SEQ ID NO: 120. In certain embodiments, the Fab may have a heavy chain with an amino acid sequence shown in SEQ ID NO: 122 and a light chain with an amino acid sequence shown in SEQ ID NO: 120.

[0153] In some embodiments, the immunotoxin may be an Fab attached to modified diphtheria toxin by a peptide linker. In some embodiments, the modified diphtheria toxin has a reduced propensity to elicit an immune response. In a preferred embodiment, the modified diphtheria toxin has a reduced propensity to activate T-cells and the modified diphtheria toxin is modified at one or more amino acid residues in a T-cell epitope. [0154] In some embodiments, the modified bouganin has a reduced propensity to elicit an immune response. In a preferred embodiment, the modified bouganin has a reduced propensity to activate T-cells and the modified bouganin is modified at one or more amino acid residues in a T-cell epitope. In some embodiments, the modified bouganin protein (deBouganin) has amino acids as shown in SEQ ID NO: 12. In some embodiments, the modified bouganin protein may have amino acids as shown in SEQ ID NO: 58. In some embodiments, the modified bouganin protein may have amino acids as shown in SEQ ID NO: 59. In some embodiments, the modified bouganin protein may have amino acids as shown in SEQ ID NO: 60. In some embodiments, the modified bouganin protein may have amino acids as shown in SEQ ID NO: 61.

[0155] In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 69 and a second polypeptide as shown in SEQ ID NO: 51. The first and the second polypeptide may be joined by one or more di-sulfide linkages. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 71. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 49 and a second polypeptide as shown in SEQ ID NO: 71. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 73 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 75. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 56 and a second polypeptide as shown in SEQ ID NO: 75. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 76 and a second polypeptide as shown in SEQ ID NO: 51. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 77. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 78 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 79. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 80 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 81 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 82 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 83 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 84 and a second polypeptide as shown in SEQ ID NO: 51. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 84 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 85. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 86 and a second polypeptide as shown in SEQ ID NO: 51. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 86 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide selected from SEQ ID NOS: 49, 53, 56, or 57, and a second polypeptide as shown in SEQ ID NO: 87. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 88 and a second polypeptide as shown in SEQ ID NO: 51. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 88 and a second polypeptide as shown in SEQ ID NO: 55. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 89 and a second polypeptide as shown in SEQ ID NO: 51. In some embodiments, the immunotoxin may be VB6-901 Fab having a first polypeptide as shown in SEQ ID NO: 89 and a second polypeptide as shown in SEQ ID NO: 55.

[0156] In some embodiments, the immunotoxin may be VB6-011 Fab having a first polypeptide as shown in SEQ ID NO: 121 and a second polypeptide as shown in SEQ ID NO: 120. In some embodiments, the immunotoxin may be VB6-011 Fab having a first polypeptide as shown in SEQ ID NO: 122 and a second polypeptide as shown in SEQ ID NO: 120.

[0157] The first and the second polypeptide disclosed herein in the above immunotoxins may be joined by one or more di-sulfide linkages.

[0158] The antibodies or the antibody fragments described herein may be conjugated to the effector molecule by any means. For example, the antibody or the antibody fragment may be attached to the toxin by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare the immunotoxin. The method used to conjugate the antibody or the antibody fragment and toxin must be capable of joining the antibody with the toxin without interfering with the ability of the antibody or the antibody fragment to bind to the target molecule.

[0159] In one embodiment, the antibody and toxin are both proteins and can be conjugated using techniques well known in the art. There are several hundred crosslinkers disclosed in the art that can conjugate two proteins. The crosslinker is generally chosen based on the reactive functional groups available or inserted on the antibody or toxin. In addition, if there are no reactive groups, a photoactivatible crosslinker can be used. In certain instances, it may be desirable to include a spacer between the antibody and the toxin. Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and bis(diazobenzidine) and the heterobifunctional agents: m- maleimidobenzoyl-N-hydrox-ysuccinimide and sulfo-m maleimidobenzoyl-N- hydroxysuccinimide.

[0160] Other crosslinkers that may be used to couple an effector molecule to the antibody fragment include TPCH(S-(2- thiopyridyl)-L-cysteine hydrazide and TPMPH ((S- (2-thiopyridyl) mercapto- propionohydrazide). TPCH and TPMPH react at the carbohydrate moieties of glycoproteins that have been previously oxidized by mild periodate treatment, thus forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate generated aldehydes. The hetero-bifunctional crosslinkers GMBS (N-gama- malimidobutyryloxy)-succinimide) and SMCC (succinimidyl 4-(N-maleimido- methyl)cyclohexane) react with primary amines, thus introducing a maleimide group onto the component. This maleimide group can subsequently react with sulfhydryls on the other component, which can be introduced by previously mentioned crosslinkers, thus forming a stable thioether bond between the components. If steric hindrance between components interferes with either component's activity, crosslinkers can be used which introduce long spacer arms between components and include derivatives, such as n-succinimidyl-3-(2- pyridyldithio)propionate (SPDP). Thus, there is an abundance of suitable crosslinkers that can be used and each of which is selected depending on the effects it has on optimal immunotoxin production.

[0161] An antibody-effector molecule fusion protein may also be prepared using recombinant DNA techniques. In such a case a DNA sequence encoding the antibody is fused to a DNA sequence encoding an effector molecule, such as a toxin, resulting in a chimeric DNA molecule. A cleavable linker can be inserted between the antibody and the effector molecule. The chimeric DNA sequence is transfected into a host cell that expresses the antibody-effector molecule fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.

[0162] In one embodiment, the cleavable linker fuses an anti-HER2/neu antibody or antibody fragment to a deBouganin toxin. In one embodiment, the cleavable linker fuses an anti-EpCAM antibody or antibody fragment to a deBouganin toxin. In another embodiment, the cleavable linker comprises a furin protease sensitive linker. In another embodiment, the furin linker has been mutated. In yet another embodiment, the mutated furin linker is deimmunized. In another embodiment, the amino acid sequence encoding the mutated furin linker is truncated compared to the wild type furin linker. In another embodiment, the amino acid sequence encoding the mutated furin linker has one or more amino acids substituted, deleted or added compared to the wild type furin linker. In one embodiment, the wild type furin linker is encoded by SEQ ID NO: 17 (amino acid sequence) and by SEQ ID NO: 16 (nucleotide sequence). In another embodiment, the mutated furin linker is encoded by SEQ ID NOs: 32-36, 119 and 124 (amino acid sequences) and by SEQ ID NOs: 38-47 (nucleotide sequences).

[0163] In a preferred embodiment, an immunotoxin that comprises an anti-EpCAM antibody or antibody fragment that binds EpCAM positive cancer cells is fused to a deimmunized Bouganin toxin by a furin linker of SEQ ID NO: 17. In another embodiment, the anti-EpCAM fragment comprises an Fab. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 49 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C- terminus of SEQ ID NO: 51 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 53 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 55 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 49 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 49 and a light chain with an amino acid sequence of SEQ ID NO: 51, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 51 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 53 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 53 and a light chain with an amino acid sequence of SEQ ID NO: 55, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 55 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124.

[0164] In a preferred embodiment, an immunotoxin that comprises an anti-HER2/neu antibody or antibody fragment that binds HER2/neu positive cancer cells is fused to a deimmunized Bouganin toxin by a furin linker of SEQ ID NO: 17. In another embodiment, the anti-HER2/neu antibody fragment comprises a diabody. In some embodiments, the diabody may have a heavy chain variable region with an amino acid sequence of SEQ ID NO: 2 and a light chain variable region with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the N-terminus of SEQ ID NO: 2 by a furin linker of SEQ ID NO: 17. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the C-terminus of SEQ ID NO: 4 by a furin linker of SEQ ID NO: 17. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the N-terminus of SEQ ID NO: 4 by a furin linker of SEQ ID NO: 17. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the C- terminus of SEQ ID NO: 2 by a furin linker of SEQ ID NO: 17. In some embodiments, the diabody may have a heavy chain variable region with an amino acid sequence of SEQ ID NO: 2 and a light chain variable region with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the N-terminus of SEQ ID NO: 2 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the diabody may have a heavy chain variable region with an amino acid sequence of SEQ ID NO: 2 and a light chain variable region with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the N-terminus of SEQ ID NO: 2 by a furin linker of SEQ ID NO: 119. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the C-terminus of SEQ ID NO: 4 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the N-terminus of SEQ ID NO: 4 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the diabody may have a heavy chain with an amino acid sequence of SEQ ID NO: 2 and a light chain with an amino acid sequence of SEQ ID NO: 4, and the modified bouganin protein is fused to the C-terminus of SEQ ID NO: 2 by a furin linker selected from SEQ ID NOs: 32-36, 119 and 124. In one embodiment, the anti- HER2/neu diabody comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10.

[0165] In a preferred embodiment, an immunotoxin that comprises an Hl l antibody or antibody fragment that binds CSA positive cancer cells is fused to a deimmunized Bouganin toxin by a furin linker of SEQ ID NO: 17. In another embodiment, the Hl l antibody fragment comprises an Fab. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 100, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the N- terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 100, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 17. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the C-terminus of SEQ ID NO: 99 by a furin linker selected from the group consisting of SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 100, and the modified bouganin is fused to the C- terminus of SEQ ID NO: 99 by a furin linker selected from the group consisting of SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 99 by a furin linker selected from the group consisting of SEQ ID NOs: 32-36, 119 and 124. In some embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 100, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 99 by a furin linker selected from the group consisting of SEQ ID NOs: 32-36, 119 and 124. In certain embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the C- terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 33. In one embodiment the 011 Fab comprises a heavy chain with an amino acid sequence of SEQ ID NO: 121 and a light chain with an amino acid sequence of SEQ ID NO: 120. In another embodiment, the 011 Fab is VB6-011-C _H -deBouganin. In certain embodiments, the Fab may have a heavy chain with an amino acid sequence of SEQ ID NO: 99 and a light chain with an amino acid sequence of SEQ ID NO: 120, and the modified bouganin is fused to the N-terminus of SEQ ID NO: 99 by a furin linker of SEQ ID NO: 119. In one embodiment the 011 Fab comprises a heavy chain with an amino acid sequence of SEQ ID NO: 122 and a light chain with an amino acid sequence of SEQ ID NO: 120. In another embodiment, the 011 Fab is VB6-011-NV _H - deBouganin. In one embodiment, the 011 Fab comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 105-110.

[0166] In one embodiment, the invention encompasses expression vectors comprising immunotoxins comprised of polypeptides of SEQ ID NOs: 49, 51, 53, 55-57, 69, 71, 73, 75- 89, 92, 94-98. In other non-limiting embodiments, the immunotoxin comprises a variant of an immunotoxin comprised of polypeptides of SEQ ID NOs: 49, 51, 53, 55-57, 69, 71, 73, 75- 89, 92, 94-98. A variant binds to the same EpCAM epitope or to a substantially similar EpCAM epitope that is bound by an immunotoxin comprised of polypeptides of SEQ ID NOs: 49, 51, 53, 55-57, 69, 71, 73, 75-89, 92, 94-98, and the variant may competitively inhibit binding to EpCAM by an immunotoxin comprised of polypeptides of SEQ ID NOs: 49, 51, 53, 55-57, 69, 71, 73, 75-89, 92, 94-98, under physiologic conditions, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. A variant may comprise the same deBouganin toxin as SEQ ID NOs: 69, 71, 73, 75- 89, 92, 94-98, or may comprise a different portion of the same toxin or a different toxin.

[0167] In another non-limiting embodiment, the immunotoxin comprises an EpCAM- binding portion comprising the variable region of an anti-EpCAM antibody or an anti- EpCAM antibody fragment, or a variant thereof. Binding of any of these immunotoxins to EPCAM may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by competition with the reference anti- EpCAM antibody or anti-EpCAM antibody fragment under physiologic conditions.

[0168] In one embodiment, the invention encompasses an expression vector comprising an immunotoxin comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27, or an immunotoxin comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 29 or SEQ ID NO: 31. In some embodiments, the disclosure encompasses an expression vector comprising an immunotoxin comprised of an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments, the disclosure encompasses an expression vector comprising an immunotoxin comprised of an amino acid sequence set forth in SEQ ID NO: 131. In other non-limiting embodiments, the immunotoxin comprises a variant of an immunotoxin comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27, or a variant of an immunotoxin comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 29 or SEQ ID NO: 31. In some embodiments the immunotoxin comprises a variant of an immunotoxin comprised of an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments the immunotoxin comprises a variant of an immunotoxin comprised of an amino acid sequence set forth in SEQ ID NO: 131. A variant binds to the same HER2/neu epitope or to a substantially similar HER2/neu epitope that is bound by an immunotoxin comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27, an immunotoxin comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 29 or SEQ ID NO: 31, or an immunotoxin comprised of an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135, and the variant may competitively inhibit binding to HER2/neu by an immunotoxin comprised of amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27, an immunotoxin comprised of amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 29 or SEQ ID NO: 31, or an immunotoxin comprised of an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135, under physiologic conditions, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. A variant may comprise the same deBouganin toxin as SEQ ID NOs: 23, 25, 27, 29, 31, 125, 127, 129, 131, 133 and 135, or may comprise a different portion of the same toxin or a different toxin.

[0169] In another non-limiting embodiment, the immunotoxin comprises a HER2/neu-binding portion comprising the variable region of an anti-HER2/neu antibody or an anti-HER2/neu antibody fragment, or a variant thereof. Binding of any of these immunotoxins to HER2/neu may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by competition with the reference anti-HER2/neu antibody or anti-HER2/neu antibody fragment under physiologic conditions.

[0170] In one embodiment, the invention encompasses expression vectors comprising immunotoxins comprised of polypeptides of SEQ ID NOs: 99-101, 103, 120-123. In other non-limiting embodiments, the immunotoxin comprises a variant of an immunotoxin comprised of polypeptides of SEQ ID NOs: 99-101, 103, 120-123. A variant binds to the same CSA epitope or to a substantially similar CSA epitope that is bound by an immunotoxin comprised of polypeptides of SEQ ID NOs: 99-101, 103, 120-123 and the variant may competitively inhibit binding to CSA by an immunotoxin comprised of polypeptides of SEQ ID NOs: 99-101, 103, 120-123 under physiologic conditions, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. A variant may comprise the same deimmunized diptheria toxin, or may comprise a different portion of the same toxin or a different toxin.

[0171] In another non-limiting embodiment, the immunotoxin comprises a CSA- binding portion comprising the variable region of an HI 1 antibody or an HI 1 antibody fragment, or a variant thereof. Binding of any of these immunotoxins to CSA may be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by competition with the reference Hl l antibody or Hl l antibody fragment under physiologic conditions.

[0172] The skilled artisan would appreciate that specificity determining residues can be identified. The term "specificity determining residue," also known as "SDR," refers to a residue that forms part of the paratope of an antibody, particularly CDR residues, the individual substitution of which by alanine, independently of any other mutations, diminishes the affinity of the antibody for the epitope by at least 10 fold, preferably by at least 100 fold, more preferably by at least 1000 fold. This loss in affinity underscores that residue's importance in the ability of the antibody to bind the epitope. See, e.g., Tamura et al, 2000, "Structural correlates of an anticarcinoma antibody: identification of specificity-determining residues (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only," J. Immunol. 164(3): 1432-1441.

[0173] The effect of single or multiple mutations on binding activity, particularly on binding affinity, may be evaluated contemporaneously to assess the importance of a particular series of amino acids on the binding interaction (e.g., the contribution of the light or heavy chain CDR2 to binding). Effects of an amino acid mutation may also be evaluated sequentially to assess the contribution of a single amino acid when assessed individually. Such evaluations can be performed, for example, by in vitro saturation scanning (see, e.g., U.S. Pat. No. 6,180,341 ; Hilton et al, 1996, "Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor," J Biol Chem. 271 : 4699-4708) and site-directed mutagenesis (see, e.g., Cunningham and Wells, 1989, "High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis," Science 244: 1081-1085; Bass et al., 1991, "A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor," Proc Natl Acad Sci. USA 88: 4498-4502). In the alanine-scanning mutagenesis technique, single alanine mutations are introduced at multiple residues in the molecule, and the resultant mutant molecules are tested for biological activity to identify amino acid residues that are critical to the activity of the molecule.

[0174] Sites of ligand-receptor or other biological interaction can also be identified by physical analysis of structure as determined by, for example, nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids (see, e.g., de Vos et al, 1992, "Human growth hormone and extracellular domain of its receptor: crystal structure of the complex," Science 255: 306- 312; Smith et al, 1992, "Human interleukin 4. The solution structure of a four-helix bundle protein," J Mol Biol. 224: 899-904; Wlodaver et al, 1992, "Crystal structure of human recombinant interleukin-4 at 2.25 A resolution," FEBS Lett. 309: 59-64). Additionally, the importance of particular individual amino acids, or series of amino acids, may be evaluated by comparison with the amino acid sequence of related polypeptides or analogous binding sites.

[0175] Furthermore, the skilled artisan would appreciate that increased avidity may compensate for lower binding affinity. The avidity of an immunotoxin for a cancer cell antigen is a measure of the strength of the immunotoxin's binding of the cancer cell antigen, which can have multiple binding sites. The functional binding strength between the cancer cell antigen and the immunotoxin represents the sum strength of all the affinity bonds, and thus an individual component may bind with relatively low affinity, but a multimer of such components may demonstrate potent biological effect. In fact, the multiple interactions between immunotoxin and cancer cell antigen epitopes may demonstrate much greater than additive biological effect, i.e., the advantage of multivalence can be many orders of magnitude with respect to the equilibrium constant.

[0176] In one non-limiting embodiment, the portion of the HER2/neu-binding protein that binds a HER2/neu epitope has a structure substantially similar to that of an anti- HER2/neu antibody. The substantially similar structure can be characterized by reference to epitope maps that reflect the binding points of the immunotoxin's HER2/neu-binding portion to a HER2/neu molecule.

[0177] In a preferred embodiment, an immunotoxin comprises an anti-HER2/neu diabody. In another embodiment, the anti-HER2/neu diabody comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 5-10.

[0178] In another non-limiting embodiment, the portion of the EpCAM-binding protein that binds an EpCAM epitope has a structure substantially similar to that of an anti- EpCAM antibody. The substantially similar structure can be characterized by reference to epitope maps that reflect the binding points of the immunotoxin's EpCAM-binding portion to an EpCAM molecule. In a preferred embodiment, an immunotoxin comprises an anti- EpCAM Fab.

[0179] In one non-limiting embodiment, the portion of the CSA-binding protein that binds a CSA epitope has a structure substantially similar to that of an HI 1 antibody. The substantially similar structure can be characterized by reference to epitope maps that reflect the binding points of the immunotoxin's CSA-binding portion to a CSA molecule.

[0180] In a preferred embodiment, an immunotoxin comprises an HI 1 Fab. In another embodiment, the Hl l Fab comprises the complementarity determining region (CDR) sequences of SEQ ID NOs: 105-110.

[0181] The antibody portion of an immunotoxin may be immunoglobulin derived, i.e., can be traced to a starting molecule that is an immunoglobulin (or antibody). For example, the antibody may be produced by modification of an immunoglobulin scaffold using standard techniques known in the art. In another, non-limiting example, immunoglobulin domains (e.g., variable heavy and/or light chains) may be linked to a non- immunoglobulin scaffold. Further, the antibody may be developed by, without limitation, chemical reaction or genetic design. Accordingly, in a non-limiting example, an immunotoxin may comprise an immunoglobulin-derived polypeptide (e.g., an antibody selected from an antibody library), or variant thereof, that specifically binds to liver cancer cells; and a toxin or variant thereof. Such immunoglobulin polypeptides can be re-designed to affect their binding characteristics to a target a tumor associated molecule, or to improve their physical characteristics, for example.

[0182] The antibody portion of the immunotoxin need not be immunoglobulin based. Accordingly, an immunotoxin may comprise a non-immunoglobulin polypeptide (e.g., Affibody®), or variant thereof, that specifically binds to liver cancer cells; and a toxin or variant thereof. Such non-immunoglobulin polypeptide can be designed to bind to a target tumor associated molecule. Moreover, non-immunoglobulin polypeptide can be engineered to a desired affinity or avidity and can be designed to tolerate a variety of physical conditions, including extreme pH ranges and relatively high temperature.

[0183] Indeed, for use in a pharmaceutical composition, the design of a non- immunoglobulin polypeptide with a relatively long half-life at physiological conditions (e.g., 37° C. in the presence of peptidases) can be advantageous. Furthermore, such molecules, or variants thereof, may demonstrate good solubility, small size, proper folding and can be expressed in readily available, low-cost bacterial systems, and thus manufactured in commercially reasonable quantities. The ability to design a non-immunoglobulin polypeptide is within the skill of the ordinary artisan.

[0184] Examples of epitope-binding polypeptides include, without limitation, ligands comprising a fibronectin type III domain, binding molecules based on assembly of repeat protein domains comprising Pleckstrin-Homology (PH) domains, ankyrin repeats, and the like. Other epitope-binding polypeptides or domains include a Kunitz protease inhibitor domain, a lipocalin domain, a thioredoxin, a cell surface receptor A domain, and/or a cysteine-rich knottin peptide.

[0185] In other embodiments, the immunotoxin comprises a variant that has amino acid sequences, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to deimmunized bouganin as shown in SEQ ID NOs: 12, 58, 59, 60 or 61.

[0186] In other embodiments, the immunotoxin comprises a variant that has amino acid sequences, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to deimmunized diphtheria toxin. Deimmunized Bouganin toxin

[0187] A variety of toxins may be used to design an EpCAM-targeted immunotoxin, a HER2/neu-targeted immunotoxin or a CSA-targeted immunotoxin according to the invention. In preferred embodiments, the toxin comprises a polypeptide having ribosome- inactivating activity including, without limitation, gelonin, bouganin, saporin, ricin A chain, bryodin, diphtheria toxin, restrictocin, and variants thereof. When the protein is a ribosome- inactivating protein, the immunotoxin must be internalized upon binding to the cancer cell in order for the toxin to be cytotoxic to the cells.

[0188] In one embodiment, the toxin portion comprises at least a toxic portion of bouganin toxin, or a variant thereof. In a preferred embodiment, the toxin comprises a deimmunized Bouganin toxin ("deBouganin"). DeBouganin is a type 1 Ribosome Inactivating Protein (RIP) isolated from Bougainvillea spectabilis willd that has been de- immunized for systemic delivery. In a particular embodiment, the deimmunized Bouganin toxin comprises SEQ ID NO: 12 (amino acid sequence) and SEQ ID NO: 11 (nucleotide sequence). In another embodiment, the deimmunized Bouganin toxin comprises SEQ ID NOs: 58, 59, 60 or 61. It is understood that one of skill in the art can codon optimize the deimmunized Bouganin toxin to optimize expression in a cell. A codon optimized deBouganin sequence is exemplified by SEQ ID NO: 13. Modified bouganin proteins are described in WO 2005/090579, which is incorporated herein by reference.

[0189] In some embodiments, the modified bouganin protein has reduced propensity to activate human T cells compared to a non-modified bouganin protein and has a biological activity that is comparable to non-modified bouganin protein. In some embodiments, the modified bouganin protein has reduced propensity to activate human T cells compared to a non-modified bouganin protein and has biological activity that is lower than the non-modified bouganin protein. In yet another embodiment, the disclosure provides a modified bouganin protein wherein the modified bouganin protein has reduced propensity to activate human T cells and no biological activity.

[0190] In some embodiments, the modified bouganin peptide is modified at one or more T-cell epitopes in the bouganin protein sequence.

[0191] The term "T-cell epitope" means an amino acid sequence which is able to bind major histocompatibility complex (MHC) class II, able to stimulate T-cells and/or also able to bind (without necessarily measurably activating) T-cells in complex with MHC class II. [0192] In one embodiment, a method that can be used to generate the modified bouganin proteins with modified T-cell epitopes comprises the following steps: (i) determining the amino acid sequence of the protein or part thereof; (ii) identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by methods such as determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays; (iii) designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in turn, modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope; (iv) constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties according to well-known recombinant techniques; and (v) optionally repeating steps (ii) to (iv). In an example, step (iii) is carried out by substitution, addition or deletion of amino acid residues in any of the T-cell epitopes in the non-modified bouganin protein. In another example, the method to make the modified bouganin protein is made with reference to the homologous protein sequence and/or in silico modeling.

[0193] In an embodiment of the invention, the modified bouganin protein has at least one T-cell epitope removed. In another embodiment, the modified bouganin protein of the invention has one, two or three T-cell epitopes removed. The invention also contemplates a modified bouganin protein wherein 1 to 9 amino acid residues are modified, preferably in the T-cell epitope. In another embodiment, 1 to 5 amino acid residues are modified. In another embodiment the modified bouganin protein has a biological activity, such as cell cytotoxicity.

[0194] For the elimination of T-cell epitopes, amino acid substitutions are made at appropriate points within the peptide sequence predicted to achieve substantial reduction or elimination of the activity of the T-cell epitope. In practice an appropriate point will in one embodiment equate to an amino acid residue binding within one of the pockets provided within the MHC class II binding groove.

[0195] In one embodiment, the epitopes are compromised by mutation to result in sequences no longer able to function as T-cell epitopes. It is possible to use recombinant DNA methods to achieve directed mutagenesis of the target sequences and many such techniques are available and well known in the art. In practice a number of modified bouganin proteins will be produced and tested for the desired immune and functional characteristic. It is particularly important when conducting modifications to the protein sequence that the contemplated changes do not introduce new immunogenic epitopes. This event is avoided in practice by re-testing the contemplated sequence for the presence of epitopes and/or of MHC class II ligands by any suitable means.

[0196] The modified bouganin proteins of the invention may also contain or be used to obtain or design "peptide mimetics." "Peptide mimetics" are structures which serve as substitutes for peptides in interactions between molecules. Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of the modified bouganin protein, including biological activity and a reduced propensity to activate human T cells. Peptide mimetics also include peptoids and oligopeptoids.

[0197] Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule. Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of the secondary structures of the proteins of the invention. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids, which are oligomers of N-substituted amino acids, can be used as motifs for the generation of chemically diverse libraries of novel molecules.

Deimmunized Diptheria toxin

[0198] A variety of toxins may be used to design an EpCAM-targeted immunotoxin, a HER2/neu-targeted immunotoxin or a CSA-targeted immunotoxin according to the invention. In preferred embodiments, the toxin comprises a polypeptide having ribosome- inactivating activity including, without limitation, gelonin, bouganin, saporin, ricin A chain, bryodin, diphtheria toxin, restrictocin, and variants thereof. When the protein is a ribosome- inactivating protein, the immunotoxin must be internalized upon binding to the cancer cell in order for the toxin to be cytotoxic to the cells. [0199] In a particular preferred embodiment, the toxin portion comprises at least a toxic portion of diphtheria toxin ("DT"), or a variant thereof. Diphtheria toxin is a member of the mono-ADP-ribosylating toxin family which further includes such toxins as cholera toxin, pseudomonas exotoxin A, pertussis toxin, and Clostridium C3-like toxin. Members of this family contain many similar protein domains and motifs, in particular the catalytic site of the toxins. DT is composed of three domains: a catalytic domain; a transmembrane domain; and a receptor binding domain. The nucleic acid and amino acid sequences of native DT were described by Greenfield et al. PNAS (1983) 80: 6853-6857. Native DT is targeted to cells that express heparin binding epidermal growth factor-like receptors (Naglish et at., Cell, 69: 1051 -1061 (1992)). Once the toxin, toxin conjugate or fusion toxin has bound to the cell surface receptor the cell internalizes the toxin bound receptor via endocytic vesicles. As the vesicles are processed they become acidified and the translocation domain of the DT toxophore undergoes a structural reorganization which inserts the nine transmembrane segments of the toxin into the membrane of the endocytic vesicle. This event triggers the formation of a productive pore through which the catalytic domain of the toxin is threaded. Once translocated the catalytic domain which possess the ADP-ribosyltransferase activity is released into the cytosol of the targeted cell where it is free to poison translation thus effecting the death of the cell (reviewed in vanderSpek et al., Methods in Molecular Biology, Bacterial Toxins: methods and Protocols, 145 : 89-99, Humana press, Totowa, N.J., (2000)).

[0200] In a specific embodiment, the cytotoxic portion comprises a DT variant that, when administered alone, is substantially unable to bind to cells. In a further, specific embodiment, the cytotoxic portion comprises DT. The cytotoxic portion may comprises one or more diphtheria toxins known in the art (see U. S. Patent No. 8,470,314, which is incorporated herein by reference in its entirety), or variants thereof.

[0201] Modifications to toxin can include modifications to produce polypeptides that exhibit reduced immunogenicity (T-cell, B-cell, or both) and reduced capacity to cause vascular leak syndrome (VLS) (i.e., reduced binding to endothelial cells and vascular endothelial cells and reduced disruption of endothelial cell junctions and other indications of vascular leak syndrome).

[0202] To produce a toxin (e.g., diphtheria toxin) that has modifications in T-cell epitopes, B-cell epitopes, VLS motifs, domain deletions and combinations thereof, the amino acid residue modifications made for each type of modification are considered in light of the other modifications. In one non-limiting example, modification of both T-cell epitopes and VLS motifs is desired. When modifying both T-cell epitopes and VLS motifs, modification of at least one amino acid within at least one T-cell epitope within the toxin should not create a VLS motif. Similarly, the modification of at least one amino acid within at least one VLS motif within the toxin should not create a T-cell epitope. Additionally, the modification of a T-cell epitope or a VLS motif should not re-introduce a previously modified T-cell or VLS motif. Furthermore, modification of such polypeptides can also take into consideration the modification of B-cell epitopes, which when desired, should also not create or re-introduce Tcell epitopes or VLS motifs.

[0203] Modifications that affect the cytotoxicity of the toxins can also be made. For instance, if one or more modifications to one or more of a T cell epitope, a VLS motif and/or a B cell epitope are made and the cytotoxicity of the modified toxin is less than the unmodified toxin, it is contemplated herein that one or more modifications could be made to restore the cytotoxicity to a level comparable to that of the unmodified toxin or to an effective level.

[0204] The membrane-inserting domain (translocation domain) of diphtheria toxin (T) contains an amphipathic region that is involved in the delivery of the catalytic (C) domain to the cytosol of a cell. In one non-limiting example, replacement of one or more amino acid residues of the amphipathic region with one or more different amino acid residues that retain the helical structure of the region can maintain cytotoxicity. Consideration of the composition and distribution of the charged and hydrophobic amino acid residues within the amphipathic region and modification thereof is also contemplated herein. Examples of charged amino residues include Glu, Asp, Asn, Gin, Lys, Arg and His; hydrophobic amino acids include, but are not limited to, alanine and phenylalanine.

[0205] Modification of the binding cleft of the catalytic domains of the ADPribosylating protein family can also affect cytotoxicity. In one non-limiting example, modification of one or more amino acid residues of the F/Y -X-S-T-X motif of the diphtheria toxin C domain can affect the cytotoxicity of the polypeptide. Replacement of one or more amino acid residues in this region with one or more different amino acid residues that retain the catalytic domain can maintain cytotoxicity. Comparison with related members of the ADP-ribosylating protein family (e.g., Pseudomonas aeruginosa exotoxin A) can provide further guidance on amino acid residue modification and composition of domains such as the diphtheria toxin C domain. Modifications such as these are also contemplated herein.

Methods of Use [0206] Disclosed are methods of using immunotoxins described herein, wherein the immunotoxins comprise a binding protein linked to a toxin by a deimmunized furin protease sensitive linker. The present invention contemplates methods of treating or preventing cancer comprising administering an effective amount of said immunotoxins to a subject in need thereof.

[0207] In each of the foregoing embodiments, the antibodies, the antibody fragments, and/or the immunotoxins used may include any of the de-immunized antibodies/antibody fragments disclosed herein, wherein the antibody/antibody fragment comprises a heavy chain having an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55.

[0208] In some embodiments, a method of treating a subject with cancer may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain having an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In another embodiment, the method may involve administering an immunotoxin comprising a heavy chain having an amino acid sequence of SEQ ID NO: 49, and a light chain having an amino acid sequence of SEQ ID NO: 51. In another embodiment, the method may involve administering an immunotoxin comprising a heavy chain having an amino acid sequence of SEQ ID NO: 53, and a light chain having an amino acid sequence of SEQ ID NO: 55. In another embodiment, the method may involve administering an immunotoxin comprising a heavy chain having an amino acid sequence of SEQ ID NO: 56, and a light chain having an amino acid sequence of SEQ ID NO: 55. In another embodiment, the method may involve administering an immunotoxin comprising a heavy chain having an amino acid sequence of SEQ ID NO: 57, and a light chain having an amino acid sequence of SEQ ID NO: 55.

[0209] In some embodiments, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 2, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 4. In another embodiment, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, and a light chain variable region having an amino acid sequence of SEQ ID NO: 4. In another embodiment, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by an amino acid sequence set forth in SEQ ID NO: 131.

[0210] In some embodiments, a method of treating or preventing cancer in a subject in need thereof may involve administering a therapeutically effective amount of an immunotoxin, wherein the immunotoxin comprises a heavy chain variable region and a heavy chain constant region. In another embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 99. In a further embodiment, the heavy chain variable region and the heavy chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 99. In another embodiment, the heavy chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 101. In a further embodiment, the heavy chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 101. In one embodiment, the HI 1 antibody or antibody fragment comprises a light chain variable region and a light chain constant region. In another embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 100. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 100. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 103. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 103. In some embodiments, the light chain variable region and the light chain constant region are encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 120. In a further embodiment, the light chain variable region and the light chain constant region are encoded by an amino acid sequence shown in SEQ ID NO: 120. In another embodiment, the light chain variable region is encoded by an amino acid sequence sharing at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 123. In a further embodiment, the light chain variable region is encoded by an amino acid sequence shown in SEQ ID NO: 123. In certain embodiments, the Hl l antibody or Hl l antibody fragment comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 99 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 121 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 122 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0211] The immunotoxin may comprise an antibody fragment, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, multimers, and any combination thereof. In some embodiments described herein, the effector molecule may be a radioisotope, an antineoplastic agent, an immunomodulator, a biological response modifier, lectin, a toxin, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof. In a preferred embodiment, the effector molecule comprises a deimmunized Bouganin toxin. In another preferred embodiment, the effector molecule comprises a deimmunized diphtheria toxin.

[0212] In some embodiments, the antibodies and immunotoxins may be used to treat cancer, such as lung cancer, gastric cancer, renal cancer, thyroid cancer, breast cancer, bladder cancer, ovarian cancer, colorectal cancer, head and neck cancer, hepatocellular carcinoma, esophageal, pancreas, and prostate cancer. Cancers originating from any epithelial cell may also be targeted by these immunotoxins and antibodies.

[0213] In preferred non-limiting embodiments, the cancer is amenable to treatment by direct administration of the antibody or immunotoxin to the cancer site. For example, a target tumor mass may be close to the surface of the skin. In another example, a diseased tissue may be encapsulated by a cyst, or is found in a substantially enclosed cavity including, without limitation, a lumen. In other embodiments, the cancer is amenable to treatment by intravenous administration of the antibody or immunotoxin.

[0214] In some embodiments, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain having an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55, and instructions for the use thereof. [0215] In some embodiments, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 2, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 4, attached to an effector molecule and instructions for the use thereof. In another embodiment, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, and a light chain variable region having an amino acid sequence of SEQ ID NO: 4, attached to an effector molecule and instructions for the use thereof. In another embodiment, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region and a light chain variable region encoded by an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135.

[0216] In some embodiments, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 103, attached to an effector molecule and instructions for the use thereof. In another embodiment, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 103, attached to an effector molecule and instructions for the use thereof. In some embodiments, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 123, attached to an effector molecule and instructions for the use thereof. In another embodiment, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 123, attached to an effector molecule and instructions for the use thereof. In another embodiment, a kit for diagnosing cancer may include an immunotoxin comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 121 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 122 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0217] In some embodiments, the kit for detecting cancer may include an anti- EpCAM antibody fragment, and preferably further include a reagent containing a labeled anti-Ig antibody, for example, an anti-Ig antibody linked with an enzyme such as alkaline phosphatase or a radiolabeled anti-Ig antibody. In some embodiments, the anti-EpCAM antibody fragment may be attached to a chromophore, a fluorophore or a radiolabelled ligand.

[0218] In some embodiments, the kit for detecting cancer may include an anti- HER2/neu antibody fragment, and preferably further include a reagent containing a labeled anti-Ig antibody, for example, an anti-Ig antibody linked with an enzyme such as alkaline phosphatase or a radiolabeled anti-Ig antibody. In some embodiments, the anti-HER2/neu antibody fragment may be attached to a chromophore, a fluorophore or a radiolabelled ligand.

[0219] In some embodiments, the kit for detecting cancer may include an Hl l antibody fragment, and preferably further include a reagent containing a labeled anti- Ig antibody, for example, an anti-Ig antibody linked with an enzyme such as alkaline phosphatase or a radiolabeled anti-Ig antibody. In some embodiments, the Hl l antibody fragment may be attached to a chromophore, a fluorophore or a radiolabelled ligand.

[0220] The immunotoxins disclosed herein may also be used to detect or monitor in a subject. In some embodiments, a method of detecting or monitoring cancer in a subject may involve contacting a test sample taken from the subject with an immunotoxin to form an immunotoxin-antigen complex; measuring the amount of the immunotoxin-antigen complex in the test sample; and normalizing the results against a control. The test sample may be serum, lymph, ascitic exudate, intercellular fluid, tissue lysate, saliva, tissue sections, cells, biopsy samples, and the like. The immunotoxin-antigen complex may be detected by any means, such as for example, dot-blot method, Western blot method, ELISA method, or sandwich ELISA method. Also, the immunotoxin-antigen complex can be detected by use according to multistage reactions, such as reaction with a biotin-bound anti-Ig antibody and then with an avidin-bound material. In one embodiment, the immunotoxin comprises a heavy chain amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 2, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, and a light chain variable region having an amino acid sequence of SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by an amino acid sequence set forth in SEQ ID NO: 131. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 123. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 123. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 121 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 122 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0221] In another embodiment, a method of detecting or monitoring cancer in a subject may involve administering to the subject an immunotoxin and detecting the immunotoxin. In one embodiment, the immunotoxin comprises a heavy chain amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 2, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, and a light chain variable region having an amino acid sequence of SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by an amino acid sequence set forth in SEQ ID NO: 131. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 123. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 123. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 121 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 122 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120.

[0222] In some embodiments, the immunotoxins disclosed herein may be used for imaging a tumor in a subject. In some embodiments, a method of imaging a tumor in a subject may involve administering to the subject an immunotoxin and detecting the immunotoxin by in vivo imaging. In one embodiment, the immunotoxin comprises a heavy chain amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57; and a light chain having an amino acid sequence of SEQ ID NO: 51 or SEQ ID NO: 55. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 2, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2, and a light chain variable region having an amino acid sequence of SEQ ID NO: 4. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by amino acid sequences set forth in SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In another embodiment, the immunotoxin comprises a heavy chain variable region and a light chain variable region encoded by an amino acid sequence set forth in SEQ ID NO: 131. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 103. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence sharing at least 90% homology with SEQ ID NO: 101, and a light chain variable region sharing at least 90% homology with SEQ ID NO: 123. In another embodiment, the immunotoxin comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 101, and a light chain variable region having an amino acid sequence of SEQ ID NO: 123. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 121 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. In certain embodiments, the immunotoxin comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 122 and a light chain having an amino acid sequence as shown in SEQ ID NO: 120. The immunotoxin may further include an effector molecule.

[0223] In some embodiments, the effector molecule utilized for detecting cancer or imaging a tumor may be a radioisotope, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof. The in vivo imaging may be performed by any known technique in the art, such as near-infrared fluorescence imaging (NIRF), fluorescence reflectance imaging (FRI), fluorescence-mediated tomography (FMT), positron emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance imaging (MRI), PET with concurrent computed tomography imaging (PET/CT), PET with concurrent magnetic resonance imaging (PET/MRI), and any combination thereof.

[0224] In some embodiments, the method may further include resecting cancerous tissue, such as a tumor or a part of an organ, after in vivo imaging of the subject. Surgical resection can be performed by any technique known in the art. In some embodiments, the method may further include administering the antibody or immunotoxin after resection to measure the completeness of tumor resection.

[0225] In certain embodiments, the antibodies as described herein are labeled with a radiotracer. A radiotracer is typically a substance containing a radioisotope that allows for easy detection and measurement. A number of different forms of hydrogen, carbon, phosphorous, sulfur and iodine are commonly used in medical diagnostics. The antibodies of the present invention may be labeled with any suitable radiotracer. Preferred radiotracers include radiotracers for medical imaging. Common radiotracers used include ¹⁸ F, ⁶⁷ Ga, ^81m Kr, ⁸² Rb, ^99m Tc, ^m In, ¹² I, ^m I, ¹ Xe, ²⁰¹ T1 and ⁹⁰ Y. Preferably, the antibodies as described herein are labeled with ¹⁸ F, ^{12 /1 1} I, ^m In, ⁹⁰ Y or ^99m Tc. [0226] The antibodies of the present invention may also be labeled with any fluorescent probes known in the art. Non-limiting examples include fluorescein, amino coumarin acetic acid, tetramethylchodomine isocyanate, Texas Red, Cy 3.0, Cy 5.0, green fluorescent protein, and the like.

[0227] In another preferred embodiment, the antibodies as described herein are labeled with a contrast agent. A contrast agent is a substance used to increase or modify the contrast of organs, fluids or anatomical structures in the human or animal body. The antibodies of the present invention may be labeled with any suitable contrast agent. Preferred contrast agents include contrast agents for medical imaging. Preferably, the antibodies of the present invention are labeled with an MRI (magnetic resonance imaging) contrast agent such as a superparamagnetic contrast agent or a paramagnetic contrast agent. MRI contrast agents are typically chelated metals or colloids. The most commonly used contrast agents include gadolinium (Gd) based contrast agents such as gadolinium-DTPA, iron oxide based contrast agents such as superparamagnetic Small Particles of Iron Oxide (SPIO) and superparamagnetic Ultrasmall Small Particles of Iron Oxide (USPIO) and paramagnetic contrast agents based on manganese chelates such as Mn-DPDP.

[0228] The invention also provides methods for reducing the risk of post-surgical complications comprising administering an effective amount of an immunotoxin before, during, or after surgery, and in specific non-limiting embodiments, surgery to treat cancer.

[0229] The invention also provides methods for preventing occurrence, preventing or delaying recurrence, or reducing the rate of recurrence of cancer comprising directly administering to a patient in need thereof an effective amount of an immunotoxin.

[0230] The invention also provides methods for sensitizing a tumor or cancer to one or more other cancer therapeutics comprising administering an immunotoxin of the invention. In a non-limiting embodiment, the other cancer therapeutic comprises another EpCAM- targeted immunotoxin. In another embodiment, the other cancer therapeutic comprises another anti-HER2/neu immunotoxin. In another embodiment, the other cancer therapeutic comprises another Hl l immunotoxin. In another non-limiting embodiment, the other cancer therapeutic comprises radiation. The other cancer therapeutic may be administered prior to, overlapping with, concurrently, and/or after administration of the immunotoxin. When administered concurrently, the immunotoxin and the other cancer therapeutic may be administered in a single formulation or in separate formulations, and if separately, then optionally, by different modes of administration. Accordingly, the combination of one or more immunotoxins and one or more other cancer therapeutics may synergistically act to combat the tumor or cancer.

[0231] Where an immunotoxin of the invention is administered in addition to one or more other therapeutic agents, these other cancer therapeutics may include, without limitation, 2,2',2"trichlorotriethylamine, 6-azauridine, 6-diazo-5-oxo-L-norleucine, 6- mercaptopurine, aceglarone, aclacinomycinsa actinomycin, altretamine, aminoglutethimide, aminoglutethimide, amsacrine, anastrozole, ancitabine, angiogenin antisense oligonucleotide, anthramycin, azacitidine, azaserine, aziridine, batimastar, bcl-2 antisense oligonucleotide, benzodepa, bicalutamide, bisantrene, bleomycin, buserelin, busulfan, cactinomycin, calusterone, carboplatin, carboquone, carmofur, carmustine, carubicin, carzinophilin, chlorambucil, chloraphazine, chlormadinone acetate, chlorozotocin, chromomycins, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, defosfamide, demecolcine, denopterin, diaziquone, docetaxel, doxifluridine, doxorubicin, droloxifene, dromostanolone, edatrexate, eflornithine, elliptinium acetate, emitefur, enocitabune, epirubicin, epitiostanol, estramustine, etoglucid, etoposide, fadrozole, fenretinide, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosfestrol, fotemustine, gallium nitrate, gemcitabine, goserelin, hexestrol, hydroxyurea, idarubicin, ifosfamide, improsulfan, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, L-asparaginase, lentinan, letrozole, leuprolide, lomustine, lonidamine, mannomustine, mechlorethamine, mechlorethamine oxide hydrochloride, medroxyprogesterone, megestrol acetate, melengestrol, melphalan, menogaril, mepitiostane, methotrexate, meturedepa, miboplatin, miltefosine, mitobronitol, mitoguazone, mitolactol, mitomycins, mitotane, mitoxantrone, mopidamol, mycophenolic acid, nilutamide, nimustine, nitracine, nogalamycin, novembichin, olivomycins, oxaliplatin, paclitaxel, pentostain, peplomycin, perfosfamide, phenamet, phenesterine, pipobroman, piposulfan, pirarubicin, piritrexim, plicamycin, podophyllinic acid 2-ethyl-hydrazide, polyestradiol phosphate, porfimer sodium, porfiromycin, prednimustine, procabazine, propagermanium, PSK, pteropterin, puromycin, ranimustine, razoxane, roquinimex, sizofican, sobuzoxane, spirogermanium, streptonigrin, streptozocin, tamoxifen, tegafur, temozolomide, teniposide, tenuzonic acid, testolacone, thiamiprine, thioguanine, Tomudex, topotecan, toremifene, triaziquone, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trilostane, trimetrexate, triptorelin, trofosfamide, trontecan, tubercidin, ubenimex, uracil mustard, uredepa, urethan, vinblastine, vincristine, zinostatin, and zorubicin, cytosine arabinoside, gemtuzumab, thioepa, cyclothosphamide, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozoamide), hexamethylmelamine, LYSODREN, nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan (CAMPTOSAR,CPT- 11), vincristine, vinca alkyloids such as vinblastine.) podophyllotoxin, epipodophyllotoxin, VP-16 (etoposide), cytochalasin B, gramicidin D, ethidium bromide, emetine, anthracyclines (e.g., daunorubicin), doxorubicin liposomal, dihydroxyanthracindione, mithramycin, actinomycin D, aldesleukin, allutamine, biaomycin, capecitabine, carboplain, chlorabusin, cyclarabine, daclinomycin, floxuridhe, lauprolide acetate, levamisole, lomusline, mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab, campath- 1, straplozocin, tretinoin, VEGF antisense oligonucleotide, vindesine, and vinorelbine. Compositions comprising one or more cancer therapeutics (e.g., FLAG, CHOP) are also contemplated by the present invention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. For a full listing of cancer therapeutics known in the art, see, e.g., the latest editions of The Merck Index and the Physician's Desk Reference. Likewise, the immunotoxin of the invention may be used in conjunction with radiation therapy or other known cancer therapeutic modalities.

[0232] An immunotoxin of the present invention can be administered with a cancer therapeutic modality such as an antibody drug conjugate (ADC). An ADC comprises a monoclonal antibody or antibody fragment, a cytotoxic payload or drug and a stable, chemical linker with labile bonds connecting the payload to the antibody. ADCs approved by the FDA and routinely used in the treatment of various cancers include gemtuzumab ozogamicin (Mylotarg ^® ), ibritumomab tiuxetan (Zevalin ^® ), tositumomab (Bexxar ^® ), ado- trastuzumab emtansine (Kadcyla) and Brentuximab Vedotin (Adcetris ^® ).

[0233] An immunotoxin of the present invention can be administered with a cancer therapeutic modality such as immune checkpoint inhibitors. By checkpoint inhibitor it is meant that the compound inhibits one or more proteins in a number of inhibitory pathways that usually serve to modulate an immune response. The pathways are co-opted by tumors to evade the immune system and proliferate. Proteins in the checkpoint signaling pathways include for example, PD-1, PD-L1, PD-L2, TIM3, LAG3 and CTLA-4. Checkpoint inhibitors are known in the art. For example, the checkpoint inhibitor can be a small molecule. A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight in the range of less than about 5 kD to 50 kD, for example less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, less than 750 daltons, less than 500 daltons, less than about 450 daltons, less than about 400 daltons, less than about 350 daltons, less than 300 daltons, less than 250 daltons, less than about 200 daltons, less than about 150 daltons, less than about 100 daltons. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Alternatively, the checkpoint inhibitor is an antibody or antibody fragment thereof. For example, the antibody or antibody fragment thereof is specific to a protein in a checkpoint signaling pathway, such as PD-1, PD-L1, PD-L2, LAG3, TIM3 or CTLA-4.

[0234] In another embodiment, methods of treating cancer comprising administering an immunotoxin in combination with a regimen of radiation therapy are provided. The therapy may also comprise surgery and/or chemotherapy. For example, the immunotoxin may be administered in combination with radiation therapy and cisplatin (Platinol), fluo- rouracil (5-FU, Adrucil), carboplatin (Paraplatin), and/or paclitaxel (Taxol). Treatment with the immunotoxin may allow use according to lower doses of radiation and/or less frequent radiation treatments, which may for example, reduce the incidence of severe sore throat that impedes swallowing function potentially resulting in undesired weight loss or dehydration.

[0235] Pharmaceutical compositions for combination therapy may also include, without limitation, antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin), asparaginase, BCG protein, diphtheria toxin, procaine, tetracaine, lidocaine, propranolol, antimitotic agents, abrin, ricin A, Pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, antihistaminic agents, anti-nausea agents, etc.

[0236] Indeed, direct administration of an effective amount of an immunotoxin to a patient in need of such treatment may result in reduced doses of another anticancer agent having clinically significant efficacy. Such efficacy of the reduced dose of the other anticancer agent may not be observed absent administration with an immunotoxin. Accordingly, the present invention provides methods for treating a tumor or cancer comprising administering a reduced dose of one or more other anticancer agents.

[0237] Moreover, combination therapy comprising an immunotoxin to a patient in need of such treatment may permit relatively short treatment times when compared to the duration or number of cycles of standard treatment regimens. Accordingly, the present invention provides methods for treating a tumor or cancer comprising administering one or more other anticancer agents for relatively short duration and/or in fewer treatment cycles.

[0238] Thus, in accordance with the present invention, combination therapies comprising an immunotoxin and another anticancer agent may reduce toxicity (i.e., side effects) of the overall cancer treatment. For example, reduced toxicity, when compared to a monotherapy or another combination therapy, may be observed when delivering a reduced dose of immunotoxin and/or other anticancer agent, and/or when reducing the duration of a cycle (i.e., the period of a single administration or the period of a series of such administrations), and/or when reducing the number of cycles.

[0239] Clinical outcomes of cancer treatments using an immunotoxin of the invention are readily discernible by one of skill in the relevant art, such as a physician. For example, standard medical tests to measure clinical markers of cancer may be strong indicators of the treatment's efficacy. Such tests may include, without limitation, physical examination, performance scales, disease markers, 12-lead ECG, tumor measurements, tissue biopsy, cytoscopy, cytology, longest diameter of tumor calculations, radiography, digital imaging of the tumor, vital signs, weight, recordation of adverse events, assessment of infectious episodes, assessment of concomitant medications, pain assessment, blood or serum chemistry, detecting serum markers, urinalysis, CT scan, and pharmacokinetic analysis. Furthermore, synergistic effects of a combination therapy comprising the immunotoxin and another anticancer agent may be determined by comparative studies with patients undergoing monotherapy.

[0240] The effective dose of immunotoxin to be administered during a cycle varies according to the mode of administration. Direct administration (e.g., intratumoral injection) requires much smaller total body doses of immunotoxin as compared to systemic, intravenous administration of the immunotoxin. It will be evident to the skilled artisan that local administration can result in lower body doses, and in those circumstances, and resulting low circulating plasma level of immunotoxin would be expected and desired.

[0241] In one embodiment, the effective dose by direct administration of antibody or immunotoxin may range from about 10 to 3000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 60 to 500, 70 to 400, 80 to 300, 90 to 200, or 100 to 150 micrograms/tumor/day. In other embodiments, the dose may range from approximately 10 to 20, 21 to 40, 41 to 80, 81 to 100, 101 to 130, 131 to 150, 151 to 200, 201 to 280, 281 to 350, 351 to 500, 501 to 1000, 1001 to 2000, or 2001 to 3000 micrograms/tumor/day. In specific embodiments, the dose may be at least approximately 20, 40, 80, 130, 200, 280, 400, 500, 750, 1000, 2000, or 3000 micrograms/tumor/ day .

[0242] In other embodiments, the antibody or immunotoxin administration is at a dosage of about 0.01 mg/kg/dose to about 2000 mg/kg/dose. [0243] In another embodiment, the effective dose of antibody or immunotoxin may range from about 100 to 5000, 200 to 4000, 300 to 3000, 400 to 2000, 500 to 1000, 600 to 900, or 700 to 1500 micrograms/tumor/month. In other embodiments, the dose may range from approximately 100 to 199, 200 to 399, 400 to 649, 650 to 999, 1000 to 1799, 1800 to 2499, 2500 to 3499, 3500 to 4999, 5000 to 7499, 7500 to 10000, or 10001 to 20000 micrograms/tumor/month. In specific embodiments, the dose may be at least approximately 100, 200, 400, 650, 1000, 1400, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 10000, or 20000 micrograms/tumor/month.

[0244] In another embodiment, the antibody or immunotoxin is administered intratumourally at a total dose per cycle equivalent to, or below the maximum tolerated dose established in a safety trial but the dosage is standardized in relation to the tumor volume. For example, subjects will receive between 1 microgram per cm ³ and 500 microgram per cm ³ tumor or a dose sufficient to reach about between 14 picomole and 7 nanomole per cm ³ tumor tissue. The dose will be administered in a volume not exceeding about 20-50% of the tumor volume. The immunotoxin will be diluted in a suitable salt solution. For example, for a tumor of estimated volume of 3 cm ³ , a target dose of 14 picomoles (1 microgram per cm ³ ), and a maximum injection relative volume of about 1/3 of the tumor, 3 microgram of immunotoxin will be diluted into about 1 ml of diluent.

[0245] The effective dose of another cancer therapeutic to be administered together with an immunotoxin during a cycle also varies according to the mode of administration. The one or more anticancer agent may be delivered intratumorally, or by other modes of administration. Typically, chemotherapeutic agents are administered systemically. Standard dosage and treatment regimens are known in the art (see, e.g., the latest editions of the Merck Index and the Physician's Desk Reference).

[0246] For example, in one embodiment, the additional anticancer agent comprises dacarbazine at a dose ranging from approximately 200 to 4000 mg/m ² /cycle. In a preferred embodiment, the dose ranges from 700 to 1000 mg/m ² /cycle.

[0247] In another embodiment, the additional anticancer agent comprises fludarabine at a dose ranging from approximately 25 to 50 mg/m ² /cycle.

[0248] In another embodiment, the additional anticancer agent comprises cytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to 2000 mg/m ² /cycle.

[0249] In another embodiment, the additional anticancer agent comprises docetaxel at a dose ranging from approximately 1.5 to 7.5 mg/kg/cycle. [0250] In another embodiment, the additional anticancer agent comprises paclitaxel at a dose ranging from approximately 5 to 15 mg/kg/cycle.

[0251] In yet another embodiment, the additional anticancer agent comprises cisplatin at a dose ranging from approximately 5 to 20 mg/kg/cycle.

[0252] In yet another embodiment, the additional anticancer agent comprises 5- fluorouracil at a dose ranging from approximately 5 to 20 mg/kg/cycle.

[0253] In yet another embodiment, the additional anticancer agent comprises doxorubicin at a dose ranging from approximately 2 to 8 mg/kg/cycle.

[0254] In yet another embodiment, the additional anticancer agent comprises epipodophyllotoxin at a dose ranging from approximately 40 to 160 mg/kg/cycle.

[0255] In yet another embodiment, the additional anticancer agent comprises cyclophosphamide at a dose ranging from approximately 50 to 200 mg/kg/cycle.

[0256] In yet another embodiment, the additional anticancer agent comprises irinotecan at a dose ranging from approximately 50 to 75, 75 to 100, 100 to 125, or 125 to 150 mg/m ² /cycle.

[0257] In yet another embodiment, the anticancer agent comprises vinblastine at a dose ranging from approximately 3.7 to 5.4, 5.5 to 7.4, 7.5 to 1 1, or 11 to 18.5 mg/m ² /cycle.

[0258] In yet another embodiment, the additional anticancer agent comprises vincristine at a dose ranging from approximately 0.7 to 1.4, or 1.5 to 2 mg/m ² /cycle.

[0259] In yet another embodiment, the additional anticancer agent comprises methotrexate at a dose ranging from approximately 3.3 to 5, 5 to 10, 10 to 100, or 100 to 1000 mg/m ² /cycle.

[0260] Combination therapy with an immunotoxin may sensitize the cancer or tumor to administration of an additional cancer therapeutic. Accordingly, the present invention contemplates combination therapies for preventing, treating, and/or preventing recurrence of cancer comprising administering an effective amount of an immunotoxin prior to, subsequently, or concurrently with a reduced dose of a cancer therapeutic. For example, initial treatment with an immunotoxin may increase the sensitivity of a cancer or tumor to subsequent challenge with a dose of cancer therapeutic. This dose is near, or below, the low range of standard dosages when the cancer therapeutic is administered alone, or in the absence of an immunotoxin. When concurrently administered, the immunotoxin may be administered separately from the cancer therapeutic, and optionally, via a different mode of administration. [0261] Accordingly, in one embodiment, the additional cancer therapeutic comprises cisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at a dose ranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75 mg/m ² / cycle.

[0262] In another embodiment, the additional cancer therapeutic comprises carboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging from approximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75 mg/m ² /cycle.

[0263] In another embodiment, the additional cancer therapeutic comprises cyclophosphamide, e.g., CYTOXAN (Bristol Myers Squibb), at a dose ranging from approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to 10, 11 to 20, or 21 to 40 mg/kg/cycle.

[0264] In another embodiment, the additional cancer therapeutic comprises cytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging from approximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to 100 mg/m ² /cycle. In another embodiment, the additional anticancer agent comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.), at a dose ranging from approximately 5 to 50 mg/m ² /cycle.

[0265] In another embodiment, the additional cancer therapeutic comprises dacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose ranging from approximately 15 to 250 mg/m2/cycle or ranging from approximately 0.2 to 2 mg/kg/cycle.

[0266] In another embodiment, the additional cancer therapeutic comprises topotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging from approximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5 mg/m ² /Cycle.

[0267] In another embodiment, the additional cancer therapeutic comprises irinotecan, e.g., CAMPTOSAR (Pharmacia & Upjohn), at a dose ranging from approximately 5 to 9, 10 to 25, or 26 to 50 mg/m ² /cycle.

[0268] In another embodiment, the additional cancer therapeutic comprises fludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging from approximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m ² /cycle.

[0269] In another embodiment, the additional cancer therapeutic comprises cytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to 2000 mg/m2/cycle, 300 to 1000 mg/m2/cycle, 400 to 800 mg/m2/cycle, or 500 to 700 mg/m ² /cycle.

[0270] In another embodiment, the additional cancer therapeutic comprises docetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging from approximately 6 to 10, 11 to 30, or 31 to 60 mg/m ² /cycle. [0271] In another embodiment, the additional cancer therapeutic comprises paclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging from approximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

[0272] In another embodiment, the additional cancer therapeutic comprises 5- fiuorouracil at a dose ranging from approximately 0.5 to 5 mg/kg/cycle, 1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

[0273] In another embodiment, the additional cancer therapeutic comprises doxorubicin, e.g., ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX (Bristol Myers Squibb), at a dose ranging from approximately 2 to 4, 5 to 8, 9 to 15, 16 to 30, or 31 to 60 mg/kg/cycle.

[0274] In another embodiment, the additional cancer therapeutic comprises etoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging from approximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m ² /cycle.

[0275] In another embodiment, the additional cancer therapeutic comprises vinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging from approximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6 mg/m2/cycle.

[0276] In another embodiment, the additional cancer therapeutic comprises vincristine, e.g., ONCOVIN (Eli Lilly), at a dose ranging from approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 mg/m ² /cycle.

[0277] In another embodiment, the additional cancer therapeutic comprises methotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6 to 10, or 11 to 20 mg/m ² / cycle.

[0278] In another embodiment, an immunotoxin is administered in combination with at least one other immunotherapeutic which includes, without limitation, rituxan, rituximab, campath-1, gemtuzumab, and trastuzumab.

[0279] In another embodiment, an immunotoxin is administered in combination with one or more anti-angiogenic agents which include, without limitation, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor), anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13 amino acid peptide corresponding to a fragment of platelet factor-4, a 14-amino acid peptide corresponding to a fragment of collagen I, a 19 amino acid peptide corresponding to a fragment of thrombospondin I, a 20-amino acid peptide corresponding to a fragment of SPARC, and a variant thereof, including a pharmaceutically acceptable salt thereof. [0280] In another embodiment, an immunotoxin is administered in combination with a regimen of radiation therapy. The therapy may also comprise surgery and/or chemotherapy. For example, the immunotoxin may be administered in combination with radiation therapy and cisplatin (Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), and/or paclitaxel (Taxol). Treatment with the immunotoxin may allow use of lower doses of radiation and/or less frequent radiation treatments, which may for example, reduce the incidence of severe sore throat that impedes swallowing function potentially resulting in undesired weight loss or dehydration.

[0281] In another embodiment, an antibody or immunotoxin is administered in combination with one or more cytokines which include, without limitation, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokine, lymphotoxin, interferon, macrophage inflammatory protein, granulocyte monocyte colony stimulating factor, interleukin (including, without limitation, interleukin-1 , interleukin-2, interleukin-6, interleukin-12, interleukin- 15, interleukin-18), and a variant thereof, including a pharmaceutically acceptable salt thereof.

[0282] In yet another embodiment, an immunotoxin is administered in combination with a cancer vaccine including, without limitation, autologous cells or tissues, non- autologous cells or tissues, carcinoembryonic antigen, alpha-feto-protein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins, and mutated, tumor-specific antigens.

[0283] In yet another embodiment, an immunotoxin is administered in association with hormonal therapy. Hormonal therapeutics include, without limitation, a hormonal agonist, hormonal antagonist (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroid (e.g., dexamethasone, retinoid, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid, mineralocorticoid, estrogen, testosterone, progestin).

[0284] In yet another embodiment, an immunotoxin is administered in association with a gene therapy program to treat or prevent cancer.

[0285] In yet another embodiment, an EpCAM-targeted immunotoxin is administered in combination with one or more agents that increase expression of EpCAM in the tumor cells of interest. EpCAM expression preferably is increased so that a greater number of EpCAM molecules are expressed on the tumor cell surface. For example, the agent may inhibit the normal cycles of EpCAM antigen endocytosis. Such combination treatment may improve the clinical efficacy of the EpCAM-targeted immunotoxin alone, or with other anticancer agents or radiation therapy. In specific, nonlimiting embodiments, the agent which increases EpCAM expression in the tumor cells is vinorelbine tartrate (Navelbine) and/or paclitaxel (Taxol).

[0286] In yet another embodiment, a HER2/neu-targeted immunotoxin is administered in combination with one or more agents that increase expression of HER2/neu in the tumor cells of interest. HER2/neu expression preferably is increased so that a greater number of HER2/neu molecules are expressed on the tumor cell surface. For example, the agent may inhibit the normal cycles of HER2/neu endocytosis. Such combination treatment may improve the clinical efficacy of the Her2/neu-targeted immunotoxin alone, or with other cancer therapeutics or radiation therapy.

[0287] In yet another embodiment, a CSA-targeted immunotoxin is administered in combination with one or more agents that increase expression of CSA in the tumor cells of interest. CSA expression preferably is increased so that a greater number of CSA molecules are expressed on the tumor cell surface. For example, the agent may inhibit the normal cycles of CSA endocytosis. Such combination treatment may improve the clinical efficacy of the CSA-targeted immunotoxin alone, or with other cancer therapeutics or radiation therapy.

[0288] Combination therapy may thus increase the sensitivity of the cancer or tumor to the administered immunotoxin and/or additional cancer therapeutic. In this manner, shorter treatment cycles may be possible thereby reducing toxic events. Accordingly, the invention provides a method for treating or preventing cancer comprising administering to a patient in need thereof an effective amount of an immunotoxin and at least one other cancer therapeutic for a short treatment cycle. The cycle duration may range from approximately 1 to 30, 2 to 27, 3 to 15, 4 to 12, 5 to 9, or 6-8 days. The cycle duration may vary according to the specific cancer therapeutic in use. The invention also contemplates continuous or discontinuous administration, or daily doses divided into several partial administrations. An appropriate cycle duration for a specific cancer therapeutic will be appreciated by the skilled artisan, and the invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic. Specific guidelines for the skilled artisan are known in the art. See, e.g., Therasse et al., 2000, "New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada," J Natl Cancer Inst. February 2; 92(3):205-16.

[0289] Alternatively, longer treatment cycles may be desired. Accordingly, the cycle duration may range from approximately 10 to 56, 12 to 48, 14 to 28, 16 to 24, or 18 to 20 days. The cycle duration may vary according to the specific cancer therapeutic in use. Routes of Administration

[0290] The immunotoxins described herein may be administered to the patient via any suitable route. The immunotoxins may be administered by injection into the vascular system or by injection into an organ. Preferred administration routes include parenteral, intravascular and/or intravenous injection. Parenteral administration includes subcutaneous, intramuscular, intraperitoneal, intracavity, intrathecal, intratumoral, transdermal and intravenous injection. In a preferred embodiment, the antibodies and/or immunotoxins are administered intravenously as a bolus or by continuous infusion over a period of time. In another embodiment, the immunotoxin is administered by intracranial infusion. In other embodiments, the antibodies and/or immunotoxins may be administered directly to the cancer site.

[0291] The immunotoxin and antibodies of the present invention can be administered in the conventional manner by any route where they are active. Administration can be systemic, parenteral, topical, or oral. For example, administration can be, but is not limited to, parenteral, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants. Thus, modes of administration for the antibodies/immunotoxins of the present invention (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use according to vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.

[0292] In accordance with one aspect of the present invention, the immunotoxin and/or other anticancer agent is delivered to the patient by direct administration. Accordingly, the immunotoxin and/or other anticancer agent may be administered, without limitation, by one or more direct injections into the tumor, by continuous or discontinuous perfusion into the tumor, by introduction of a reservoir of the immunotoxin, by introduction of a slow-release apparatus into the tumor, by introduction of a slow-release formulation into the tumor, and/or by direct application onto the tumor. By the mode of administration into the tumor, introduction of the immunotoxin and/or other anticancer agent to the area of the tumor, or into a blood vessel or lymphatic vessel that substantially directly flows into the area of the tumor, is also contemplated. In each case, the pharmaceutical composition is administered in at least an amount sufficient to achieve the endpoint, and if necessary, comprises a pharmaceutically acceptable carrier. [0293] It is contemplated that the immunotoxins may be administered intratumorally, whereas any other anticancer agent may be delivered to the patient by other modes of administration (e.g., intravenously). Additionally, where multiple anticancer agents are intended to be delivered to a patient, the immunotoxin and one or more of the other anticancer agent may be delivered intratumorally, whereas other anticancer agents may be delivered by other modes of administration (e.g., intravenously and orally).

[0294] In some embodiments, a composition may be an antibody described herein and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer. In some embodiments, a composition may be an immunotoxin described herein and a pharmaceutically acceptable excipient, carrier, buffer or stabilizer. An immunotoxin according to the invention may be comprised in a pharmaceutical composition or medicament. Pharmaceutical compositions adapted for direct administration include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Immunotoxin may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.

[0295] Pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(l(2,3-dioleyloxy) propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.

[0296] In another embodiment, a pharmaceutical composition comprises an antibody or immunotoxin and one or more additional anticancer agent, optionally in a pharmaceutically acceptable carrier.

[0297] The composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.

[0298] In various embodiments of the invention, the pharmaceutical composition is directly administered to the area of the tumor(s) by, for example, local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), injection, means of a catheter, means of a suppository, or means of an implant. An implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Suppositories generally contain active ingredients in the range of 0.5% to 10% by weight.

[0299] In other embodiments, a controlled release system can be placed in proximity of the target tumor. For example, a micropump may deliver controlled doses directly into the area of the tumor, thereby finely regulating the timing and concentration of the pharmaceutical composition.

[0300] In some embodiments, the pharmaceutical carrier may include, without limitation, binders, coating, disintegrants, fillers, diluents, flavors, colors, lubricants, glidants, preservatives, sorbents, sweeteners, conjugated linoleic acid (CLA), gelatin, beeswax, purified water, glycerol, any type of oil, including, without limitation, fish oil or soybean oil, or the like. Pharmaceutical compositions of the antibodies/immunotoxins also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.

[0301] For oral administration, the immunotoxins and antibodies can be formulated readily by combining these immunotoxins/antibodies with pharmaceutically acceptable carriers well known in the art. Such carriers enable the immunotoxins of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0302] Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of antibodies/immunotoxins doses.

[0303] Pharmaceutical preparations which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the immunotoxins/antibodies can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.

[0304] For buccal administration, the compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.

[0305] For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use according to a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the immunotoxins/antibodies and a suitable powder base such as lactose or starch.

[0306] The compositions of the present invention can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0307] In addition to the formulations described previously, the compositions of the present invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.

[0308] Depot injections can be administered at about 1 to about 6 months or longer intervals. Thus, for example, the immunotoxins can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0309] In transdermal administration, the compositions of the present invention, for example, can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism.

[0310] The compositions of the present invention can also be administered in combination with other active ingredients, such as, for example, adjuvants, protease inhibitors, or other compatible drugs or compounds where such combination is seen to be desirable or advantageous in achieving the desired effects of the methods described herein.

[0311] In some embodiments, the disintegrant component comprises one or more of croscarmellose sodium, carmellose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, an ion exchange resin, an effervescent system based on food acids and an alkaline carbonate component, clay, talc, starch, pregelatinized starch, sodium starch glycolate, cellulose floe, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, a metal carbonate, sodium bicarbonate, calcium citrate, or calcium phosphate.

[0312] In some embodiments, the diluent component comprises one or more of mannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powdered cellulose, microcrystalline cellulose, carboxymethylcellulose, carboxyethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodium starch glycolate, pregelatinized starch, a calcium phosphate, a metal carbonate, a metal oxide, or a metal aluminosilicate.

[0313] In some embodiments, the optional lubricant component, when present, comprises one or more of stearic acid, metallic stearate, sodium stearyl fumarate, fatty acid, fatty alcohol, fatty acid ester, glyceryl behenate, mineral oil, vegetable oil, paraffin, leucine, silica, silicic acid, talc, propylene glycol fatty acid ester, polyethoxylated castor oil, polyethylene glycol, polypropylene glycol, polyalkylene glycol, polyoxyethylene-glycerol fatty ester, polyoxyethylene fatty alcohol ether, polyethoxylated sterol, polyethoxylated castor oil, polyethoxylated vegetable oil, or sodium chloride. Nucleic Acid Molecules

[0314] A person skilled in the art will appreciate that the novel nucleic acid sequences of the present application can be used in a number of recombinant methods.

[0315] In some embodiments, the sequences, vectors, and constructs of the present invention are codon optimized to the organism in which they are used. In some embodiments, the codon usage in the coding sequences of the present invention is optimized to express one or more immunotoxins described herein. In some embodiments, the codons of a deimmunized Bouganin are optimized for expression in non-native bacterial, archaeal, and eukaryotic systems. An exemplary codon optimized deBouganin is shown in SEQ ID NO: 13. Methods of codon-optimization are well-known to those skilled in the art. More information about codon optimization can be found in US2008/0194511, and US2007/0292918, both of which are incorporated herein for all purposes.

[0316] Accordingly, the nucleic acid sequences of the present application may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the proteins encoded thereof. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the present application and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

[0317] The present application therefore contemplates a recombinant expression vector of the present application containing a nucleic acid molecule of the present application, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein sequence.

[0318] Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (For example, see the regulatory sequences described in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.

[0319] The recombinant expression vectors of the present application may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the present application. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, (3-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the present application and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.

[0320] The recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.

[0321] Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. The term "transformed host cell" as used herein is intended to also include cells capable of glycosylation that have been transformed with a recombinant expression vector of the present application. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium- chloride mediated transformation. For example, nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in (Sambrook et al, 2001) (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001), and other laboratory textbooks.

[0322] Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the present application may be expressed in yeast cells or mammalian cells. Other suitable host cells can be found in (Goeddel, 1990), Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991). In addition, the proteins of the present application may be expressed in prokaryotic cells, such as Escherichia coli (Zhang et al, 2004), Science 303(5656): 371-3). In addition, a Pseudomonas based expression system such as Pseudomonas fluorescens can be used (US Patent Application Publication No. US 2005/0186666, (Schneider et al, 2005)).

[0323] Yeast and fungi host cells suitable for carrying out the present application include, but are not limited to Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl ((Baldari et al, 1987), Embo J. 6: 229- 234), pMFa ((Kurjan and Herskowitz, 1982), Cell 30: 933-943 (1982)), pJRY88 ((Schultz et al., 1987), Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art (see (Hinnen et al., 1978) Proc. Natl. Acad. Sci. USA 75: 1929); ((Ito et al, 1983), J. Bacteriology 153: 163) and ((Cullen et al., 1987) BiolTechnology 5: 369).

[0324] Mammalian cells suitable for carrying out the present application include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 ((Seed, 1987), Nature 329: 840) and pMT2PC ((Kaufman et al, 1987), EMBO J. 6: 187-195). [0325] Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished. For example, within one embodiment, the proteins of the present application may be expressed from plant cells (see (Sinkar et al, 1987), J. Biosci (Bangalore) 11 : 47-58), which reviews the use of Agrobacterium rhizogenes vectors; see also ((Zambryski et al, 1984), Genetic Engineering, Principles and Methods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, New York), which describes the use of expression vectors for plant cells, including, among others, PAPS2022, PAPS2023, and PAPS2034).

[0326] Insect cells suitable for carrying out the present application include cells and cell lines from Bombyx, Trichoplusia or Spodotera species. Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series ((Smith et al., 1983), Mol. Cell. Biol. 3: 2156-2165) and the pVL series ((Luckow and Summers, 1989), Virology 170: 31-39). Some baculovirus-insect cell expression systems suitable for expression of the recombinant proteins of the present application are described in PCT/US/02442.

[0327] Alternatively, the proteins of the present application may also be expressed in non-human transgenic animals such as rats, rabbits, sheep and pigs ((Hammer et al, 1985). Nature 315:680-683); (Brinster et al, 1985; Palmiter and Brinster, 1985; Palmiter et al, 1983) Science 222:809-814); and ((Leder and Stewart, 1988) U.S. Pat. No. 4,736,866).

[0328] Accordingly, the present application provides a recombinant expression vector comprising one or more of the novel nucleic acid sequences as well as methods and uses of the expression vectors in the preparation of recombinant proteins. Further, the application provides a host cell comprising one or more of the novel nucleic acid sequences or expression vectors comprising one or more of the novel nucleic acid sequences.

Antibody or antibody fragments

[0329] The present application also includes antibody or an antibody fragment comprising one or more of the amino acid sequences disclosed herein (i.e. SEQ ID NOS: 2, 4, 23, 25, 27, 29, 31, 49, 51, 53, 55-57, 69, 71, 73, 75-89, 92, 94-98, 99-101, 103, 120-123, 125, 127, 129, 131, 133 and 135). In one embodiment, the antibody or antibody fragment comprises amino acids 23-535 of the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 27. In another embodiment, the antibody or antibody fragment comprises amino acids 23-529 of the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 29 or SEQ ID NO: 31. In another embodiment, the antibody or antibody fragment comprises an amino acid sequence selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In another embodiment, the antibody or antibody fragment comprises an amino acid sequence set forth in SEQ ID NO: 131.

[0330] In one embodiment, the antibody or antibody fragment comprises the VH region shown in SEQ ID NO: 2 and the VL region shown in SEQ ID NO: 4. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region encoded by amino acid sequences selected from SEQ ID NOs: 125, 127, 129, 131, 133 and 135. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region encoded by an amino acid sequence set forth in SEQ ID NO: 131. In another embodiment, the antibody or antibody fragment comprises a heavy chain having an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 56, and SEQ ID NO: 57, and a light chain having an amino acid sequence selected from SEQ ID NO: 51 and SEQ ID NO: 55. In another embodiment, the antibody or antibody fragment comprises a heavy chain having an amino acid sequence selected from SEQ ID NO: 99, SEQ ID NO: 121 and SEQ ID NO: 122, and a light chain having an amino acid sequence selected from SEQ ID NO: 100 and SEQ ID NO: 120.

[0331] The present application also includes the use of the novel nucleic acid sequences for the preparation of antibodies or antibody fragments and methods thereof.

[0332] The present application includes the use of the antibodies or antibody fragments disclosed herein in any and all applications including diagnostic and therapeutic applications. In one embodiment, the antibodies or antibody fragments are used for detecting or monitoring cancer. In another embodiment, the antibodies or antibody fragments are used for treating cancer.

[0333] The present application also includes leader sequences. In one embodiment, the leader sequence is encoded by the nucleic acid sequence shown in SEQ ID NO: 20 or comprises the amino acid sequence shown in SEQ ID NO: 21. Such leader sequences can be used to optimize the expression of recombinant proteins including immunotoxins.

[0334] The present application also includes linker sequences. In particular, the present application includes the linker sequences encoded by the amino acid sequences shown in SEQ ID NOs: 15, 17, 32-36. The linker sequences can be used in the preparation of immunotoxins. [0335] The present invention will be better understood by the following exemplary teachings. The examples set forth herein are not intended to limit the invention.

INCORPORATION BY REFERENCE

[0336] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art in any country in the world.

EXAMPLES

EXAMPLE 1: Biological characterization of VB6-901-DI-linker variants— Experimental Design

[0337] VB6-901 is a recombinant immunotoxin that comprises an anti-Epithelial Cell Adhesion Molecule (EpCAM) MOCB-Fab fragment genetically fused to a de-immunized Bouganin (deBouganin) via a furin protease sensitive linker (VBRR0405 Increased VB6-901 soluble expression via molecular re-engineering). A phase I clinical study showed that 6 out of 8 cancer patients treated with VB6-901 for 4 to 12 weeks had stable disease. The analysis of patient sera confirms that deBouganin was de-immunized as negligible to no titer was observed after 4 to 8 weeks treatment (VB6-901 clinical report, A Phase I, Escalating Dose Study of VB6-901, A Recombinant Fusion Protein Targeting EpCAM, in Patients with Advanced Solid Tumors of Epithelial Origin: Final Report (Study No. VB6-901-01-I) 04MarlO). However, a significant immune response was detected against the Fab portion in all patients. The isotyping of the immune response showed a class switch from IgM to IgG suggesting the involvement of T-cells and consequently the presence of T-cell epitopes. Therefore, the amino acid sequences of the variable domains were analyzed in-silico by Antitope and a de-immunized VB5-845-DI and VB6-901-DI were engineered and their biological activity characterized (VBRR0733 Biological characterization of the de- immunized anti-EpCAM 4D5 Fab fragment). In addition, the furin linker that links the C- terminal end of the light chain to the N-terminus end of deBouganin was also examined. The analysis revealed a weak potential T-cell epitope. Therefore, the leucine residue of the furin linker (TRHRQPRGWEQL) was mutated to H (SEQ ID NO: 32), K (SEQ ID NO: 33), N (SEQ ID NO: 34), P (SEQ ID NO: 35) or S (SEQ ID NO: 36) and the level of expression, potency and stability for each clone assessed. The VB6-901-DI with mutation L to K (SEQ ID NO: 71) was one of the clones with the highest level of expression and was slightly more potent than the other variants. In addition, serum stability analysis and storage at various temperatures showed that the K mutation did not change the stability of the VB6-901 fusion protein.

[0338] Therefore, the following describes the engineering and biological characterization of VB6-901-DI variant fusion proteins with a mutated furin linker allowing its de-immunization based on an in-silico analysis.

1) Molecular engineering

[0339] Using site-directed mutagenesis specific primers, the linker variant fragments were obtained by SOE-PCR, cloned into pCR2.1 plasmid and transformed into 10F E. coli cells for sequencing (SOP 2.1.86, Molecular engineering of a DNA fragment using the splice overlap extension polymerase chain reaction method; SOP 2.1.82, Transformation of a Recombinant Plasmid into Chemically Competent and Electrocompetent E. coli Cells; SOP 2.1.2, Purification of Plasmid DNA using a QIAprep Spin Miniprep Kit; SOP 2.1.131, Sequencing Reaction for the CEQ 8000 Genetic Analysis System). After confirming the sequence, the F-deBouganin linker variant and VB6-901-DI fragments were digested with Bsml-Xhol and EcoRI-Bsml, respectively, and ligated into the pING3302 plasmid digested with EcoRl-Xhol restriction enzymes. The resulting plasmids were then transformed into El 04 E. coli cells for expression (SOP 2.1.86, Molecular engineering of a DNA fragment using the splice overlap extension polymerase chain reaction method).

2) Small-Scale Expression

[0340] Transformed E104 cells containing the VB6-901-DI/3302 plasmid linker variants were inoculated into 5 mL 2xYT containing 25 μg/mL tetracycline and incubated at 37°C with constant shaking at 225 rpm. After 16 hours of incubation, 300 overnight seed culture was inoculated into 30 mL TB (1% inoculum), and incubated at 37°C with constant shaking at 225 rpm until an OD ₆ oo of 2.0 was attained (SOP 2.1.83, Growth and Expression of E. coli Transformed with a Recombinant pING3302 Plasmid). The culture was induced with 150 L-Arabinose (0.1% final), and incubated at 25°C with constant shaking at 225 rpm. At 16 hours post-induction, the culture supernatant was collected for analysis by Western blot. 3) Western Blot Analysis

[0341] The level of expression of the VB6-901-DI linker variants was estimated by Western blot analysis. Briefly, 16 of induced culture supernatant and 4 LDS sample buffer were loaded onto a NuPAGE 10% Bis-Tris gel (SOP 2.1.55, Electrophoresis, Transfer and Staining of SDS-Page Gels Using the Novex System). The gel was then transferred to a nitrocellulose membrane at 40V for 1 hour. After blocking and washing the membrane, the VB6-901-DI linker variant proteins were detected using an anti-kappa-horseradish peroxidase antibody (1/1000) for 2 hours at room temperature (SOP 2.1.63, Immunodetection of Electroblotted Proteins). The membrane was developed using DAB, and the level of expression of the VB6-901-DI linker proteins compared to VB6-901-DI.

4) VB 6-901-DI linker purification

[0342] Transformed E.coli cells were grown in Glycerol minimal media (15 L) in a 20 L bioreactor to an OD ₆ oo of 50 at which time the cells were induced with the addition of L- arabinose for 30 hrs. Then, the culture supernatant was microfiltered, concentrated 10-fold and buffer exchanged against 20 mM sodium phosphate (5x) for binding onto a 50 mL CM sepharose column. The product in the CM eluate was then flowed through a 25 mL Q- sepharose column and captured on a 5 mL SP-sepharose column. The SP eluate was then applied to a 2 L Sephacryl-S200 size exclusion column and the peaks were fractionated. Based on the SEC chromatograms, the product peak fractions were pooled and applied to another 5 mL SP-sepharose column. The bound protein was then eluted in 20 mM sodium phosphate, 300 mM NaCl, pH 7.5, filter sterilized and stored at -20°C.

5) Quantification by ELISA

[0343] An ELISA was used to quantify the VB6-901-DI linker variant protein present in the CM starting material (SOP 2.1.116 Quantification of VB6-901-CL Bouganin in fed- batch culture supernatant using enzyme linked immunosorbant assay (ELISA)). Briefly, an ImmunolonlB plate was coated with 10 μg/mL rabbit-anti-bouganin and incubated overnight at 4°C. After washing and blocking the plate, the CM starting materials, diluted at 1/6400 and 1/12800 as well as a standard curve prepared from the purified VB6-901 antibody (25 ng/mL-0.195 ng/mL), were added to the plate and incubated for 2 hours at 22°C. Bound VB6-901-DI linker protein was detected using an anti-kappa-horseradish peroxidase antibody (1/1000) for 1 hour at 22°C. The plate was developed using TMB. The pING3302 plasmid induced culture supernatants were used as a positive and negative control, respectively.

6) VB6-901-DI linker variants biological activity

[0344] Binding reactivity— Flow cytometry was used to measure the binding reactivity of the VB6-901-DI linker variants (SOP 2.1.101, Detection of cell-surface bound IgG/IgM by flow cytometry). Briefly, VB6-901-DI variants were incubated at 100 ng/mL with 0.2 x 10 ⁶ CAL-27 cells on ice for 2 hours. After washing with PBS-5% FBS, a biotinylated goat-anti-human IgG antibody (1/200) was added and incubated for 1 hour on ice. The cells were washed with PBS-5% FBS and streptavidin-cy chrome (1/120) was added for 30 minutes on ice to detect cell-bound VB6-901-DI linker variants. The selectivity of the binding was assessed with EpCAM-negative cell line, A-375.

[0345] Cytotoxicity— The cytotoxicity was measured with a MTS assay using EpCAM-positive CAL-27 and OVCAR-3 cells seeded at 5000 cells per well (SOP 2.1.15, MTS Assay). VB6-901 was used as a positive control. The selectivity of the potency was measured with EpCAM-negative cell line, A-375.

7) Stability

[0346] Serum— The mouse and human serum stability of VB6-901-DI linker variants (K and N) was determined by Western blot analysis. Briefly, variants were added at a concentration of 80 μg/mL in 500 of serum and incubated at 37°C, 5% CO2 for 72 hours. At 0 hour, 24, 48 and 72 hours, samples were vortexed and a 45 aliquot was taken and stored at -20°C in presence of 15 μΐ. LDS. The Western blot was performed as above, loading 200 ng/well of variant. The VB6-901-DI linker proteins were detected using a rabbit-anti-4D5 antibody (1/1000) for 45 minutes at room temperature, followed by an anti- rabbit-horseradish peroxidase antibody (1/2000) for 45 minutes at room temperature. The membrane was developed with DAB.

[0347] Storage stability— The storage stability of VB6-901-DI linker variants K and N was determined at 37°C, 4°C and -20°C. Briefly, samples were divided into 40 μΐ.

aliquots and placed at the different temperatures. After 2, 4, 8 and 13 weeks, one aliquot for each construct at different temperatures was taken and analyzed by HPLC. The HPLC profiles were compared to time zero for each construct. EXAMPLE 2: Biological characterization of VB6-901-DI-linker variants— Results

1) In-silico analysis of the furin linker and junction

[0348] The in-silico analysis of the furin linker junctions with the C-terminal end of the kappa light chain and the N-terminal end of deBouganin was performed by Antitope and revealed a moderate potential T-cell epitope. Therefore, to eliminate the potential T-cell epitope, the leucine residue corresponding to the last amino acid of the furin linker was mutated to histidine, asparagine, lysine, proline or serine.

2) VB6-901-DI linker variants small-scale expression

[0349] The mutated furin linkers were engineered by SOE-PCR and expressed as VB6-901-DI fusion proteins (NB 1394, p26-44). Selected E104 transformed with VB6-901- DI linker variants cloned into the pING3302 plasmid were induced with L-arabinose. Western blot analysis of two independent clones for each linker variant revealed a band migrating ~ 65 kDa present in the supernatant, respectively (FIG. 2). Of note, the intensity of the supernatant bands was similar to the positive control VB6-901-DI, suggesting that the mutations in the linker did not affect expression.

3) Purification, yield and biological activity of the linker variants

[0350] The VB6-901-DI linker variants were grown in a 15L fermentor using HCD conditions. The ELISA measurement of the CM starting material suggested that the K and N linker had the highest level of expression followed by H and S whereas the P linker was poorly expressed (Table 1). The poor expression of the P linker was due to degradation as indicated by the SEC chromatogram which showed a high level of Fab fragment (data not shown). The higher expression level observed from the supernatant translated into higher purified yield for the K and N linker variants. The biological activity was assessed by flow cytometry and MTS. As expected, all linker variants had similar binding reactivity against CAL-27 which was slightly lower than the VB6-901-WT (NB 1394, p71-94). However, the potency assay revealed that the K linker variant was a bit more potent than the others against both EpCAM-positive cell lines (FIG. 3A and 3B). The same trend was also observed with a second purified lot comparing linker H, K and N (data not shown). Therefore, at this stage, clone H, P and S were eliminated based on expression and recovery level. Both K and N clones were then assessed for serum stability and storage stability (NB 1394, p95). As seen in FIG. 3C and 3D, Western blot analysis showed that both clones remained intact in mouse and human sera suggesting that the linker mutations did not create potential proteolytic sites.

[0351] Table 1: Summary of linker clone selection

F.I.: Fold-increase over PBS.

ND: Not determined.

* Purified using a chelating column.

** Quantified from the CM starting material.

4) Storage stability

[0352] To ensure that the linker mutations did not affect storage stability, the variants were incubated at -20°C, 4°C and 37°C and aliquots were taken after 2, 4, 8 and 13 weeks and the percentage of monomer was assessed by HPLC. Both linker variants were stable at 4°C and -20°C as reported earlier for the WT molecule (VBRR0616 Interim report long term stability testing of VB6-901 VBI C03071 (lOmg/mL) at 12 months).

[0353] Table 2: Linker variant stability at 4°C and -20°C

[0354] The Examples describe the engineering and biological characterization of VB6-901-DI containing one point mutation within the linker leading to its de-immunization. The in silico analysis of the furin linker and deBouganin junction by Antitope identified one weak potential T-cell epitope. Therefore, based on their recommendation, 4 mutations were tested and the level of expression, biological activity, serum and storage stability were determined and used to select the final clone. The VB6-901-DI linker K was the most potent among the variants. Its expression level was one of the highest and the stability of the molecule was not altered compared to wild-type molecule. VB6-901-DI linker K (SEQ ID NOs: 49 and 71) was therefore selected as the final clone. Of note, as shown in previous reports, the mutations introduced for the de-immunization of the Fab fragment resulted in lower binding activity as compared to the wild-type molecule explaining the decreased potency of the linker variants (VBRR00676-R02, Engineering and biological testing of T-cell epitope depleted-Fab moiety of VB6-901).

EXAMPLE 3: Biological characterization of deBouganin-AvP07-17 containing de- immunized furin linker variants— Experimental Design

[0355] deBouganin-F-AvP07-17 is a recombinant immunotoxin that comprises an anti-Her2 C6.5 diabody fragment (AvP07-17) genetically linked to deBouganin via a furin (F) protease sensitive linker. In vitro study demonstrated that debouganin-F-AvP07-17 IC5 ₀ S were in the subnanomolar range with most of the Her2 3+ tumor cell lines. By engineering a fusion construct, a new junction between the furin linker and the VH domain was created. To ensure that no potential T-cell epitopes were present, the junction was assessed in-silico by Antitope (Cambridge, UK). The analysis revealed a strong potential T-cell epitope. Therefore, the leucine residue of the furin linker (TRHRQPRGWEQL, SEQ ID NO: 17) was mutated to E (SEQ ID NO: 119), P (SEQ ID NO: 35) or T (SEQ ID NO: 124) and the level of expression, potency and stability for each clone assessed. The potency and expression of all clones was similar to the parental fusion protein; however, deBouganin-F(E)-AvP07-17 with the L to E mutation was more stable in mouse and human serum. Hence, deBouganin-F(E)- AvP07-17 was selected for future pre-clinical efficacy study.

[0356] Therefore, the following describes the engineering and characterization of the biological activity of deBouganin-F-AvP07-17 fusion proteins with a mutated furin linker allowing its de-immunization based on an in-silico analysis. 1) Molecular engineering

[0357] Using site-directed mutagenesis specific primers, the linker variant fragments were obtained by SOE-PCR, cloned into pCR2.1 plasmid and transformed into 10F E. coli cells for sequencing. After confirming the sequence, the deBouganin-F linker variant inserts were digested with Spel-Bglll and ligated into the deBouganin-F-AvP07-17-His/pING3302 plasmid digested with the same restriction enzymes. The resulting plasmids were then transformed into El 04 E. coli cells for expression.

[0358] "deBouganin-F(E)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 119), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 126 (nucleotide sequence) and by SEQ ID NO: 125 (amino acid sequence). "deBouganin-F(T)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 124), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 128 (nucleotide sequence) and by SEQ ID NO: 127 (amino acid sequence). "deBouganin-F(P)-AvP07-17-His" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 35), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and a His tag at the C-terminus, and which is represented by nucleotides 132-1670 of SEQ ID NO: 130 (nucleotide sequence) and by SEQ ID NO: 129 (amino acid sequence).

[0359] A final clone without a His tag was generated by swapping the C-terminal end of AvP07-17-His with AvP07-17 using Bsu36I and Xhol restriction enzymes.

[0360] "deBouganin-F(E)-AvP07-17" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 119), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 132 (nucleotide sequence) and by SEQ ID NO: 131 (amino acid sequence). "deBouganin-F(T)- AvP07-17" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 124), an anti-HER2/neu heavy chain variable region (V _H ) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 134 (nucleotide sequence) and by SEQ ID NO: 133 (amino acid sequence). "deBouganin-F(P)-AvP07-17" refers to an antibody fragment comprised of, starting at the N-terminus: deBouganin toxin, deimmunized furin linker (SEQ ID NO: 35), an anti-HER2/neu heavy chain variable region (VH) linked to an anti-HER2/neu light chain variable region (VL), and which is represented by nucleotides 132-1652 of SEQ ID NO: 136 (nucleotide sequence) and by SEQ ID NO: 135 (amino acid sequence).

2) Small-Scale Expression

[0361] Transformed El 04 cells containing the deBouganin-F-AvP07-l 7/3302 linker variants plasmid (with or without a His tag) were inoculated into 5 mL 2xYT containing 25 μg/mL tetracycline and incubated at 37°C with constant shaking at 225 rpm. After 16 hours of incubation, 300 overnight seed culture was inoculated into 30 mL TB (1% inoculum), and incubated at 37°C with constant shaking at 225 rpm until an OD600 of 2.0 was attained. The culture was induced with 150 L-Arabinose (0.1% final), and incubated at 25°C with constant shaking at 225 rpm. At 16 hours post-induction, the culture supernatant was collected for analysis by Western blot.

3) Western Blot Analysis

[0362] The level of expression of the deBouganin-F-AvP07-17/3302 linker variants was estimated by Western blot analysis. Briefly, 16 of induced culture supernatant and 4 μΐ. LDS sample buffer were loaded onto a NuPAGE 10% Bis-Tris gel. The gel was then transferred to a nitrocellulose membrane at 40V for 1 hour. After blocking and washing the membrane, the deBouganin-F-AvP07-17 linker variant proteins were detected using a rabbit anti-deBouganin antibody followed by an anti-rabbit-HRP antibody. The membrane was developed using DAB, and the level of expression of the deBouganin-F-AvP07-17 linker proteins compared to parental molecule.

4) deBouganin-F-AvP07-l 7 linker variants cytotoxicity

[0363] The cytotoxicity was measured with an MTS assay using Her2-positive BT- 474, SkBR3 and MDA-MB-453 cells seeded at 5000 cells per well. The specificity of the potency was confirmed against a Her2 0/1+ MDA-MB-231 cell line. 5) Serum stability

[0364] The mouse and human serum stability of deBouganin-F-AvP07-17 linker variants (E and P) was determined by flow cytometry. Briefly, linker variant and parental fusion proteins were incubated at a concentration of 80 μg/mL in 500 of serum and placed at 37°C, 5% C02 for 48 hours. At 0, 24 and 48 hours, samples were vortexed and a 45 aliquot was taken and stored at -20°C. Flow cytometry was used to measure the binding reactivity of the deBouganin-F-AvP07-17 linker variants and compared to the parental protein. Briefly, deBouganin-F-AvP07-17 fusion proteins were incubated at 100 ng/mL with 0.2 x 10 ⁶ BT-474 cells on ice for 2 hours. After washing with PBS-5% FBS, a rabbit-anti- deBouganin antibody (1/100) was added and incubated for 1 hour on ice. The cells were washed with PBS-5% FBS and an anti-rabbit-FITC (1/100) was added for 30 minutes on ice to detect cell-bound deBouganin-F-AvP07-17 fusion proteins.

EXAMPLE 4: Biological characterization of deBouganin-AvP07-17 containing de- immunized furin linker variants— Results

1) In-silico analysis of the furin linker and VH junction

[0365] The in-silico analysis of junction between the furin linker (italicized) and the N-terminus end of the VH domain (underlined)

(TRHROPRGWEOLOVO VOSGAEVKK^GES KT) was performed by Antitope and revealed one high affinity potential T-cell epitope (FIG. 4). Therefore to eliminate the potential T-cell epitope, recommended mutations of the leucine residue (in bold) in order of preference were either proline (P), glutamic acid (E), threonine (T), glycine (G), aspartic acid (D) or alanine (A).

2) deBouganin-F-AvP07-l 7 linker variants small-scale expression

[0366] The first three mutations of the furin linker (P, E and T) were engineered by SOE-PCR and expressed as deBouganin-F-AvP07-17-His fusion proteins (see Molecular engineering in Example 3). Westem blot analysis of two independent clones for each linker variant revealed a deBouganin-F-AvP07-17-His band migrating ~ at 50 kDa present in the supernatant (FIG. 5A). The expression level of the linker variants P (SEQ ID NO: 129) and E (SEQ ID NO: 125) was similar to the parental fusion protein whereas linker variant T was slightly lower.

[0367] As a final fusion protein, the non-His version was engineered for P (SEQ ID NO: 135) and E (SEQ ID NO: 131) linker variants (see Molecular engineering in Example 3). As seen in FIG. 5B, the soluble expression level of both variants was similar to the parental molecule.

3) Purification, yield and biological activity of the linker variants

[0368] The deBouganin-F-AvP07-17 linker variants were grown in a 15L fermentor using HCD conditions. The final yield for both variants was lower than the parental molecule. The biological activity, assessed by MTS, revealed that the potency of both linker variants was similar to the parental molecule against Her2 3+ cell lines (Table 3, FIG. 6A and FIG. 6B).

[0369] Table 3: Yield and potency of linker variants

Linker Yield ICso (nM)

(mg) BT-474 SkBR3 MDA-MB-453 MDA-MB-231

E 88 0.067 ± 0.008 0.32 ± 0.05 0.4 ± 0 > 10

P 93 0.046 ± 0.005 0.3 ± 0 N.D. > 10

WT 110 0.058 ± 0.006 0.32 ± 0.05 0.28 ± 0 .7 > 10

4) Serum stability

[0370] Both E and P linker variants were assessed for mouse and human serum stability. As seen in FIG. 7A-7B, flow cytometry analysis showed that the reactivity of deBouganin-F(E)-AvP07-17 (SEQ ID NO: 131) remained intact after 48 hours incubation. In contrast, the biological activity of deBouganin-F(P)-AvP07-17 (SEQ ID NO: 135) and parental molecule decreased in mouse serum especially after 48 hours, suggesting some degradation. In addition, Western blot analysis confirmed the appearance of a truncated deBouganin fragment which was more intense for the linker P variant and WT compared to linker E variant (data not shown).

[0371] Examples 3 and 4 describe the engineering and biological characterization of deBouganin fused to the C6.5 diabody fragment with a furin linker containing different point mutations required for its de-immunization. The in silico analysis of the junction between the furin linker and the VH domain by Antitope identified one strong potential T-cell epitope. Therefore, based on their recommendation, 2 mutations were tested and the level of expression, biological activity and serum stability were determined and used to select the final clone. The deBouganin-F(E)-AvP07-17 (SEQ ID NO: 131) potency was similar to the parental molecule against representative Her2 3+ tumor cell lines. In addition, the serum stability of the deBouganin-F(E)-AvP07-17 fusion protein was superior to the parental and P linker variant. Therefore, the deBouganin-F(E)-AvP07-17 (SEQ ID NO: 131) was selected for preclinical study. The de-immunization of the furin linker- VH junction will be confirmed using overlapping 15-mer peptides in a T-cell proliferation assay. It is expected that the E mutation will lead to a lower stimulation index of the T-cells.

EXAMPLE 5: Analysis of furin-deBouganin and furin(K)-deBouganin for immunogenicity

[0372] NetMHCII and NetMHCIIpan were employed for the identification of potential T-helper cell epitopes for the most prevalent HLA-alleles, i.e. Alleles found in more than 1% of the population. The first 100 amino acids of Furin-deBouganin and Furin(K)- deBouganin were analyzed for sub-15mer MHC II binding peptides restricted to each of the HLA alleles by decomposing the protein to overlapping 15-mer peptides.

[0373] The mutation L12K led to a drop in the Risk Score (FIG. 8). However, Risk Score at position 12 in Sequence 1 was already very low, so the impact of this mutation in the overall immunogenicity risk is limited.